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The role of Src homology 2 domain containing 5' inositol phosphatase 1 (SHIP) in hematopoietic cells

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Title:
The role of Src homology 2 domain containing 5' inositol phosphatase 1 (SHIP) in hematopoietic cells
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English
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Desponts, Caroline
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University of South Florida
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Subjects / Keywords:
Embryonic stem cell
Hematopoietic stem cell
NK cells
Megakaryocytes
Hematopoiesis
S-SHIP
Dissertations, Academic -- Biochemistry and Molecular Biology -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: The principal isoform of Src homology (SH) 2-domain containing 5' inositol phosphatase protein 1 (SHIP) is a 145kDa protein primarily expressed by cells of the hematopoietic compartment. The enzymatic activity of SHIP is responsible for hydrolyzing the 5' phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), and thereby preventing the recruitment of pleckstrin homology domain containing effector proteins. Furthermore, SHIP contains protein-protein interaction domains, such as an SH2 domain, two NPXY and several proline-rich motifs. All of these different domains endow SHIP with the capacity to impact signaling pathways important for proliferation, survival, differentiation and activation. Therefore, we hypothesized that SHIP-deficiency could result in the loss of hematopoietic cell homeostasis and functionTo this verify this hypothesis, we first studied the effect of SHIP ablation on hematopoietic stem cell (HSC) proliferation, survival, function and hom ing. Most interestingly we observed that SHIP impacts HSC homeostasis and their ability to home appropriately to the bone marrow. Then, since SHIP was shown to be activated after engagement of the c-mpl receptor by its ligand, thrombopoietin, we studied the impact of SHIP deletion on the function of megakaryocytes, the major target cell of that cytokine. We found that SHIP is also important for homeostasis of the megakaryocyte compartment. Thirdly, we studied the role of SHIP in natural killer (NK) cells biology. We observed that F4 generation SHIP-/- mice have increased NK cells in their spleen and that these cells exhibit a disrupted receptor repertoire. We verified the hypothesis that SHIP helps shape the receptor repertoire of NK cells, mainly through regulation of cell survival and proliferation. Also included, is a study on the role of a SHIP isoform lacking the SH2-domain, called stem cell-SHIP (s-SHIP) in the biology of embryonic stem (ES) cells. To date, this isoform i s expressed by stem/progenitor cells and not by normal differentiated cells. Due to its specific expression pattern, s-SHIP has the potential to have an important role in stem cell biology.
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Dissertation (Ph.D.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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by Caroline Desponts.
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Document formatted into pages; contains 187 pages.
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Includes vita.

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The role of Src homology 2 domain containing 5' inositol phosphatase 1 (SHIP) in hematopoietic cells
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ABSTRACT: The principal isoform of Src homology (SH) 2-domain containing 5' inositol phosphatase protein 1 (SHIP) is a 145kDa protein primarily expressed by cells of the hematopoietic compartment. The enzymatic activity of SHIP is responsible for hydrolyzing the 5' phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), and thereby preventing the recruitment of pleckstrin homology domain containing effector proteins. Furthermore, SHIP contains protein-protein interaction domains, such as an SH2 domain, two NPXY and several proline-rich motifs. All of these different domains endow SHIP with the capacity to impact signaling pathways important for proliferation, survival, differentiation and activation. Therefore, we hypothesized that SHIP-deficiency could result in the loss of hematopoietic cell homeostasis and functionTo this verify this hypothesis, we first studied the effect of SHIP ablation on hematopoietic stem cell (HSC) proliferation, survival, function and hom ing. Most interestingly we observed that SHIP impacts HSC homeostasis and their ability to home appropriately to the bone marrow. Then, since SHIP was shown to be activated after engagement of the c-mpl receptor by its ligand, thrombopoietin, we studied the impact of SHIP deletion on the function of megakaryocytes, the major target cell of that cytokine. We found that SHIP is also important for homeostasis of the megakaryocyte compartment. Thirdly, we studied the role of SHIP in natural killer (NK) cells biology. We observed that F4 generation SHIP-/- mice have increased NK cells in their spleen and that these cells exhibit a disrupted receptor repertoire. We verified the hypothesis that SHIP helps shape the receptor repertoire of NK cells, mainly through regulation of cell survival and proliferation. Also included, is a study on the role of a SHIP isoform lacking the SH2-domain, called stem cell-SHIP (s-SHIP) in the biology of embryonic stem (ES) cells. To date, this isoform i s expressed by stem/progenitor cells and not by normal differentiated cells. Due to its specific expression pattern, s-SHIP has the potential to have an important role in stem cell biology.
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The Role of Src Homology 2 Domain Containing Inositol-5-Phosphatase 1 (SHIP) in Hematopoietic Cells by Caroline Desponts A dissertation submitted in partial fulfillment of the requirement s for the degree of Doctor of Philosophy Department of Biochemist ry and Molecular Biology College of Medicine University of South Florida Major Professor: Willi am G. Kerr, Ph.D. Huntington Potter Ph.D. Gary W. Reuther Ph.D. Kenneth L. Wright Ph.D. Date of Approval: June 2, 2006 Keywords: embryonic stem cell, he matopoietic stem cell, NK cells, megakaryocytes, hematopoiesis, s-SHIP Copyright 2006, Caroline Desponts

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Dedication In loving memory of Joseph McIntosh MD who touched everybodys life with his generosity, talent, and simplicity.

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Acknowledgements Thanks to my husband, Luc Aubin, and my parents, Francine and Gaston who always were believing in me, encouraging me, and giving me the strength to pursue my dream. I express sincere appreciation to John Ninos M.D., Amy L Hazen, Kim HT Paraiso M.S., Joseph Wahle, Amy Costello, Jia-Wang Wang Ph.D., Steve L. Highfill, Tomar Ghansah Ph.D., Daniela Wood, Davina Ramos and Sarah L. Highfill for their help and for making the lab an enjoyable place to work. Thanks to Lia E. Perez M.D. and Nancy Parquet for collaboration on the megakaryocyte project. I thank the members of my committee for their support. I express my deepest gratitude to William G. Kerr Ph.D. for his mentorship, patience, and resourcefulness.

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Note to Reader The original of this document contains color that is necessary for understanding the data. The original is on file with the USF library in Tampa, Florida.

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i TABLE OF CONTENTS Table of Contents ..................................................................................................i List of Tables ......................................................................................................viii List of Figures .......................................................................................................ix Abstract ...............................................................................................................xii Introduction on SH2 Domain Containing 5 Inositol Phosphatase 1 (SHIP) ..........1 SHIP Structure and Cell Signaling .............................................................5 SH2 Domain ....................................................................................5 5 Inositol Phosphatase ...................................................................8 NPXY Motifs ....................................................................................9 Proline Rich (PxxP) Region ...........................................................10 SHIP Isoforms ..........................................................................................10 Inositol Phosphatases with a Redundant Function to SHIP .....................12 SHIP2 ............................................................................................12 Phosphatase and Tensin Homolog Deleted on Chromosome Ten .........................................................................................14 Study of SHIP Function Using SHIP Knock-Out Models ..........................15 Results ...............................................................................................................18 Section I SHIP-Deficiency Enhances HSC Proliferation and Survival but Compromises Homing and Repopulation ....................................18

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ii Introduction ...................................................................................18 HSC Development ..............................................................18 Cytokines Impacting HSC Self-Renewal ............................20 BM Homing and Retention of HSC .....................................21 SHIP in HSC Biology .....................................................................22 Aims: ..................................................................................26 Results ..........................................................................................26 SHIP-/Mice Have an Expanded HSC Compartment ..........26 Induced Deletion of SHIP Du ring Adulthood Leads to an Increase in KFLS Num bers in Hematopoietic Organs ..........................................................................31 SHIP-/BM Cells Show Decreased Ability to Reconstitute the Hematopoietic Compartment of Lethally Irradiated Recipients ........................................35 SHIP-/HSC do not Exhibit Characteristics of Premature Differentiation ..............................................39 SHIP-/HSC Self-Renew to a Lesser Extent than WT HSC in Transplanted Mice ............................................43 SHIP-/HSC Have a Lower Rate of Spontaneous Apoptosis ......................................................................46 In vivo Homing of SHIP-/Stem/Progenitors to the BM is Significantly Reduced as Compared to WT ...............48

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iii Reduced Surface Expression of CXCR4 and VCAM1+ on KTLS Cells in SHIP-/BM .....................................50 Elevated Levels of Soluble VCAM-1 Levels in SHIP-/Mice Sera ......................................................................56 Discussion .....................................................................................57 Materials and Methods ..................................................................61 Mice ....................................................................................61 Cell Isolation .......................................................................62 HSC Phenotype ..................................................................62 Cell Cycle Analysis .............................................................64 CRU Assay .........................................................................65 DC Assay ...........................................................................65 Assessment of MultiLineage Reconstitution ......................66 Annexin V Assay and TUNEL Assay ..................................67 In Vivo Homing Assay ........................................................67 Measurement Cytokines and Growth Factors Levels in Mice Sera ..................................................................68 Section II: Influence of SHIP on Megakaryocytes and Megakaryocyte Progenitors ..............................................................69 Introduction ...................................................................................69 Megakaryocytes .................................................................69 The Involvement of SHIP in MK Signaling Pathway ...........72

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iv Aims: ..................................................................................76 Results ..........................................................................................76 MKP and MK are Increased in BM and Spleen of SHIP-Ablated Mice ........................................................80 Platelet Levels are Lower in SHIP-Deficient Mice as Compared to WT Mice ..................................................82 SHIP-Deficient MK are Morphologically Different than WT MK ..........................................................................83 Comparable Ploidy Distribution in SHIP-/MK as Compared to WT ...........................................................84 TPO Levels are Increased in SHIP-/Plasma as Compared to WT ...........................................................86 Discussion .....................................................................................88 Materials and Methods ..................................................................92 Mice Strains ........................................................................92 Cell Isolation .......................................................................93 Flow Cytometry Analysis and Antibodies ...........................93 Platelet Analysis .................................................................94 Histopathology ....................................................................94 Ploidy assay .......................................................................94 Measurement of Cytoki nes and Growth Factors Levels in the Sera of Experimental Mice .......................95

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v Section III: Natural Killer Cells and SHIP .................................................96 Introduction to Natural Killer Cells .................................................96 NK Cell Receptors ..............................................................98 SHIP and NK Cell Development .......................................104 Results ........................................................................................105 Spleen of SHIP-/Have Increased Number of NK Cells ....105 SHIP is found associated with Ly49 receptors where it may control the level of PI(3,4,5)P3 generated by PI3K and negatively regulate Akt phosphorylation ......110 DAP12 is Expressed by SHIP-/BM Cells and NK Cells ............................................................................112 SHIP but not Shp-1 is Found Associated with Ly49A under Physiological Conditions ...................................114 Discussion ...................................................................................117 Materials and Methods ................................................................118 FACS Analysis of the NK Ce ll Compartment and their Receptors ....................................................................118 Protein Lysis Buffers ........................................................119 Radioimmunoprecipitation (RIPA) buffer. ..............119 Digitonin cell lysis buffer. .......................................120 Biochemical Analysis of SHIP and Akt .............................121 RT-PCR for DAP10 and DAP12 .......................................123

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vi Western Blot for Shp-1 .....................................................124 Section IV: Murine ES Cells and s-SHIP ................................................126 Introduction to ES Cells ...............................................................126 ES Cells Signaling Pathways ...........................................127 LIF and ES cells. ....................................................127 STAT3 stimulates ES cell self-renewal. .................129 ERKs antagonize ES cell self-renewal. ..................129 PI3K signaling in ES cells. .....................................131 s-SHIP ..............................................................................132 s-SHIP and ES Cell ..........................................................133 Aims: ................................................................................135 Results ........................................................................................135 MEF Cells Express Full-Length SHIP, but Only ES Cells Express the s-SHIP Isoforms .............................135 SHIP-/Murine BM Cells Express s-SHIP ..........................137 s-SHIP Does Not Become Phosphorylated Following LIF Stimulation ............................................................138 s-SHIP Associates with gp130 In Vivo .............................140 Discussion ...................................................................................142 Materials and methods ................................................................144 Propagation of mES Cells ................................................144

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vii Preparation of mES Cell Lysates for Biochemical Analysis .......................................................................144 Nested Reverse-Transcription Polymerase Chain Reaction Assay for Detection of s-SHIP Expression ..................................................................145 Western blot antibodies and techniques ...........................146 Cell stimulation .................................................................147 Western blot analysis of gp130 immunoprecipitates ........148 Final Discussion ...............................................................................................150 Bibliography ......................................................................................................154 About the Author .. End Page

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viii LIST OF TABLES Table 1. Increased numbers of HSC ce lls in the BM and spleen of SHIP-/mice compared to WT littermates. ...................................................27 Table 2. Platelet and Hematocrit counts in SHIP-deficient mice. ......................82 Table 3. Functions and ligands of Ly49 NK cell receptors ................................99

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ix LIST OF FIGURES Figure 1. Signaling pathways influenced by SHIP. ...........................................4 Figure 2. Schematic representing different SH IP isoforms structure. ................7 Figure 3. Two SHIP KO models, SHIP-/and SHIP IP/ IP. ................................17 Figure 4. Hemat opoietic compartment. ...........................................................19 Figure 5. Significant increase in the percentage and absolute number of KLSCD48cells in SHIP-/BM. ....................................................30 Figure 6. Significant increase in the percentage and absolute number of HSC in SHIP-ablated BM, spleen and PB. .................................34 Figure 7. SHIP-/WBM cells have compromised reconstituting activity. ..........36 Figure 8. SHIP-/purified HSC have compromi sed reconstituting activity. ......38 Figure 9. More SHIP-/TLS cells express high levels of c-Kit as compared to WT. ........................................................................40 Figure 10. SHIP-/KTLS express similar levels of Mac1 as compared to WT KTLS. ...................................................................................42 Figure 11. SHIP-/HSC do not engraft and self-renew as well as WT HSC, although transpl anted in equal numbers. ..........................44 Figure 12. SHIP-/HSC exhibit decreased apoptotic rate. .................................47 Figure 13. SHIP-/Sca1+Lincells do not home to the BM as efficiently as WT Sca1+Lin-. .............................................................................49

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x Figure 14. SHIP-/HSC express lower levels of CXCR4 and VCAM-1 molecules as assessed by flow cytometry. .................................52 Figure 15. SHIP-/late progenitor and different iated cells express the same levels of homing molecules as assessed by flow cytometry. ...................................................................................53 Figure 16. SHIP-/early progenitors express the same levels of CXCR4 and have a reduced percentage of VCAM-1+ cells. ...................54 Figure 17. SHIP-/early progenitors express the same levels of CXCR4 and have a reduced percentage of VCAM-1+ cells. ....................55 Figure 18. sVCAM-1 levels are significantly increased in SHIP-/sera as compared to WT littermates. ......................................................56 Figure 19. Megakaryocytopoiesis and cytokines that influence the process. ......................................................................................71 Figure 20. Increased number of MKP in SHIP-deficient BM. ............................77 Figure 21. Total MKP but not tota l MK numbers are increased in SHIPdeficient mice are compared to WT. ...........................................79 Figure 22. Significant increase in the percentage of MKP cells in SHIPablated BM and spleen. ..............................................................81 Figure 23. Hematoxylin-eosin stai ning of SHIP-deficient WT BM and spleen. ........................................................................................83 Figure 24. SHIP-/MK undergo endocytosis with the same efficiency as WT MK. ......................................................................................85

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xi Figure 25. TPO levels are significantly increased in SHIP-/sera as compared to WT littermates. ......................................................87 Figure 26. Missing-self hypothesis. .................................................................103 Figure 27. Increased NK cell numbers in SHIP-/-. ...........................................106 Figure 28. MHC class I receptor s on peripheral NK cells in SHIP-/mice. .......109 Figure 29. SHIP is recruited to NK inhibitory receptors in vivo to oppose activation of Akt. .......................................................................111 Figure 30. DAP12 is expressed in SHIP-/and WT BM cells and NK cells. .....113 Figure 31. DAP10 is expressed in SHIP-/and WT BM cells. ..........................113 Figure 32. SHIP but not Shp-1 is re cruited to NK inhibitory receptor Ly49A in vivo. ...........................................................................115 Figure 33. Signaling pathways down stream of Ly49A and C that can be influenced by SHIP in NK cells. ................................................116 Figure 34. LIFR/gp130 receptor complex signal transduction pathways and how s-SHIP may impact them in pluripotent stem cells. ....128 Figure 35. MEF cells express full -length SHIP, while ES cells express only s-SHIP. .............................................................................136 Figure 36. Nested RT-PCR detecti on of s-SHIP expression in SHIP-/BM. ....137 Figure 37. ES cells express the s-SHIP protein isoform that does not become phosphorylated following growth factor stimulation. ....139 Figure 38. s-SHIP is associated with gp130 in ES cells. .................................141

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xii The Role of Src Homology 2 Domain Containing Inositol-5-Phosphatase 1 (SHIP) in Hematopoietic Cells Caroline Desponts ABSTRACT The principal isoform of Src homology (SH) 2-domain containing 5 inositol phosphatase protein 1 (SHIP) is a 145kDa protein primarily expressed by cells of the hematopoietic com partment. The enzymatic activity of SHIP is responsible for hydrolyzing the 5 phosphate of p hosphatidylinositol-3,4,5-trisphosphate (PI (3,4,5) P3), and thereby preventing the re cruitment of pleckstrin homology domain containing effector proteins. Fu rthermore, SHIP contai ns protein-protein interaction domains, such as an SH2 domai n, two NPXY and several proline-rich motifs. All of these different domains endow SHIP with the capacity to impact signaling pathways important for proliferation, surv ival, differentiation and activation. Therefore, we hypothesized t hat SHIP-deficiency could result in the loss of hematopoietic cell homeostasis and function To this verify this hypothesis, we fi rst studied the effect of SHIP ablation on hematopoietic stem cell (HSC) proliferat ion, survival, function and homing. Most interestingly we observed that SHIP impacts HSC homeostasis and their

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xiii ability to home appropriately to the bon e marrow. Then, since SHIP was shown to be activated after engagement of the c-mpl receptor by its ligand, thrombopoietin, we studied the impact of SHIP deletion on the function of megakaryocytes, the major tar get cell of that cytokine. We found that SHIP is also important for homeostasis of the megakaryocyte compartment. Thirdly, we studied the role of SHIP in natural killer (NK) cells biology. We observed that F4 generation SHIP -/mice have increased NK cells in their spleen and that these cells exhibit a disrupted receptor repertoir e. We verified the hypothesis that SHIP helps shape the receptor repertoire of NK cells, mainly through regulation of cell survival and proliferat ion. Also included, is a study on the role of a SHIP isoform lacking the SH2-domain, called stem cell-SHIP (s-SHIP) in the biology of embryonic stem (ES) cells. To date, this isoform is express ed by stem/progenitor cells and not by normal diffe rentiated cells. Due to its specific expression pattern, s-SHIP has the potential to have an important role in stem cell biology.

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INTRODUCTION ON SH2 DOMAIN CONTAINING 5 INOSITOL PHOSPHATASE 1 (SHIP) SHIP stands for Src homology (SH) 2 domain containing 5 inositol phosphatase 1 and is a protein primarily expressed by cells of the hematopoietic compartment. 1-4 In 1996, five independent gr oups cloned murine SHIP using its ability to bind; 1) the SH3 domain of growth factor receptor-bound protein 2 (Grb2), 2 2-3) the protein-tyrosine binding domain (PTB) of SH2-containing sequence protein (Shc) (also called SH and collagen gene or SH2-containing oncogene src homology protein), 3,4 4) the Fc RIIB, 5 and 5) by gene trap assay. 6 SHIP is known to hydrolyze the 5 phosphate of phosphatidylinositol-3,4,5phosphate (PI (3,4,5) P3) in vivo and inositol-1,3,4,5tetrakisphosphate (I (1,3,4,5) P4) in vitro, 2,4 two inositides phosphates import ant for cell signaling (Figure 1). 7-10 Since SHIP appears to require the presence of a phosphate on the 3 position to exert its effect, it was proposed t hat it opposes the activity of phosphatidyl inositol 3' kinase (PI3K), and by doing so, regulates several cellular signaling pathways important for proliferation, differ entiation, apoptosis and migration. 1

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SHIP was first observed as a 145kDa protein that becomes tyrosinephosphorylated after stimulation of hematopoietic human DA-ER and MO7-ER cell lines with erythropoietin (Epo). 11 In this particular model, stimulation of these cells with Epo resulted in SHIP tyro sine-phosphorylation and association with Shc. 11 Shc being implicated in several mitogenic signaling transduction pathways, 12 it became essential to determine the role of SHIP in hematopoietic cell function. It was then determined that stimulation of several receptors resulted in SHIP tyrosine-phosphorylati on and/or SHIP association with other signaling molecules such as SH2-cont aining tyrosine phosphatase (Shp-2), 13 and Grb2. 14,15 SHIP becomes activated after engagement of cytokines, chemokine and immunological receptors. The cytoki nes that have been id entify to stimulate SHIP consist of interleukin (IL)-3, 13,16-18 IL-4, 19,20 macrophage colony stimulating factor (M-CSF or c-FMS), 21 granulocyte-macrophage-CSF (GM-CSF), 16,18 granulocyte-CSF(G-CSF), 22,23 Stem Cell Factor (c-KitL or SCF), 1,16 thrombopoietin (TPO), 24,25 Fms-like tyrosine kinase 3 ligand (Flt3L). 26 The major chemokine known to induce SHIP recrui tment is stromal cell derived factor1/CXCL12 (SDF-1) which binds to the CXCR4 receptors on multiple hematopoietic cells. 27,28 The immunological receptor s known to recruit SHIP are the Fc receptor, 5,29,30 the T cell receptor, 31 and B-cell-receptor Igand Igchains. 32-34 Once SHIP is activated or recr uited by these receptors, it can dampen the levels of PI (3,4,5) P3, phosphorylation of Prot ein Kinase B (Akt or PKB), accumulation of intracellular calcium, the formation of the protein complex, 2

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composed of Shc, Grb2, and Son of Sevenless (SOS), and by doing so, SHIP negatively regulates the activation of the mitogen-activated protein kinase (MAPK or Erk) pathway (Figure 1). For exampl e, SHIP impacts pathways downstream of G-CSF receptors that tightly regulate proliferation and survival of neutrophils. 22,23 The cytoplasmic tail of the GCSF receptor contains a r egion that is responsible for recruiting PI3K and stimulating prolif eration and survival signaling pathways. 22 On the other hand, this same receptor has another region on its cytoplasmic tail that can recruit the SHIP/Shc complex, this region is associated with negative regulation of prolif erative signaling. 22 This demonstrates the great balancing act in which these molecules are involved. 3

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Figure 1. Signaling pathways influenced by SHIP. Engagement of receptor by its ligand leads to recruitment of SHIP with can impact different signaling pathway. 1) SHIP can prevent the Grb2/SOS from binding to Shc, thus preventing the activation of RAS and the downstream MAPK pathway. 2) Alternativ ely, SHIP can hydrolyze the 5 phosphate of PI (3,4,5) P3 generating PI (4,5) P2. This prevents the recruitment of pleckstrin homology (PH) containing protein to the membrane and their activation. In this manner, SHIP prevents activation of Brutons tyrosine kinase (Btk), which is responsible for phospholipase C (PLC activation, leading calcium entry and MAPK activation. SHIP also dampens the recruitment and activation of Akt, which leads to phosphorylation of proteins in the Bcl family, such as BAD, Bcl-2, BclXL, preventing apoptosis. Furthermore Akt can phosphorylate Forkhead (FH) and prevent its translocation to the nucleus. 35,36 Consequently, FH is prevented from inducing transcription of cell death related genes such as Fas ligand (FasL). 35 4

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SHIP Structure and Cell Signaling The SHIP protein sequence contains several interaction domains including an SH2 domain, a 5inositol phosphatase, several proline-rich domain, and two NPXY motifs (Figure 2A). Full length SHIP is a 145kDa protein, 11 which has several isoforms that have different molecular weight incl uding an 130kDa or 135kDa isoform in human and murine cells, respectively, and an 110kDa isoform found in both human and mu rine cells (Figure 2). 4,37 All of these isoforms appear to have lost a specific protein domain wh en compared to full length SHIP, which could lead to having a different function in the cell. SH2 Domain The SH2 domain of SHIP, stands for Src homology domain 2, a structure that has the potential to bind to phosphotyrosine. Using this domain SHIP can bind to phosphorylated immuno-rec eptor tyrosine based activation or inhibition motifs (ITAMs or ITIMs) present in the cytoplasmic tail of several receptors, such as Fc RIIB (ITIM) on B cells, Ly49A and C receptors on natural killer (NK) cells, and FcR I (ITAM) on mast cells. 38,39 5,40 5,41 42,43 40,44 The SH2 domain of SHIP was also shown to interact with the Src kinase Lyn, 45 which can phosphorylate SHIP. 46 This stabilizes SHIP near the membrane and enhances 5

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its capacity to regulate the Akt signaling pathway. 45 It is important to note that location more than phosphorylation contri butes to SHIP phosphatase activity. 46 Furthermore, it was shown that the SH2 do main of SHIP interacts with tyrosinephosphorylated Shp-2. Since a SHIP mutant, with a deleted SH2 domain, can not be phosphorylated or bind to Shc nor Shp-2 after stimulation with IL-3, it was suggested that this domain is crucial for SHIP function. Consistent with this observation, we found that a SHIP isoform lacking the SH2 domain called stem cell-SHIP (s-SHIP) (Figur e 2) can not get phosphorylated and is unable to associate with Shc after stimulation of embryonic stem (ES) cells with leukemia inhibitory factor (LIF). The SH2 domain of full-length SHIP appears to compete with Grb2 for interaction with the same phosphorylated tyrosine on Shc. Interaction of Shc with Grb2 would lead to the recruitment of a protein complex including SOS, which catalyzes th e exchange of GDP to GTP on RAS, resulting in the activation of the MAPK/Erk pathway. Thus, SHIP prevents formation of this complex and negatively regulates MAPK activation (Figure 1). Although this hypothesis is compelling, it is important to keep in mind that Shc could associate with SHIP through a differ ent mechanism. For example, it has been proposed that Shc PTB domain can bind the phosphorylated tyrosine Y917 and Y1020 on the NPXY motifs of SHIP. Studies using a SH2 domain deleted SHIP mutant revealed that this domain is necessary for the NPXY motifs to become phosphorylated and for SHIP to interact with Shc. Therefore, it becomes difficult to exactly determi ne which domain betw een the SH2 and NPXY domain is used by SHIP to bind to Sh c. Another protein with which SHIP 13,17 13,47,48 49 1 1 50 48 50 6

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interacts through its SH2 domain is Gab1. However, in this case it is impossible to rule out if SHIP and Gab1 associate directly or through intermediary molecules, since they are f ound in a multi-protein complex involving phosphorylated Shp-2, PI3K, and Shc. 51 51,52 Figure 2. Schematic representing different SHIP isoforms structure. (A) Full-length SHIP, is SHIP and 145kDa. (B)SHIP is app. 130kDa and lacks 183 amino acids between the NPXY motifs, resulting in the deletion of a prolinerich domain and the amino acids adjacent to the first NPXY motif, IGM, which would be important for p85 subunit binding. (C) SHIP is the result of the deletion of all the proline-rich regions and the last NPXY motifs. The last 62 amino acids at the cterminal are changed from the full length SHIP, caused by an out-of-frame 230 amino acids deletion. Depending on the reports the SHIP and SHIP are either the result of proteolytic cleavage 37 or alternative splicing. 53,54 (D) Stem cell-SHIP (s-SHIP) is expressed from the same chromosomal region but has an alternative promoter/start site than the SHIP situated in the intron5/6 of SHIP This was first proposed in a paper we published in 2001 49 and then confirmed by Rohrschneider et al 55 A 125kDa isoform that is probably generated through truncation of the 145 full-length SHIP c-terminal exists, however this isoform has not been sequenced, thus the structure is not available. 7

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5 Inositol Phosphatase PI3K catalyzes the addition of a phosphate at the 3 position on Phosphatidyl Inositol(4,5) Phosphate (PI )P2), creating PI P3, this molecule allows the recruitment of pleckstrin hom ology (PH) domain containing proteins (e.g.: Akt, phosphoinositide dependent kinase 1 (PDK1) Brutons tyrosine kinase (Btk)), which can then stimulate prol iferation, survival, and activation pathways. SHIP, by removing the 5 phosphate of PI P3, can prevent the recruitment of these effector molecu les and negatively regula te some of PI3K downstream signaling pathways. In particular, SHIP has been shown to decrease Akt recruitment and phosphorylation, leading to a decrease in survival and proliferati on signaling. Furthermore, the degradation of PI P3 by SHIP after it is recruited to co -ligated B cell rec eptor (BCR) and Fc RIIB1 can lead to a reduction in Btk membrane lo calization, which would prevent its activation, resulting in a subsequent decrease in PLC 2 activity and stopping of calcium flux. On the other hand, the ability of SHIP to remove the 5 phosphate of I P4 could limit the extracel lular entry of calcium and negatively control cell activation. There is no requirement for SHIP to be phosphorylated before it can hydr olyze the 5 phosphate of PI P3 or I P4, suggesting that localization of SHIP in proximity to the target molecule is the determining mechanism of activation. (4,5 (3,4,5) 10,56-59 (3,4,5) 10,56-59 60,61 (3,4,5) 62,63 (1,3,4,5) 64-66 (4,3,5) (1,3,4,5) 54 8

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NPXY Motifs NPXY stands for arginine (N), proline (P), any amino acid (X), and tyrosine (Y), and there are two on the carboxyl-termi nus of SHIP (Figure 2). This motif can become tyrosine phosphoryl ated upon activation, whic h becomes a potential binding site for some PT B domain containing protein. 67-69 In this manner, it was proposed that the PTB domai n of Shc could interact with the phosphorylated tyrosines of the NPXY motifs on SHIP, in particular Y917 and Y1020. 2,50 Furthermore, a SHIP isoform, SHIP in which the 2nd NPXY motif and all the poly-proline rich stretch has be en deleted can not bind to Shc. 52 However, as mentioned earlier, depending on the cell context, it appear s that SHIP can also interact with Shc, through its SH2 domain binding to the phosphorylated Y187 on Shc, a site for which SHIP and Grb2 are competing in order to exert opposite effects on the Ras/MAPK pathway. 1 Although the nature of these conflicting results will not be further di scussed in this dissertation, they are worth mentioning to emphasize that more work needs to be done to fully understand the function of SHIP in the different cell context. Another protein that has been found to interact with the NPXY motifs of SHIP is the p85 subunit of PI3K. 14,70,71 Of the two NPXY motifs present on the carboxyl terminal region of SHIP, the first one is followed by a consensus sequence for the binding of the p85 s ubunit of PI3K, isoleucine-glycine9

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methionine (IGM). 53,71,72 It was shown that p85 PI3K subunit interacts with SHIP only when the first NPXY region is tyrosine-phosphorylated. 53,71 Proline Rich (PxxP) Region PxxP motifs are spread between the 1st and 2nd and after the 2nd NPXY motifs within the carboxyl terminus of SHIP (Figure 2). PxxP motifs are known to interact with SH3 motifs, 73 To this effect, SHIP can associate with Grb2, a protein containing two SH3 domains flanking an SH2 domain on each side. Immunoprecipitation assays have shown th at Grb2 can associate with SHIP and s-SHIP. 2,4,49 SHIP Isoforms As mentioned above, seve ral SHIP isoforms have been identified, mostly through immunoblots. In humans it was shown that peripheral blood (PB) cells expressed a 100kDa version of SHIP, while CD34+ acute myeloblastic leukemia cells expressed 130kDa and 145kDa. 74 In the murine system, most of the lower molecular weight isoforms the 110, 124, and 135kDa isof orms bear a deletion in the c-terminus region, and appear to be generated from the full-length 145kDa SHIP, 3,5,53 either by proteolytic cleavage 37,75,76 or alternative splicing. 52 Importantly, the 110kDa SHIP isoform (Figure 2C), whic h lacks the proline-rich 10

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region and the 2 nd NPXY motifs at the carbox yl terminus, exhibit reduced phosphorylation and interacti on with Shc after stimulation of FD-FMS cells by MCSF. 52 Lucas and Rohrschneider (1999) reported the co-expression of a 135kDa and 145kDa SHIP isoforms in a series of macrophage, B cell progenitor, and Tcell cell lines. 53 This 135kDa isoform results from the internal del etion of a PxxP containing stretch between t he 2 NPXY motifs due to al ternative splicing. This isoform, which was called SHIP 183, can still bind to Shc and Grb2 but has reduced affinity for the p85 subunit of PI3K. 53 This is explained by the deletion of an important consensus sequence after th e first NPXY motif, leading to a change in the following amino acids from Y IGM to Y IAN. The elimination of the methionine at the +3 position after the tyrosine reduces the ability of p85 PI3K subunit to bind SHIP 183. 53 Even though most of the isoforms isol ated seem to involve cleavage or alternative splicing in the carboxyl-te rminal of SHIP, another 110kDa isoform lacking the SH2 domain was identified in human 4 and in mice. 49 The human 110kDa isoform, called SIP-11 0, isolated by Kavanaugh et al lacks 214 amino acids at the amino-terminus, resulting in the absence of the SH2 domain. 4 It was proposed at the time that SIP-110 isoform resulted from alternative splicing of the full length SIP-145 mRNA. 4 In 2001, our laboratory pub lished that murine ES cells express a 110kDa SHIP isoform (calculated MW 104kDa), called s-SHIP. This isoform lacks 263 amino acids at the amino-terminus. 49 In that study, we 11

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proposed that s-SHIP and SIP-110 were hom ologues, and were both the result of an alternative start site. The promoter r egion of that isoform being present in the in the intron 5/6. 49 This was confirmed in a study by Rohrschneider et al which showed that intron 5/6 had pr omoter activity able to drive the expression of a reporter gene in stem/early progenitor cells in vivo 55 Importantly, s-SHIP is expressed in ES cell and hematopoietic stem cells (HSC) but not in normal lineage differentiated cells. 49,55 This characteristic ma kes it a very important isoform that certainly should be investigated further for its possible role in stem cell biology. Inositol Phosphatases with a Redundant Function to SHIP SHIP2 A 150-155kDa protein with 38% amino acid sequence homology to SHIP1 was identified by Pasesse et al (1997), and was named SHIP2. It was later found that this protein had already been isolated as 51C protein also called inositol polyphosphate-like-pr otein1 (INPPL-1), which is an inositol phosphatase with the potential to complement t he Fanconi anemia group A gene defect. While murine SHIP1 is expressed from chromosome 1 and its human homologue from chromosome 2, SHIP2 is expressed from Chromosome 7 in mouse and 8 in human. However, although SHIP2 reside on a different chromosome, it 77 78 79 12

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contains most of the hallmark domains of SHIP1. For example, SHIP2 has an amino-terminal SH2-domain with 54% i dentity and a 5inosit ol phosphatase with 64% homology to the one found in SHIP1. SHIP2 also has several poly-proline rich stretches in its carboxyl terminal (up to 8), where SH3-domain containing protein can bind and one NPXY motif, a site where PTB domain can bind. This could account for SHIP2 abi lity to interact with Shc. Whereas SHIP1 expression appears to be restricted to he matopoietic cells, it was reported that SHIP2 is ubiquitously expr essed, being observed in hema topoietic cells, such as T cells, B cells, thymus and spleen and in non-hematopoietic tissues such as brain and skeletal muscle. Like SHIP1, SHIP2 has the potential to influence many signaling pathways. For exampl e, like SHIP1, SHIP2 has been shown to associate with the ITIM domain of Fc RIIB in B cells un der negative signaling. Furthermore, IL-3, c-KitL, and GM-CSF stimulated the ph osphorylation and interaction of SHIP2 with Shc in hematopoietic cells. Another example of the importance of SHIP2, is it s ability to be recruited to epidermal growth factor (EGF) receptor where it hydr olyzes the 5 phosphate of PI P3 after EGF stimulation of COS-7 cells. More importantly, SHIP2 has been shown to control insulin receptor signaling in vitro and in vivo in a knock-out model. Different groups have shown that SHIP2 over-expression led to a decrease in insulin signaling whereas a SHIP2 deficiency led to an increase in insulin sensitivity. It is important to m ention that since SHIP2 interacts with different molecules than SHIP1 and that it c an not hydrolyze t he 5 phosphate of 77 80 81 77,82 83 80 (3,4,5) 84-87 88 84-89 13

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I P4, even though these molecules have great homology they are not simply redundant molecules, but have defined purpose in the cells. (1,3,4,5) Phosphatase and Tensin Homolog Deleted on Chromosome Ten The defect observed in SHIP -/mice is severe, but at the same time is probably compensated for by different phos phates such as SHIP2, s-SHIP or phosphatase and tensin homolog deleted on chromosome ten (PTEN). PTEN is a 54kDa ubiquitously expressed tumor s uppressor inositol phosphatase that can hydrolyze the 3 phosphate of PI (3,4,5) P3, thus, counteract PI3K activity and contribute to the control of Akt activation. 90 PTEN also has a modest capacity to dephosphorylate tyrosine phos phorylated proteins. 91 PTEN is one of the most commonly inactivated genes in several type of cancers 92 and a key regulator of cell growth and apoptosis. 93 The major function of PTEN appears to be the reduction of basal PI (3,4,5) P3 levels and Akt activity in a constitutive manner, 90 and not upon stimulation by growth factor like SH IP. Therefore, its ablation leads to a more severe phenotype than in SHIP -/mice, in fact PTEN -/are embryonic lethal, 94 further suggesting that PTEN is im portant for normal development. Interestingly, PTEN +/and SHIP -/mice share many characteristics, suggesting that the phenotype observed in both mice re sult mainly from the accumulation of PI (3,4,5) P3. 95,96 14

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Study of SHIP Function Using SHIP Knock-Out Models The study of SHIP knock-out (KO) mouse models has given great insight into SHIP function in the hematopoietic system. 43,95,97 Although SHIP expression is detectable during embryogene sis at day 7.5 post-coitus, 98 SHIP KO mice are viable. 43,95,97 However, they suffer from a myel oproliferative disorder, that causes an increase in macrophage progenitor and mature macrophage numbers in the BM, spleen and periphery. 95 These macrophages infilt rate the lung of SHIP -/mice, and this is the suspected cause fo r their demise at 8 to 12 weeks of age. 95 Using SHIP KO mouse model, it was shown that SHIP negatively regulates the phosphorylation and activation of Akt through downregulation of PI (3,4,5) P3 in B cells, 61 in myeloid cells, 18 and NK cells. 43 Huber et al also showed that through binding to Fc RI in mast cells, 40,44 SHIP can downregul ate their degranulation after stimulation of c-Ki t receptor with its ligand. 99 The stimulation of mast cells leads to the activation of PI3K and the production of PI (3,4,5) P3, which can then stimulate the entry of extracellular calcium, 99,100 and the release of granule content. Interestingly, SHIP KO mi ce were also shown to suffer from osteoporosis caused by hype r-resorptive osteoclasts. 101 This could disrupt the BM matrix and perturb the HSC niche. Most of our studies were done usi ng the SHIP-deficient mouse model generated in our laboratory. These mice were created by targeting the promoter 15

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and exon 1 of the SHIP gene using the Crelox P strategy and we refer to it as SHIP -/(Figure 3). 43 In this model, the region to be deleted is flanked (floxed) by two recombinase (Cre) recognition sites, called lox P, which were introduced by homologous recombination in ES cells. 102 In order to get deletion of the targeted region, the 34 base pair lox P sites need to be inserted in the same orientation. 103 Once a germline flox/flox or flox/+ animal is obtained, it is crossed with a Cre +/+ mouse, which result in t he deletion of the targeted re gion. The resulting mice can then be used to generate germline k nockout mice. This system allows deleting or targeting t he gene of interest either in t he entire animal or specific cell type dependent on the promoter that is used for Cre ex pression. The other SHIP KO model used in our l aboratory was a kind gift from Dr. Ravetch at the Rockefeller University in New York. His team produced that model by targeting the region that encodes part of the inositol phosphat ase region using the CRElox P strategy (Figure 3). 97 Consequently, this mouse m odel will be referred to as SHIP IP/ IP The SHIP IP/ IP mice were mainly used le ss frequently and mainly to confirm the results obtained with SHIP -/mice to ensure that different mutations of the same gene lead to the same phenotype. 16

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Figure 3. Two SHIP KO models, SHIP -/and SHIP IP/IP (A) Our laboratory created the SHIP -/model using 129SvJ ES cells, Crelox P recombinant technology. The promoter region and the exon 1 of SHIP were targeted. 43 The SHIP IP/ IP model was created using the same technology but targeting the exon 10 and 11, which form part of the inositol phosphatase region. 97 (B) Both of these knockout mice do not express SHIP. (i) Western blot done on littermates of a mating between heterozygote male and female, harboring a deletion of the promoter and first exon, SHIP -/. 43 (ii) Western blot done on littermates of a mating between a heterozygote male and female, harboring a deletion of exon 10 and 11, SHIP IP/ IP 97 The later mouse model was generated in the laboratory of Dr. Ravetch at Rockefeller University, New York, USA. 97 17

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RESULTS Section I SHIP-Deficiency Enhances HSC Proliferation and Survival but Compromises Homing and Repopulation Introduction HSC Development HSC are responsible for the maintenance of the hematopoietic compartment throughout an organisms lifespan (Figure 4). These cells have the ability to self-renew, differentiate into all hematopoiet ic lineage and to reconstitute a lethally irradiated host. In the mouse, the em bryonic or primitive HSC appear in the yolk sac at day E7 of embryonic development. 104,105 These cells participate in transi ent hematopoiesis during em bryogenesis giving rise mainly to nucleat ed erythrocytes, 106 although some studies have shown that putative yolk sac HSC have long-te rm multilineage repopulation capacity, 107 suggesting that they have he potential to differentiate into several hematopoietic lineage. At day E10, the definiti ve HSC is found in the aorta-gonadmesonephros (AGM). 108,109 Only these HSC will have the ability to establish definitive hematopoiesis and to reconsti tute a lethally i rradiated adult host. 104,108110 Later during fetal development the defin itive HSC migrate to the fetal liver, to the spleen and shortly thereafter birth all hematopoiesis is concentrated mainly in 18

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the BM. 104 The maintenance of the hemat opoietic compartment depends on HSC homeostasis, the balance between se lf-renewal, proliferation, apoptosis, and migration (i.e. homing and mobilization) of HSC. Se veral factors responsible for HSC homeostasis have been identified, nevertheless more information is needed. Figure 4. Hematopoietic compartment. HSC have the potential to self-renew without limits, differentiate and produce all the cells necessary for a functional hematopoietic compartment. This differentiation occurs through a series of intermediaries (often called progenitors), which usually have the potential to self-renew with limitations and to differentiate into a defined cell type. 19

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Cytokines Impacting HSC Self-Renewal It is well accepted that the niche in which the HSC reside in the BM is responsible for the retention of HSC and their ability to self-renew, differentiate, or apoptose. Cell-to-cell contact and external signaling plays a major role in that process. Several cytokines have been sh own to influence HS C proliferation and self-renewal in vitro and in vivo In fact, several in vitro studies have shown that TPO alone or in combination with c-KitL, IL-3, or Flt3L 111-115 can stimulate the proliferation of early human and murine hematopoietic progenitors. 116-127 Most importantly, expansion of early progenitor cells in th e presence of TPO with cKitL or Flt3L confer these cells with the ability to self-renew, to retain a primitive phenotype and maintain the capacity for multilineage differentiation for a defined number of cell division. 116,119-121,123-125 It was also shown that tumor necrosis factor(TNF) can stimulate the self-renewal and proliferat ion of human CD34++CD38in vitro in the presence of IL-3, potentially in a synergistic manner. 128 Most importantly, ma intenance of hematopoiesis in vivo depends on the presence of osteoblast and stromal cells that will form the niche. This microenvironment will promot e HSC homeostasis. Furt hermore, several factors that are important for ma intenance of HSC have also been shown to impact homing and retention of HSC to the BM niche. 20

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BM Homing and Retention of HSC The homing process was characterized as a 3 step process; 1) Loose or rolling interaction of cells with vascula r endothelium, 2) Firm adhesion to the vessel or sinus, 3) Diapedesis through the endothelial layer. 129 This process first requires the presence of specific cytokines and of cell adhesion molecule (CAM) that will mediate the associ ate of HSC with the stromal cells and osteoblasts that compose the niche in the endosteal region in the BM that support HSC homeostasis. 130-135 To this effect, HSC can intera ct with a series of molecules produced by the BM stroma such as ex tracellular matrix (ECM) components, CAM mediating cell-cell adhesions, and endothelial selectins. The ECM components are fibronectin, 136,137 hyaluronic acid, 138 laminins, 139 collagen, and thrombospondin. 140 The CAM mediating cellcell adhesions include the intracellular cell adhesion molecule-1 (ICAM-1/CD54) and vascular cell endothelial molecule-1 (VCAM-1 or CD107). 141,142 The endothelial selectins are P-selectin/CD62P and E-selectin/CD62E. 143,144 All of these molecules are ligands for receptors pres ent on HSC. These recept ors, once engaged will lead to HSC attachment and establishment in the BM niche and thus, contribute to HSC homeostasis. For exam ple, very-late-antigen 4 ( 4 1 CD49d/CD29 (VLA4)) is expressed by hematopoietic cells including HSC and can bind fibronectin and VCAM-1 present on endothelial cells. VLA-4 can promote initial capture, rolling and firm cell adhesion to the endothelial cells. 145 In previous studies, in vitro stimulation of murine BM stem/progeni tor cells with different cytokines was 21

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shown to induce cell cycle, decrease VLA-4 expression a nd resulting in compromised engraftment. 146,147 Most importantly it was shown that engagement of VLA-4 an VLA-5 receptors can lead to HSC quiescence, 148 an important aspect of HSC biology. Most importantly, HSC quiescence protects the compartment against external insult and insure the organism that the hematopoietic compartment will be replenish once the insult is cleared. Proper homing of the HSC to the BM also depends on the presence of a chemokine, SDF-1 also called CXCL-12 and the engagement of its receptor on HSC, CXCR4. The CXCR4 receptor induces cell migration towards an increasing gradi ent of SDF-1. 149 Treatment of NOD/SCID mice with SDF-1 leads to the mobilization of HSC to PB 150 and treatment of hum an HSC with antiCXCR4 antibody prior to transplantation in NOD/SCID mice results in HSC engraftment failure. 151 Furthermore, CXCR4 and SDF-1-deficient mice are embryonic lethal since HSC fail to migrat e from the fetal liver to the BM, where definitive hematopoiesis would take place. Interestingly, SHIP -/myeloid progenitor cells have been shown to migr ate more efficiently towards SDF-1 compared to WT myeloid progenitors. 152 Furthermore, SHIP appears to be recruited to the membrane upon CXCR4 engagement by SDF-1. 27 SHIP in HSC Biology 22

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As mentioned earlier, the survival of an organism is dependent upon the ability of HSC to replenish the blood comp artment on a daily basis (Figure 4). To accomplish this task, HSC must mainta in a fine balance bet ween three possible fates: self-renewal, differentiation or s enescence. Although the decision process that determines the fate of an HSC clone remains to be completely defined, several molecules are already known to pl ay a role in this process, including components of the HSC microenv ironment or niche. This niche consists of both extra-cellular matrix mole cules and cells, such as osteoblasts and stromal cells that produce cytokines and chemokines important fo r the maintenance of the HSC pool. 131-135 These microenvironmental or external cues engage receptors on HSC, leading to the activation of signaling pathways governing cell proliferation, selfrenewal, differentiation, mob ilization and BM retention. 135 Some of these pathways, such as those initiated by c-KitL, 153 SDF-1, 154 IL-3, 155 Flt-3L, 111-115 and TPO, 17 result in the activation of PI3K and the formation of PI (3,4,5) P3. Therefore, SHIP may influence these pathways in HSC. 47,152 SHIP is a 145kDa protein primarily expressed by cells of the hematopoietic system, 3 including HSC, 49 that can associate with various adapter prot eins, scaffold proteins, or receptors following activation of hematopoietic cells. 18,152 Formation of these complexes enables SHIP to hydrol yze the 5phosphate on PI (3,4,5) P3, 2,3 thus preventing membrane recruitment and activation of pl eckstrin homology domain containing kinases that serve as effect ors of PI3K signalin g. This activity permits SHIP to 23

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limit the survival, activation, differentiati on and/or proliferat ion of hematopoietic cells. 47 Thus, we hypothesized that SHIP might also influence these processes in the HSC compartment. Previ ous studies reported that SHIP -/whole bone marrow (WBM) cells do not reconstitute le thally irradiated mi ce as well as wildtype (WT) WBM in a non competitive setting. 156 Furthermore it was reported that SHIP -/WBM has comparable numbers of co mpetitive-repopulating-units (CRU) relative to WT littermates in a limiting-dilution assay, which uses compromised competitor cells. 156 However, because these analyses were performed with WBM rather than purified HSC, t hey did not assess whether SHIP -/HSC are defective for repopulation in a WT environ ment. Thus, it is not possible from these previous studies to conclude that SHIP plays a direct role in signaling pathways essential for HSC function. To study the role of SHIP in HSC we used SHIP -/mice generated by a Crelox mutation strategy. 43 We found that SHIP -/mice contain significantly more HSC in their BM, spleen, and blood as measured by flow cytometry. We also observed that a greater proportion of SHIP -/HSC enter the cell cycle compared to WT HSC. Since it was shown in previous studies that SHIP -/BM contains reduced HSC activity relative to WT BM, 156 it became important to assess the function of SHIP -/HSC in different assays. We found that when purified SHIP -/HSC or WBM are transplanted into lethally irradiated mice, they failed to compete effectively with WT HS C or BM cells for long-term multi-lineage repopulation of the hematopoietic compartment. These results might have 24

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suggested that the absence of SHIP causes accelerated senescence of the HSC compartment. However, we obse rved fewer apoptotic HSC in SHIP -/BM as compared to WT BM. We then assessed the ability of SHIP -/HSC to reach the BM niche, where they encount er the proper environment to support their function. In vivo homing studies performed using purified stem/progenitor cells suggest that SHIP -/cells home to the BM with a decreased efficiency compared to WT cells. Most interestingly, we also observed that SHIP -/HSC have significantly lower surface expression of CXCR4 and VCAM-1, key receptors for homing and retention of hematopoiet ic cells in the BM. 151,157,158 Therefore, SHIP plays an important role in regulati ng HSC proliferation, survival, self-renewal, as well as BM homing and retention. 25

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Aims: 1) Quantitate the HSC compartment in different hema topoietic organs of SHIPdeficient mice as compared to WT by flow cytometry. 2) Assess if SHIP -/HSC are functional in a transplant assay. This is the only in vivo assay to measure HSC ability to self-renew and differentiate into cells of the hematopoietic compartment. 3) Determine why SHIP -/HSC do not engraft as efficiently as WT HSC; Are they short-term HSC?, do they lack the abilit y to differentiate in a transplant setting?, are they more prone to apopt osis due to the osteoporotic phenotype observed in SHIP -/BM?, are they deficient in their ability to engraft? Results SHIP -/Mice Have an Expanded HSC Compartment We initially quantitated HSC numbers based on a phenotype established by Morrison et al., Lin -/low Thy1 + c-Kit + Sca1 + (KTLS), 159 which are highly enriched for long-term repopulating HSC (LT-HSC) activity. 159 We found that SHIP -/mice have an increased percentage and absolute number of KTLS cells in their BM, spleen (Table 1) and PB (data not shown). 26

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Table 1. Increased numbers of HSC cells in the BM and spleen of SHIP -/mice compared to WT littermates. n at least 3 for each genotype, spleen % over WBM cells Abs. #/pair of femur + tibia or Abs#/spleen (x10 3 ) Population SHIP IP/ IP WT p SHIP IP/ IP WT p Lin Sca1 + c-Kit + Thy1 low BM 0.276.024 0.041.008 0.0005 46.24.01 16.95.18 0.0343 spl 0.1767.0481 0.01.001 0.0255 89.2.2 2.93.282 0.0375 Population SHIP -/WT p SHIP -/WT p Lin Sca1 + c-Kit + Thy1 low BM 0.4114.0353 0.0725.025 <0.001 205.3.9 37.9.3 0.001 spl 0.0957.0131 0.0142.0040 0.004 50.90.25 5.80.92 0.01 Lin Sca1 + c-Kit + Flk2 BM 0.2860.0398 0.1340.0202 0.01 221.4.9 103.0.2 0.03 spl 0.0827.0141 0.027.002 0.02 52.10.89 13.58.75 0.008 Lin Sca1 + c-Kit + CD48 BM 0.019.002 0.0068.0006 0.001 7.84.95 2.76.30 0.001 spl 0.0020.0003 0.0002.0001 0.005 5.53.073 0.289.087 0.002 Lin Sca1 + c-Kit + CD48 CD150 + BM 0.0008.0001 0.0004.0001 0.04 0.35.019 0.164.062 0.045 Side population (SP) BM 0.0565.0044 0.0290.0044 0.004 26.7.7 12.5.4 0.035 BM of SHIP P -/mice contained 6-fold more KTLS cells than WT controls (Table 1). In the spleen of SHIP -/mice, we observed a greater than 6-fold increase in the percentage and absolute numbers of KT LS cells (Table 1). A comparable scenario was seen in the PB, where KTLS cell percentage was increased by 2.5fold as compared to WT littermates (data not shown). A similar expansion of the KTLS com partment was observed in SHIP IP/ IP mice, another germline SHIP mutant model wit h a different genetic background. 97 As for the SHIP -/, SHIP IP/ IP BM and spleen exhibited a signi ficant increase in KTLS cells when compared to WT control (Table 1). The percentage of KTLS present in SHIP IP/ IP BM and spleen were 6.7 and 2.7-fo ld higher than observed in WT 27

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control. Furthermore, KTLS absolute numbers were increased 18.2-fold in the BM and 30.4-fold in spleen of SHIP IP/ IP mice as compared to WT controls. Although Thy1.2 expression can be detected on HSC from C57BL6/J mice, surface expression is lower, maki ng negative and positive distinction more difficult than for the Thy1.1 allele. 160,161 Thus, we also quantified HSC numbers using different phenotypes, includi ng the immunophenotype defined by Christensen et al : 160 Lin -/low Flk2 c-Kit + Sca1 + (KFLS). KFLS cells, as opposed to KTLS, can be used regardless of genetic background. 160 We found that SHIP -/mice also exhibited an in creased percentage and absol ute number of KFLS cells in their BM and spleen (Table 1). KFLS cell numbers are expanded by 2-fold in the BM of SHIP -/mice relative to WT mice. We also observed that both the percentage and absolute number s of KFLS cells in SHIP -/spleens were increased by more than 3-fold as compared to WT spleens (Table 1). In addition, Kiel et al recently published a new phenotype to identify HSC that relies on the differential expression of the SLAM family receptor s, CD48 and CD150, by HSC and MPP. 162 We observe a 2.8-fold increase in the number of KLSCD48 cells in SHIP -/BM compared to WT BM (Table 1, Figure 5A, B). Moreover, the percentage and absolute numbers of KLSCD48 cells are increased by 8.7 and 19-fold, respectively in SHIP -/relative to WT spleen (Table 1, Figure. 5A, B). When using the KLSCD48 CD150 + immunophenotype we detect a 2-fold increase in the number of HSC in the BM of SHIP -/BM as compared to WT 28

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(Table1). In order to confirm our obs ervations made using immunophenotypes relying on surface marker expression, we assessed t he level of HSC using the side population (SP) phenotype. Identification of SP cells relies on the function of a transporter protein that exclude the dye Hoechst 33342, 163,164 -these cells are enriched ~1000-fold for LT-HSC activity compared to WBM cells. 163 When comparing SHIP -/vs. WT BM, we observed a 2-fold increase in the percentage and absolute number of SP cells in SHIP -/BM (Table 1). Thus, analysis of five different HSC phenotypes 159,160,162,163 demonstrated a prefer ential expansion of the LT-HSC compartment in SHIP -/mice. These observations implicate SHIP in the control of HSC co mpartment homeostasis. 29

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Figure 5. Significant increase in the percentage and absolute number of KLSCD48 cells in SHIP -/BM. (A) Representative FACS plots showing detection of KLSCD48 HSC in SHIP -/and WT BM. (B) Percentage and absolute number of KLSCD48 cells in the BM (per femur and tibia pair) and spleen (Spl) of SHIP -/(square) and WT (triangle) mice. Data acquired on a FACS Aria with DiVa (BD Biosciences, San Jose, CA) software, analyzed with FlowJo (Tree Star Inc., Ashland, Oregon). (C) Histogram of DNA content in SHIP -/and WT KTLS cells. (D) Bar graph showing the percentage of SHIP -/(black) or WT (grey) KTLS/KFLS cells in each stage of cell cycle as calculated using the Watson Pragmatic model in the FlowJo cell cycle platform. Data acquired on FACS Vantage with DiVa software (BD Biosciences). Significance was established using the unpaired student t test (Prism 4 (GraphPad Software, San Diego, CA, USA)). ***p<0.0001, **p<0.001, *p<0.01, and +++p<0.0005. (mean SEM, n 3). 30

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Since SHIP has been shown to negatively regulate pr oliferation of different cell types, 18 its deficiency could lead to an increase HSC cell cycle activity. In agreement with this, we observed that SHIP -/BM contained a greater proportion of KTLS in the S/G2 phase, 23.2.5% as compared with 14.1.1% for WT BM (Figure 5C). This study dire ctly demonstrates that SHIP -/HSC themselves have increased cycling activity relative to WT. This is consistent with findings of Helgason et al that CRU in SHIP -/BM are more sensitive to 5-fluorouracil treatment. 156 In SHIP P -/BM, we also observed a significant decrease in the proportion of HSC in the quie scent (G0/G1) stage: a subs et of cells enriched for long-term multi-lineage engraftment relative to those that have entered the cell cycle. 147,165-167 Induced Deletion of SHIP During Adulth ood Leads to an Increase in KFLS Numbers in Hematopoietic Organs In the previous section, we studied t he impact of SHIP ablation in germline knockout mice. Although this model is very useful, SHIP is ablated during embryogenesis, which could lead to the disruption of cell function or signaling pathways necessary for normal development. Thus, we also assessed the levels of HSC in an inducible Mx1-Crelox P SHIP knockout model, where SHIP is deleted only during adul thood, allowing for the mice to develop normally. In this model, the region to be targeted is flanked by lox P sites, and the Cre expression is under the control of the Type 1 interferon-inducible Mx1 promoter. 43,102,103 31

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Treatment of these mice with a double stranded RNA homolog, polyinositolic polycytidylic acid (polyIC), leads to the induction of an endogenous anti-viral Type 1 interferon response, which result s in Mx1 promoted Cr e expression and Cre-mediated recombinati on of the target gene, in this case the SHIP promoter and first exon. 102,103 This deletion results in the ablation of SHIP expression. Twenty-one days after polyIC treatment, the mice were euthanatized and HSC were quantitated by flow cytometry (Figur e 6). As a negative control, we used MxCremice treated with polyIC, howev er, since the MxCre promoter/gene is missing, no rearrangement of the lox P site can take place. In the three hematopoietic organs examined, BM, PB a nd spleen, we observed a significant increase in the percentage of KFLS cells in MxCre+ fl/fl mice as compared to MxCrefl/fl In the BM of SHIP-ablated mice, we observed a subtle but statistically significant increase of 1.5-fold in t he percentage and absolut e number of KFLS cells as compared to MxCremice (Figure 6 a, b). In terestingly, the spleen and the PB of SHIP-ablated mice exhibited t he greatest increase in the percentage of KFLS cells. Indeed, there was a 9.8 and 16. 7-fold increase, respectively, in the percentage and in the absolute numbers of KFLS cells in SHIP-ablated spleen as compared to control (Figure 6 b). In t he PB of SHIP-ablated mice, we observed a 12.6-fold increase in the percentage of KFLS cells (Figure 6 b). This result is consistent with these two organs bei ng more dynamic than the BM, and therefore, responding more rapidly to the absence of SHIP. Furthermore, in another experiment, we determined that th e percentage and absolute number of KLSCD48 cells were significantly increased 3. 2-fold and 5.6-fold, respectively, in 32

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SHIP-ablated BM. In these same mice, we observed a 3.9-fo ld augmentation in KLSCD48 cells in the PB. KLSCD48 cells were not increased to the same levels than KFLS in the PB, this might result from the fact that CD48 appears to be present on cycling HSC. 168 Since most of the HSC that are mobilized to the PB usually are pro liferating, it is difficult to find the KLSCD48 cells in the PB. Nevertheless, we observed that SHIP ablation during adulthoo d results in an enlargement of the HSC compartment, suggesting that the HSC increase observed in SHIP -/germline mice is not the result of a developmental defect. These results also suggest that the ef fect of SHIP ablation on the HSC compartment is intrinsic to the HSC. 33

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Figure 6. Significant increase in the percentage and absolute number of HSC in SHIPablated BM, spleen and PB. MxCre+ and MxCremice with floxed SHIP alleles were treated 3 times with 625 g of polyIC in the course of a week, 21 days prior to being analyzed. (A) Representative FACS plots showing detection of KFLS HSC in SHIPablated and MxCreBM. (B) Percentage of KFLS cells in the BM, spleen and PB of SHIP-ablated (black) and MxCre(grey) mice, and absolute number of KFLS cells in BM (per femur and tibia pair) and spleen. (C) Representative FACS plots showing detection of KLSCD48 HSC in SHIP-ablated and MxCreBM. (D) Percentage of KLSCD48 cells in the BM and PB of SHIP-ablated (black) and MxCre(grey) mice, and absolute number of KLSCD48 cells in BM (per femur and tibia pair). Data acquired on a FACS Calibur with CellQuest software (BD Biosciences), analyzed with FlowJo. Significance was established using the unpaired student t test (Prism 4). *p<0.01, ++ p<0.005, and +p<0.05. (mean SEM, n 3). 34

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SHIP -/BM Cells Show Decreased Ability to Reconstitute the Hematopoietic Compartment of Lethally Irradiated Recipients It was previously reported that SHIP -/BM cells have a lower ability to reconstitute irradiated mi ce after transplantation, but contains similar CRU activity as compared to WT BM when measured by limiting-dilution assay. 156 Since these results are inconsistent with our finding that SHIP -/BM contains increased number of HSC (Tab le 1, Figure 5), we addressed this discrepancy by measuring the ability of SHIP -/HSC to accomplish long-term multi-lineage repopulating activity. We first performed a well-defined CRU assay as described by Harrison 169 to assess the level of repopulation activity in WBM. WBM is used for the CRU assay and therefore is not dependent on isolation of HSC based on cell surface markers, whose expression can be altered by genetic mutation of unrelated loci. 170 Using this assay, we observed that SHIP -/BM cells did not reconstitute recipients as efficiently as WT littermates, with a significant reduction of 4.4-fold in CRU numbers in primar y transplant recipients (Figure 7A, B). 35

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Figure 7. SHIP -/WBM cells have compromised reconstituting activity. (A) CRU activity calculated based on the percentage of global repopulation in the PB by donor WBM cells (SHIP -/(black); WT (grey)). (B) Percentage of global repopulation of PB by SHIP -/(black) and WT (grey) donor in a CRU assay. (C) Percentage of lymphoid and myeloid PB cells derived from SHIP -/(black) or WT (grey) WBM 16 weeks after transplantation in a CRU assay. Data acquired using FACS Calibur, CellQuest software (BD Biosciences) and analysis done with FlowJo. Significance was established using the unpaired student t test (Prism 4): ***p<0.0001, **p<0.001, and +p<0.05. (mean SEM). SHIP -/BM cells showed a significant dec rease in long-term reconstitution of both the lymphoid and myeloid cell li neages. For example, 16 weeks after transplantation only 13.2.7% of B cells were SHIP -/derived (CD45.2 + ) compared to 60.1.8% for WT (CD45.1 + ) WBM, T cell repopulation were 21.2.7% SHIP -/derived vs. 39.7.1 for WT and 34.7.2% of myeloid cells were SHIP -/BM derived vs. 57.4.8% from WT (Figure 7C). The reduction in the SHIP -/B cell representation observed in the CRU assay agrees with other studies showing that SH IP deficiency negatively impacts early B-lineage development. 171 We did not observe that SHIP -/BM cells had enhanced myeloid repopulation relative to WT BM cells (Fi gure 7C). However, we did observe a bias toward myeloid differentiati on within cells derived from SHIP -/BM, where 59.7.7% of the CD45.2 + in the SHIP -/WBM recipients were Mac1 + /Gr1 + 36

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compared to 24.1% for WT BM recipients. This occurred at the expense of the B cell lineage, for which only 25.3 1.5% of CD45.2+ cells were B220 + compared to 61.8.1% for WT (data not shown). The overall reduction in long-term multilineage repopulation by SHIP -/BM cells demonstrates that despite the increase in HSC numbers observed (Table 1 and Figure 5A, B), HSC activity is significantly compromised in these mice (Figure 7A, C). Measuring HSC activity by transplantat ion of WBM from mutant mice can be confounded by the fact that t he mutation can endow lineage-committed progenitor cells with properties that may obscure the observation of compromised HSC function. 172 This is a concern when working with SHIP -/BM as the SHIP mutation enhances myelopoiesis, causing an increase in myeloid progenitors, which have enhanced survival. 95 Therefore, analysis of HSC activity in SHIP -/mice must also be assessed with purified HSC to rule out these potential experimental artifact s. To directly assess wh ether SHIP expression by HSC is required for long-term multi-lineage repopulation and self-renewal, we directly compared the repopulating potential of purified HSC from SHIP -/and WT mice. Thus, CD45.2 SHIP -/KTLS or KFLS cells were transplanted into lethally irradiated CD45.1/CD45.2 WT recipients with an equivalent number of CD45.1 WT KTLS or KFLS and Sca1 CD45.1/CD45.2 support ce lls. This assay, the direct competition (DC) assay, 173 directly compares the capacity of geneticallymodified HSC to compete with WT HS C for engraftment and long-term multilineage repopulation. The le vel of repopulation in reci pients PB was assessed at 37

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regular intervals (Figure 8A), and at si xteen to twenty weeks post-transplant, we observed that global repopulation by SHIP -/KTLS was reduced by 15-fold relative to WT KTLS (Figure 8B). Figure 8. SHIP -/purified HSC have compromised reconstituting activity. (A) FACS plots show the level of PB reconstitution 16-weeks after KTLS transplantation in a representative DC assay mouse using sorted SHIP -/and WT KTLS. (B) Percentage of global reconstitution 16 weeks after transplantation of sorted KTLS (SHIP -/(black) and WT (grey)) (n=11 over 2 different experiments). (C) Proportion of lymphoid and myeloid PB cells derived from SHIP -/(black) or WT (grey) WBM 16-weeks after KTLS transplantation. (D) Percentage of global reconstitution 12 weeks after sorted KFLS transplantation (SHIP -/(black); WT (grey)) (n=7). Data acquired using FACS Calibur, CellQuest software (BD Biosciences) and analysis done with FlowJo. Significance was established using the unpaired student t test (Prism 4): *p<0.01, ++p<0.005 and +p<0.05. (mean SEM). 38

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Reconstitution of different lineage by SHIP -/KTLS was also significantly reduced (Figure 8C). Thus, SHIP -/KTLS are capable of multi-lineage reconstitution, but are unable to sustain th is activity for ext ended periods when in direct competition with WT KTLS (Figure 8C). Furthermore, SHIP -/cells did not dominate the myeloid compartment as mi ght have been expected from previous studies, 95,156 with only 1% of Mac1 + /Gr1 + cells being derived from SHIP -/KTLS vs. 21% from WT KTLS (Figure 8C). This indicates that although SHIP -/myeloid lineage progenitors have enhanced survival and can prevail over seriallytransplanted competitors, 95,156 they are, nonetheless, unable to out-compete normal WT myeloid progenitors when derived from purified HSC in a chimeric transplant setting. As with KTLS cells, long-term global r epopulation by SHIP -/KFLS is significantly reduced (Figure 8D). These results demonstrate that SHIP expression is required by HSC in or der to sustain long-term multi-lineage repopulation. SHIP -/HSC do not Exhibit Characterist ics of Premature Differentiation As HSC go down the path of differentiati on, they lose the ability to selfrenew and exhibit a higher proliferative state. Si nce we observed that SHIP -/HSC proliferation is incr eased and engraftment is per turbed, we assessed by flow cytometry if SHIP -/KTLS exhibited immunophenotypic characteristic of early differentiation. It has been shown that KTLS cells with high levels of c-Kit (c-Kit hi ) are enriched for LT-HSC activity, as opposed to the c-Kit lo population that is not 39

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able to support long-term multi-lineage repopulation. 174 This assay revealed that a significantly great er proportion of SHIP -/TLS were c-Kit hi as compared to WT KTLS cells (Figure 9A, B). There was a greater than 3 fold increase in the percentage of c-Kit hi TLS in the SHIP -/BM as compared to WT BM (Figure B). Figure 9. More SHIP -/TLS cells express high levels of c-Kit as compared to WT. (A) Representative FACS plots showing detection of Lin Thy1 + Sca1 + c-Kit high and Lin Thy1 + Sca1 + c-Kit low (B) Percentage (left column) and absolute number (right column) of Lin Thy1 + Sca1 + c-Kit high (top row) and Lin Thy + Sca1 + c-Kit low cells). Absolute numbers calculated for 1 pair of femur and tibia. Data acquired on a FACS Aria with DiVa software, analyzed with FlowJo. Significance was established using the unpaired student t test (Prism 4). ***p<0.0001 and ++p<0.005. (mean SEM, n=4). 40

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In fact, in SHIP -/mice 95% of KTLS cells expressed high levels of c-Kit while only 78% of KTLS in WT mice (Figur e 9A). This analysis revealed that the percentage and the mean abs olute number of c-Kit hi TLS cells in SHIP -/WBM cells were 4 and 4.6 fold higher, respec tively, as compared to WT littermates (Figure 9B). As LT-HSC differentiate towards short term (ST)-HSC phenotype, they begin expressing increasing amoun t of Mac-1 on their surface, they proliferate at a faster rate and hav e a reduced ability to self-renew. 159 Therefore, we assessed the level Mac1 expression on SHIP -/KTLS cells in order to better characterize the HSC compartment in these mice. After gating on live KTLS cells, we looked at the level of Mac1 expression on SHIP -/and WT HSC by flow cytometry. Since the histogram for Mac1 on KTLS was unimodal, we assessed the level of Mac1 expression using the mean fluorescence intensity (MFI) instead of percentages (Figure 10A, B). Our results revealed that SHIP -/KTLS cells express comparable levels of Mac1 mo lecules on their surface as WT KTLS (Figure 10A, B), suggesting that the pr oportion of ST-HSC contained in SHIP -/KTLS fraction is not increased as compared to WT control. Thus, according to this phenotypic assay, SHIP -/KTLS do not exhibit characteristic of premature differentiation. 41

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Figure 10. SHIP -/KTLS express similar levels of Mac1 as compared to WT KTLS. (A) Representative FACS plots showing detection of Mac1 expression (histogram) on KTLS. (B) Two representative overlay histogram of Mac1 expression for SHIP -/(black) and WT (grey) KTLS. (C) Graph comparing the MFI of Mac1 expression for SHIP -/and WT KTLS. Data acquired on a FACS Aria with DiVa software, analyzed with FlowJo. Statistical analysis was done using the unpaired student t test (Prism 4). (mean SEM, n=4). 42

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SHIP -/HSC Self-Renew to a Lesser Extent than WT HSC in Transplanted Mice Our data suggest that SHIP-deficient mice have increase in HSC number and proliferation. However, once transplanted these cell s did not reconstitute the host was well as WT HSC. One of t he hypothesis proposed to explain these observation was that SHIP -/HSC might be unable to differ entiate. In that case, SHIP -/HSC would be present in the BM and self-renew wi thout having the ability to differentiate and reconstitute the host PB. Therefore, we assessed the level of reconstitution of the different hematopoietic organs of transplanted mice at least 6 months after transplantation. Three of the CRU transplanted mice were sacrificed and their spleen, BM and PB were assessed for granulocyte/macrophage and lymphoid reconstitution. This assay revealed that every lineage in every organ except for Mac1Gr1 in PB was significantly less reconstituted by SHIP P -/WBM cells as compared to WT WBM cells (Figure 11A). The majority of the lineages were reconstituted to less than 25% by SHIP -/WBM cells. 43

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Figure 11. SHIP -/HSC do not engraft and self-renew as well as WT HSC, although transplanted in equal numbers. (A) CD45.1 mice were irradiated (1000 rads) and transplanted with 1 million CD45.2 SHIP -/and 1 million CD45.1 WT WBM. Percentage of lymphoid and myeloid PB, spleen and BM cells derived from SHIP -/(black) or WT (grey) WBM >16 weeks after transplantation. (B) Percentage of KTLS cells in the BM that are derived from SHIP -/(black) or WT (grey) WBM. (C) CD45.1xCD45.2 mice were irradiated (600 and 400 rads at 3-hour interval) and transplanted with 2x10 5 CD45.2 SHIP -/and 2x10 5 CD45.1 WT KTLS cells with 4x10 5 CD45.1xCD45.2 Sca1 cells. First bar graph shows the percentage of KTLS cells derived from SHIP P -/(black) or WT (grey) KTLS cells. Second bar graph Absolute numbers of SHIP -/and WT KTLS found in 1 pair of femur and tibia SHIP -/(black) or WT (grey) KTLS cells. Third bar graph compares fold expansion for SHIP -/and WT KTLS cells SHIP -/(black) or WT (grey) KTLS cells. Data acquired using FACS Vantage, DiVa software and analysis done with FlowJo. Significance was established using the unpaired student t test (Prism 4): *p<0.01, +++p<0.0005, ++p<0.005 and +p<0.05. (mean SEM, n=3) 44

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We then had to consider that the SHIP -/HSC could self-renew but not differentiate as efficiently as WT. Thus, we observed the level of reconstitution of the KTLS compartment. Briefly, BM cells were enriched for HSC by lineage depletion using the AutoMACS (Miltenyi Biotec, Auburn, CA, USA), and then stained for KTLS, CD45.1 and CD45.2 in the presence of DAPI. This assay revealed that the per centage of KTLSCD45.2 + (SHIP -/) over the total amount of KTLS cells was 13.8 4.8%, while 57.3 7.2% of KTLS were CD45.1 + (WT) (Figure 11B). Therefor e, the capacity of SHIP -/WBM cells to reconstitute the HSC compartment of a trans planted mouse is signific antly lower than he one of WT WBM cells, p=0.007 (Figure 11B). Th is result correlates with the lower reconstitution of the different hematopoietic organs by SHIP -/CD45.2 cells (Figure 11A). In the CRU assay the recipi ent is CD45.1, therefore, we could not totally exclude that a portion of CD45.1+ KTLS came from the host. To address this issue, we assessed the level of KTLS reconstitution in DCA transplanted mice more than 12 months after transplantati on. In this case the recipients are CD45.1xCD45.2, thus WT (CD45.1) and SHIP -/(CD45.2) cells can be differentiated from the host. In these transplanted mice, it was also obvious that CD45.2 SHIP -/KTLS cells composed a significant ly reduced portion of the KTLS compartment as compared to WT (Figure 11Ci). The absolute numbers of SHIP /KTLS cells contained in one hind limb were reduced 6.4-fold as compared to the number of WT KTLS (Figure 11Cii). Furthermore, the fold expansion of KTLS cells calculated based on the amount of transplanted KTLS cells and the total amount of KTLS recovered in the recipients revealed that SHIP -/KTLS only 45

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expanded 44 25-fold compared to 287 56 fold for WT KTLS. Thus, SHIP -/KTLS did no self-renew with the same e fficiency than WT KTLS p=0.008 (Figure 11Ciii). These results confirm that SHIP -/WBM and KTLS cells cannot reconstitute the hematopoietic system of a lethally irradiated mouse as efficiently as WT cells. The higher pr oliferative profile of SHIP -/HSC should have given them an advantage in the reconstitution assa y. Therefore, we had to further investigate why SHIP -/HSC are not able to accomp lish long-term reconstitution. SHIP -/HSC Have a Lower Rate of Spontaneous Apoptosis It was reported that SHIP -/mice have increased osteoporosis caused by an increase in osteoclast activity. 101 The HSC niche is dependent on the presence of osteoblasts, the counter part of osteoclasts, to properly sustain the hematopoietic compartment. 132 Thus, any disruption of the balance between osteoblasts and osteoclasts could destroy the niche necessary for HSC survival, resulting in anoikis-asso ciated apoptosis of the HSC compartment. If SHIP -/HSC exhibited increased apoptosis, this c ould contribute to the decreased ability of SHIP -/HSC to accomplish long-term repopula tion. Therefore, we assessed the level of apoptosis in SHIP-deficient HSC and we found that a significantly lower proportion of SHIP -/HSC were apoptotic relative to WT HSC as measured by Annexin V staining (Figure 12A). In fact, 11.1% of WT HSC (KTLSFlk2-) stained with Annexin V wh ile only 1.1% of SHIP -/HSC did. TUNEL analysis 46

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done on SHIP IP/ IP HSC also revealed that SHIP-deficient HSC were undergoing apoptosis at half the rate of WT HSC in the BM (Figure 12B). Thus, the lack of long-term repopulating activity of SHIP -/HSC activity appears not to be associated with anoikis-induced apopt osis of the compartment. Figure 12. SHIP -/HSC exhibit decreased apoptotic rate. (A) Representative FACS plots of DAPI vs. Annexin V after gating on KTLSFlk2 cells. Bar graph shows the percentage of KTLSFlk2 that are apoptotic based on the Annexin V + and DAPI staining, SHIP -/(black); WT (grey). (B) Representative FACS plots for TUNEL staining after gating on KTLS cells. Bar graph represents the percentage of KTLS cells positive for TUNEL staining, SHIP -/(black); WT (grey). Data acquired using FACS Aria, DiVaII software and analysis done with FlowJo. Significance was established using the unpaired student t test (Prism 4): *p<0.01 and ++p<0.005. (mean SEM, n 3). 47

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In vivo Homing of SHIP -/Stem/Progenitors to the BM is Significantly Reduced as Compared to WT We considered that the dimini shed repopulation observed for SHIP -/HSC could result from an inefficiency of SHIP -/HSC to home and be retained in the BM niche. Thus, we assess ed the homing capacity of SHIP -/stem/progenitor cells compared to WT cells in an in vivo homing assay. 147,175 Sca1 + Lin cells from the BM of SHIP -/and WT mice were isolated, stained with fluorescent dyes, and injected into irradiated recipi ent mice. The frequency of SHIP -/and WT Sca1 + Lin cells present in the recipient BM and spleen was later assessed by flow cytometry (Figure 13A). Using a tota l of 12 recipients for each genotype, we found that SHIP -/stem/progenitor cells reached the BM with a significantly reduced efficiency relative to WT st em/progenitor cells (Figure 13B). Furthermore, we observed that SHIP -/Sca1 + Lin cells did not home to the spleen as well as WT Sca1 + Lin cells (Figure 13C). Thes e results suggest that SHIP -/HSC are impaired in their ability to home and be retained in hematopoietic organs. This deficiency likely contributes to their inability to engraft and sustain multi-lineage hematopoiesis. 48

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Figure 13. SHIP -/Sca1 + Lin cells do not home to the BM as efficiently as WT Sca1 + Lin (A) Representative FACS plot of BM from transplant recipients after gating on live cells. (I) Shows BM from control recipient 12-14 hours after transplantation with 0.5x10 6 WBM stained with 1 M DDAO (II) Shows BM from control recipient 12-14 hours after transplantation with 0.5x10 6 WBM stained with 0.5 M CFSE. (III) Representative FACS plot of BM from a mouse 12-14 hours after transplantation with 2x10 5 SHIP -/Lin Sca1 + cells stained with 0.5 M CFSE and 2x10 5 WT Lin Sca1 + cells stained with 1 M DDAO. (B) Left: Percentage of dye + SHIP -/(black) or WT (grey) Sca1 + Lin cells found in the recipient BM 12-14 hours after transplantation. Right: Percentage of SHIP -/(black) or WT (grey) Sca1 + Lin cells that trafficked to and was retained in BM of recipients over the total number of cells injected, 12-14 hours after transplantation. (C) Left: Percentage of stained SHIP -/(black) or WT (grey) Sca1 + Lin cells found in the spleen of recipients 12-14 hours after transplantation. Right: Percentage of SHIP -/(black) or WT (grey) Sca1 + Lin cells that reached the spleen of recipients over the total number of cells injected, 12-14 hours after transplantation. Data acquired using FACS ARIA, DiVaII and analysis done with FlowJo. Significance was established using the stratified Wilcoxon-Mann Whitney test using StatXact (Cytel Software Corporation, Cambridge, MA, USA): +p<0.05, *p<0.01 and ++p<0.005. (mean SEM, n=12). 49

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Reduced Surface Expression of CXCR4 and VCAM-1 + on KTLS Cells in SHIP -/BM CXCR4, VCAM-1 and VLA-4 are all known to play prominent roles in homing and trafficking of HSC and other hematopoietic cells to BM. 151,158,176 Thus, we decided to examine the level of expression of these markers on SHIP -/HSC cells compared to WT HSC. By flow cytometry, we observed a 2.5-fold reduction in the surface expressi on of the CXCR4 receptor on SHIP -/KTLS relative to WT KTLS (Figure 14A, B). Flow cytometry analysis showed a reduction in VCAM-1 surface expression on SHIP -/HSC cells as compared to WT HSC (Figure 14A). We found that 22.1.5% of SHIP -/KTLS were positive for VCAM-1 while 50.8.9% of WT KTLS were (Figure 14A, B). The VCAM-1 MFI values for SHIP -/KTLS were also reduced by 3-fold as compared to WT KTLS (Figure 14B). However, not a ll homing molecules showed a reduced level of expression. For exampl e, the percentage of KTLS cells expressing VLA-4 as well as their MFI values in SHIP -/BM cells were unchanged compared to WT BM. Furthermore, contrary to SHIP -/KTLS, SHIP -/Lin + c-Kit and Lin c-Kit + cells did not exhibit a significant differenc e in the level of VCAM-1 and CXCR4 expression as compared to WT Lin + c-Kit cells (Figure 15). We also observed the level of VCAM-1 and CXCR4 expr ession in the KLS and KLSThy1populations to evaluate if SHIP defici ency impacted the expression of these markers in different progenitor populatio n or only in the HSC enriched KTLS population. We observed that the reduction in CXCR-4 expression is specific to 50

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the KTLS population (Figure 14). The KLS population, which contains HSC and mostly early and late progenitor cells did not exhibit a significant difference in their ability to express CXCR4 in the absence of SHIP as compared to WT controls (Figure 16Bi-Ci). Furthermore, we observed no significant difference in the level of CXCR4 expression by SHIP -/KLSThy1 cells as compared to WT control (Figure 17Bi-Ci). Ho wever, percentage of VCAM-1 + KTL (Figure 16Cii) and KLSThy1 (Figure 17Cii) were both reduced by 2-fold in SHIP -/mice BM as compared to WT control. These results suggest that SHIP might impact VCAM-1 expression in different cell types, includi ng KTLS (Figure 14), KLS (Figure 16), and KLSThy1 cells (Figure 17), while SHIP a ppears to affect CXCR4 expression mostly in KTLS cells (Figure 14 17). In terestingly, the late progenitor cells (lin cKit + ) and differentiated cells (Lin + cKit ) (Figure 15) in SHIP -/mice had a comparable level of CXCR4 and VCAM-1 expression as compared to WT. Reduction of VCAM-1 and CX CR4 expression on the SHIP -/HSC suggests these cells may be hampered in their abilit y to traffic and be retained in the BM, consistent with the decreased BM homing and retention we observe (Figure 13). 51

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Figure 14. SHIP -/HSC express lower levels of CXCR4 and VCAM-1 molecules as assessed by flow cytometry. (A) A representative histogram of CXCR4 (left) and VCAM1 (right) expression on live (DAPI ) KTLS cells (isotype control (light grey), SHIP -/(black), and WT (grey)). (B) The plots on the left represent the mean fluorescence intensity (MFI) of KTLS cells for CXCR4, VCAM-1, and VLA-4, respectively and the plots on the right show the percentage of KTLS cells positive for VCAM-1 and VLA-4, respectively (SHIP -/(square); WT (triangle)). Data acquired using FACS ARIA, DiVaII and analysis done with FlowJo. Significance was established using the unpaired student t test (Prism 4): +++p<0.0005 and +p<0.05. (mean SEM, n 3). 52

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Figure 15. SHIP -/late progenitor and differentiated cells express the same levels of homing molecules as assessed by flow cytometry. (A) A representative histogram of CXCR4 (left) and VCAM-1 (right) expression on live (DAPI ) Lin + c-Kit (isotype control (light grey), SHIP -/(black), and WT (grey)). (B) The plots on the left represent the mean fluorescence intensity (MFI) of Lin c-Kit + (top) and Lin + c-Kit (bottom) cells for CXCR4, VCAM-1, and VLA-4, and the plots on the right show the percentage of Lin c-Kit + (top) and Lin + c-Kit (bottom) cells positive for CXCR4, VCAM-1 and VLA-4. (SHIP -/(black); WT (grey)). Data acquired using FACS ARIA, DiVaII and analysis done with FlowJo. Statistical analysis was done using the unpaired student t test (Prism 4). (mean SEM, n 3). 53

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Figure 16. SHIP -/early progenitors express the same levels of CXCR4 and have a reduced percentage of VCAM-1+ cells. (A) FACS plot representing gating strategy for the analysis of CXCR4 and VCAM-1 expression on KLS cells for (i) SHIP -/and (ii) WT BM cells. (B) A representative histogram of CXCR4 (i) and VCAM-1 (ii) expression on live (DAPI ) Lin c-Kit + Sca1 + (isotype control (light grey), SHIP -/(black), and WT (grey)). (C) (i) Bar graphs representing the percentage and MFI of Lin c-Kit + Sca1 + cells for CXCR4 expression. (ii) Bar graphs representing the percentage and MFI of Lin cKit + Sca1 + cells for VCAM-1 expression. Data acquired using FACS ARIA, DiVaII and analysis done with FlowJo. Statistical analysis was done using the unpaired student t test (Prism 4). (mean SEM, n 3). 54

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Figure 17. SHIP -/early progenitors express the same levels of CXCR4 and have a reduced percentage of VCAM-1 + cells. (A) FACS plot representing gating strategy for the analysis of CXCR4 and VCAM-1 expression on KLSThy1 cells for (i) SHIP -/and (ii) WT BM cells. (B) A representative histogram of CXCR4 (i) and VCAM-1 (ii) expression on live (DAPI ) Lin c-Kit + Sca1 + Thy1 (KLSThy1 ) (isotype control (light grey), SHIP P -/(black), and WT (grey)). (C) (i) Bar graphs representing the percentage and MFI of Linc-Kit+Sca1+ cells for CXCR4 expression. (ii) Bar graphs representing the percentage and MFI of KLSThy1 cells for VCAM-1 expression. Data acquired using FACS ARIA, DiVaII and analysis done with FlowJo. Statistical analysis was done using the unpaired student t test (Prism 4). (mean SEM, n 3). 55

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Elevated Levels of Soluble VCAM-1 Levels in SHIP -/Mice Sera Interestingly, we observed that SHIP -/mice sera contained an increased level of soluble VCAM-1 (sVCAM-1) (Figure 18). This molecule once released in the circulation through prot eolytic cleavage remains active and can still bind VLA4 receptor. 177,178 Thus, sVCAM-1 has the potential to hinder the interaction of leukocyte and HSC with endothelium. 179,180 Therefore, elevated sVCAM-1 could potentially bind VLA-4 receptor on HS C and prevent its interaction with endothelium-bound VCAM-1 and pr event its homing to the BM. This particular scenario could in part explain why we see an increase in HSC number in the periphery and maybe why we see a decrease in the homing prop erties of these cells. One theory for the latter hypothesis would be that sVCA M1 bind the VLA-4 receptor, preventing their interaction with membrane-bound VCAM-1. Figure 18. sVCAM-1 levels are significantly increased in SHIP -/sera as compared to WT littermates. Blood was obtained by sub-mandibular bleed and collected in regular Eppendorf tube to allow coagulation. The sera was then collected and sent for analysis by ELISA (Charles River Laboratories Inc.). ELISA was then performed to assess the level of different cytokines. Significance was established using the unpaired student t test (Prism 4): ++p<0.005 (mean SEM, n 3). Collaboration with D. Woods and WG Kerr Ph.D. 56

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Discussion In this study, we show that SHIP -/mice had increased HSC in their BM, spleen and PB than WT littermates bas ed on analysis using multiple HSC phenotypes. An expansion of the HSC compartment was also observed in a second SHIP-deficient model, in whic h the inositol phosphatase region was targeted, the SHIP IP/ IP 97 This latter model is deficient for SHIP expression and, presumably s-SHIP expressi on, contrary to the SHIP -/model. s-SHIP is an isoform expressed in ES cells and HSC but not in differentiated cells. 49 Our studies show that SHIP IP/ IP HSC have a similar phenotype to the SHIP -/HSC as compared to WT showing an expans ion of the HSC compartment, decreased apoptosis rate and decreased level of CX CR4 expression. According to our data, the ablation of both isoforms did not result in a more severe HSC phenotype. However, additional studies should be done to further assess the role of s-SHIP in this cell type. The increase in HSC numbers observed in SHIP-deficient mice may partly result from the increased proliferat ion and decreased apoptotic frequency observed in the SHIP -/HSC compartment. These results are consistent with SHIP being a negative regulator of seve ral signaling pathways downstream of receptors that impact HSC proliferation and survival, such as c-mpl IL-3R, CXCR4, Flt3 and c-Kit, which induce cell proliferation and/or survival through 57

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activation of the MAPK and PI3K/Akt pathways. 15,25,47,181-187 Moreover, SHIP -/myeloid and hematopoietic progenitor cells are hyper-responsive to several of these factors. 18 The enhanced survival an d proliferation of SHIP -/HSC could partially explain the decreased repopulating ability of these cells as it was shown that cycling HSC engraft less efficiently than resting HSC. 146,165,167 Homing and eventual engraftment of HSC following transplantation has been shown to rely on the expression and activity of several surface molecules, including VLA-4, VLA-5 and CXCR4. 151,188,189 The CXCR4 receptor induces cell migration towards an increasing gradient of SDF-1/CXCL12. 149 Treatment of NOD/SCID mice with SDF-1 leads to the mobilization of HSC to PB 150 and treatment of human HSC wit h anti-CXCR4 antibody prior to transplantation in NOD/SCID mice results in HSC engraftment failure. 151 Furthermore, CXCR4 and SDF-1-deficient mice are em bryonic lethal since HSC fa il to migrate from the FL to the BM, where definitive hematopoiesis would take place. In addition, one of the ligands for VLA-4, VCAM-1, which is usually found on stromal/endothelial cells, has been shown to be expressed in hematopoietic cells such as Mac-1 + B220 + and, more interestingly, c-Kit + cells. 158 Even though the role of VCAM-1 on hematopoietic stem/progenitor cells rema ins to be fully elucidated, there is evidence that it is involved in their re tention in the BM, as shown for B cell progenitors. 158 With that information in mind, the reduction in the percentage of HSC expressing CXCR4 and VC AM-1 receptors in SHIP -/BM can provide a basis for the decrease in SHIP -/HSC ability to home to the BM, as shown by our 58

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in vivo homing assays (Figure 13). This inefficiency of SHIP -/HSC to reach and be retained in the HSC nich e could contribute to their impaired self-renewal and long-term repopulating capacity. In previous studies, in vitro stimulation of murine BM stem/progenitor cells with different cytokines was shown to induce cell cycle, decrease VLA-4 expre ssion and compromise engraftment. 146 In our study, the increase in HSC cell cycle observed in vivo did not necessarily correlate with a decrease in VLA-4 expre ssion, but caused a decline in long-term engraftment capacity. We observ ed that decreased CXCR4 and VCAM-1 expression on KTLS cells correlated wit h a reduction in the homing process. Moreover, CXCR4 has been shown to partici pate in the activation of VLA-4 and VLA-5 to promote human HSC a ttachment and extravasati on, resulting in homing of HSC to the BM niche. 189 The reduction in the CX CR4 receptor seen on SHIP -/HSC might also dampen this process and, thus, HSC homing. Furthermore, it has been shown that CXCR4 and VCAM-1 ar e important for the retention of HSC or hematopoietic pro genitors in the BM. 157,158 Interestingly, we find that SHIP -/mice have an increased number of HSC cells in their spleen (Table 1 and Figure 5A, B) and peripheral blood (data not shown), consistent with the down modulation of CXCR4 and VCAM-1 on SHIP -/HSC. On the other hand, the increased level of sVCAM-1 in the plasma of SHIP -/mice could hinder the interaction between HSC a nd endothelium and dampen the homing and retention process of these cells. 59

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Even though we observed a reduction in engraftment by both the DC and CRU assay, the defect seems greater in the DC assay, where equal numbers of purified SHIP -/and WT HSC are co-tr ansplanted. This sugge sts that in the CRU assay, where WBM cells are used, t he increased number of HSC in SHIP -/BM might partially compensate for the reduced levels of CXCR4 and VCAM-1 on SHIP -/HSC, resulting in be tter engraftment by SHIP -/HSC in the CRU assay as compared to the DC assay. The CRU assay included in this st udy showed a decrease in the repopulating ability of SHIP -/WBM, while previous studies revealed that SHIP -/WBM had comparable repopulating activity in a limiting-dilution CRU assay. 156 The different results observed between the two CRUs might stem from the variation in the assays themselves. In the Harrison CRU assay, healthy competitor cells are utilized, while the limiting-diluti on CRU assay requires the use of competitor WBM weakened by two serial transplantations. 190 Thus, the compromised WBM competitors in the latter CRU assay may be unable to compete for the niche as efficiently as normal WT WBM against SHIP -/WBM. Furthermore, the calculation method for th e Harrison CRU assay differs from the one used for the limiting-dilution assay, which might result in a different assessment of repopulating acti vity by the two assays. In summary, our findings demonstrate a role for SHIP in the maintenance of the HSC compartment. SHIP appears to be critical for the negative control of 60

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HSC proliferation and surviv al as well as impacting t he ability of HSC to home and be retained in the BM niche. Materials and Methods Mice SHIP -/mice were generated by deletion of the promot er and first exon of SHIP via a Crelox P strategy and then backcrossed to the C57BL6/J background. 43 SHIP P IP/ IP mice were created using a Crelox P strategy targeting the inositol phosphatase encoding region. SHIP IP/ IP mice are on a 129SvJ background and were kindly provided by Dr. Jeffrey Ravetch (Rockefeller University, NY, USA). 97 For all experiments, germline SHIP-deficient and WT mice were 6-8 weeks of age. For the inducible model, SHIP fl/fl mice were backcrossed to MxCre germline. The re sulting SHIP fl/fl MxCre+ and MxCremice were treated 3 times with 625 g polyIC in the course of 7 days. At least 20 days after the last treatment, the mice were sacrificed and experiment was performed. As recipients for transplant ation experiments we used 8-12 weeks old B6.SJLPtprca Pep3b/BoyJ (CD45.1) (Jackson Laboratory Bar Harbor, Maine, USA) and a CD45.1x CD45.2 strain generated by intercrossing C57BL6/J (CD45.2) and B6.SJLPtprca Pep3b /BoyJ (CD45.1) mice. Animal experiments 61

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were conducted in compliance with institut ional guidelines at the University of South Florida. Cell Isolation BM cells were flushed from intact femurs and tibia. Splenocytes were isolated by crushing the spleen with a syringe plunger. All cells were collected in tissue media (TM) composed of RPMI, 3% fetal bovine serum (FBS), and 10mM HEPES (Gibco BRL/Invitrogen, Carlsbad, CA USA). Cells we re then filtered through a 70 m strainer (BD Biosciences, San Jose, CA) and red blood cells (RBC) were lysed at room temperature (R T) for 2-5 minutes in 1xRBC lysis buffer (eBioscience, San Diego, Ca, USA). The remaining cells were then centrifuged and resuspended in staining media (SM) composed of 1xDulbecco-Phosphate buffered saline (D-PBS), 3% FBS, and 10mM HEPES (Gibco BRL/Invitrogen). PB was obtained by retro-orbital (RO) or sub-mandibular bleeding, collected in microtainers with K 2 EDTA (BD, Franklin Lakes, NJ, USA), and RBC lysed twice to obtain PB mononuclear cells (PBMC), which were resuspended in SM. HSC Phenotype All antibodies were from BD Bioscienc es except where noted. All flow cytometry data were analyze d with FlowJo Software (T ree Star, Inc. Ashland, 62

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OR, USA). BM cells, splenocytes or PBMC were treated with Fc block (2.4G2) and then stained on ice for Lineage -/low c-Kit + Sca1 + Thy1 + (KTLS) phenotype. 159 The stain included, Sca1PE (E13-161.7), c-Kit-APC (2 B8), Thy1.2-Cychrome (53-1.2) and a lineage (Lin) panel-FITC: CD2(RM2-5), CD3 (145-2C11), CD4(GK1.5), CD5(53-7.3), CD8 (53-6.7), B220(RA3-6B2) Gr-1(RB6-8C5), Mac1(M1/70), NK1.1(PK136), and Ter119(TER-119) (eBi oscience). For Lin ckit + Sca1 + CD48 CD150 + (KLSCD48 CD150 + ), 162 the same Lin panel was used with the addition of CD34F ITC (RAM34) along with Sca1-biotin (E13-161.7), cKit-APCCy7 (2B8) (eBioscience), CD48-PE (HM48-1), CD150-Alexa647 (9D1) (Serotec Inc., Raleigh, NC, USA), followed with streptavidin-PeCy7. The cells were then resuspended in SM cont aining 75ng/ml of 4',6-diamidino-2phenylindole dihydrochloride (DAPI) for dead cell exclusion (Sigma-Aldrich, St.Louis, MO, USA). Analysis was done on a FACS Vantage or FACS Aria using DiVaII software (BD Biosciences). For the c-Kit + Flk2 Lin -/low Sca1 + (KFLS) phenotype, 160 we used Sca1-FITC, c-Kit-APC and the above mentioned Lin panel-PE with the additi on of the Flk2 antibody (A2F. 10-1). After staining, the cells were washed and resuspended in SM containing 7-amino-actinomycin D (7AAD) (BD Biosciences) for dead cell exclusion. Acquisition was done on a FACS Calibur with CellQuest software (BD Biosci ences). For the side population (SP) analysis, BM cells were stained following standard procedure. 163 Analysis was done on a FACS Vantage using DiVaII. For the analysis of Mac 1 expression on KTLS, the same Lin panel (FITC) as t he one defined above was used with the 63

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exception of Mac1. The same antibody clones as the one m entioned above were used, but they were conjugated to di fferent fluorochromes: Sca1-PE, c-KitPeCy7, Thy-APC, Ma1-ApcCy7. DAPI wa s used for dead cell exclusion and the data acquired on FACS Aria with DiVaII software. For homing marker analysis, Lin-depletion of BM cells was done on the AutoMACS (Miltenyi Biotec) using t he Lin-PE panel described above, with the exclusion of CD2, anti-PE m agnetic beads (Miltenyi Biot ec). The Lin-depleted fraction was stained for KTLS and CXCR 4 (2B11/CXCR4), VCAM-1 (429), or VLA-4 (9C10) on biotin, followed by staini ng with streptavidin-PeCy7. DAPI was used for dead cell exclusion. Data was acquired on a FACS Aria using DiVaII. Cell Cycle Analysis BM cells were enriched either for Sca1 + or c-Kit + cells using AutoMACS (Miltenyi Biotec). Sca1 + or c-Kit + cells were then stained for the KTLS or KFLS, then fixed in Cytofix/Cytoperm solution (BD Biosciences) for 30-min on ice, washed in Perm/Wash buffer and incubat ed with 4-10ng/ml Hoechst dye 33342 (Sigma-Aldrich) in Perm/Wash buffer overnight at 4 o C. The DNA content was measured 24-hrs later on the FACS Vantage and analyzed using FlowJo software. 64

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CRU Assay CD45.1 recipient mice were given ant ibiotic water -sulfamethaxazole and trimethorprim oral suspensio n 40mg/ml (Hi-Tech Pharma cal CO., INC. Amityville, NY, USA) prior to receiving a si ngle dose of 9.5-Gy 3-hours before transplantation. One million WBM cells from CD45.2 SHIP -/or WT mice along with 1x10 6 CD45.1 WBM cells were injected retroorbitally into recipients, as per Harrison. 169 Transplanted mice were later assessed for PB multi-lineage reconstitution. Repopulating units (RU) were calculated as per Harrison et al.: 169 RU = (10 CD45.2 repopulation(% ))/(100 CD45.2 repopulation(%)). 169 DC Assay The DC assay was modified from Domen et al 173 Donor BM cells were magnetically enriched for Sca1 + cells on AutoMACS (Miltenyi Biotec). The Sca1 + fraction was stained for Lin -/low c-Kit + Sca1 + Thy1 low or Lin -/low Flk2 c-Kit + Sca1 + and sorted twice in the presence of DAPI using the FACS Vantage DiVAII. For the KTLS transplantation, 200 CD45.1 WT and 200 CD45.2 SHIP -/KTLS cells were injected with 4x10 4 Sca1 CD45.1/45.2 WBM cells. For the KFLS transplantation, 100 CD45.1 WT and 100 CD45.2 SHIP -/KFLS cells were injected with 4x10 5 CD45.1/45.2 Sca1 cells. CD45.1/45.2 recipients had been previously irradiated 65

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with a split dose of 10Gy at 3-hour interval and given antibiotic water, as mentioned above. Assessment of MultiLineage Reconstitution For the first 4 months after transplant ation the level of PB reconstitution was assessed, every 4 weeks, as a measure of engraftment of SHIP -/and WT HSC. PBMC were treated with Fc block, then stained with CD45.1-PE(A20), CD45.2-FITC (104), and APCconjugated Lin markers; B220, CD3, or Mac1/Gr1 (clones as previously mentioned). The cells were then was hed and resuspended in SM containing 7-AAD to exclude dead cells. Data acquired on FACSCalibur with CellQuest and analyzed with FlowJo. To assess the level of reconstitution of differentiated cells in the BM, spleen and PB 6 to 8 months after transplantation, we used the same antibody clones mentioned above except that Mac1 was conjugated to APCCy7 and Gr1 to APC. Furthermore, DAPI was used for dead cell exclusion and data was acqui red on a FACSAria using DiVaII software. To assess the level of KTLS re constitution, BM cells were enriched for Sca1 on the AutoMACS (Miltenyi Biotec) using the Sca1-Biotin and anti-biotin magnetic beads (Miltenyi Biotec). Sca1 + cells were then stained for Lin-FITC, Sca1-biotin, c-Kit-APCCy7, Thy1-APC, CD45.1-PE and CD45.2-PerCPCy5.5. The Sca1-biotin was revealed using Str eptavidin-PeCy7. DAPI was used for dead cell exclusion. Data was acquir ed on FACS Vantage with DiVaII software. 66

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Annexin V Assay and TUNEL Assay BM cells were isolated stained with c-Kit-APCCy7, Thy1.2-APC, Lin-FITC, Sca1-Biotin and Flk2-PE on ic e, biotin was revealed by streptavidin-PeCy7. The cells were then incubated with Annexin V (BD Biosciences) according to the manufacturers protocol. Analysis was done on a FACSAria using DAPI for dead cell exclusion. TUNEL assay was performed using the In situ cell death detection Kit following manufacturers in struction (Roche Applied Science, Indianapolis, IN, USA). First, BM cells were depleted of Lin + cells using Lin-PE, anti-PE beads, and the autoMAC S (Miltenyi Biotec), Lin cells were then stained for KTLS and TUNEL. In Vivo Homing Assay Homing assays were optimized from previous protocols. 147,175 BM cells from 6-8 week old SHIP -/and WT mice were isolated from hind/fore limbs and from vertebral column. BM cells were then RBC lysed, Fc blocked for 15 minutes, then depleted of Lin + cells using Lin-PE, followed by anti-PE beads, using autoMACS (Miltenyi Biotec). Lin cells were then stained for Lin-PE, Sca1FITC and DAPI and Sca1 + Lin cells were sorted on the FACS Aria. After sorting, the cells were stained with 1 M of 9-H-(1,3-dichloro-9, 9-dimethylacridin-2-one-767

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yl) phosphate (DDAO-SE) (Molecular Pr obe/Invitrogen, Carlsbad, CA, USA) and/or 0.5 M carboxyfluoroscein succinimidyl ester (CFSE) (Molecular Probe/Invitrogen) for 15 minutes at 37 o C, washed and centrifuged at 300xg for 7 minutes at 4 o C. Cells were then resuspend ed in PBS with 1mM HEPES and an equal number of live Sca1 + Lin SHIP -/and WT cells (2x10 5 cells of each genotype) were then injected into irradiat ed recipient mice (1000 rads 3 hours prior to transplantation). For some experiments, both CFSE stained SHIP -/and WT Sca1 + Lin cells were injected in different recipients. In others, CFSE stained SHIP -/and DDAO stained WT Sca1 + Lin were injected in the same recipients. Twelve to 14 hours after injection, BM ce lls and splenocytes were isolated from recipients and directly analyzed on a FACS Ar ia in the presence of DAPI. Five million events were collected for each recipient. Measurement Cytokines and Growth Factors Levels in Mice Sera Blood was obtained by sub-mandibula r bleed, collected in a regular Eppendorf tube and left at RT for 4 hours to allow coagulation. The blood was then stored at 4 o C overnight. The following day, blood clots were removed using a wooden stick and the remaining blood was centrifuged at 4000 RPM for 10 minutes at 4 o C. The sera was then isolated by taking the supernatant and sent for analysis at a custom based service at Charles Rivers Laboratories Inc., where ELISA for multiple cytokines and growth factors was performed. 68

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Section II: Influence of SHIP on Megak aryocytes and Megakaryocyte Progenitors Introduction Megakaryocytes Platelets are responsible for repair of vascular damages, wound repair, innate immune response, and metastatic tumor cell biology. 191 In human, 1 x 10 11 platelets are produced each day in order to maintain proper balance, and during time of crisis, this number can increase up to 10 fold. 192 Mature megakaryocytes (MK) that are responsible for the production of platelets arise from the differentiation and maturation of megakaryocyte progenitors (MKP). In turn, MKP are derived from early myeloi d progenitors, which also give rise to granulocytes/monocytes and erythrocytes (Figure 4). The different stages of MKP differentiation, from M KP proliferation to platelet shedding, and the diverse cytokines influencing that process, ar e depicted in Figure 19. From early progenitor proliferation, MKP maturation to platelet shedding by MK, the megakaryocyte lineage has a unique way to control its homeostasis, mainly through chemokines, growth factors, and cytokines. 193-196 ; 197-210 As it is shown on Figure 19, some cytokines are more important for the early development or 69

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proliferation of MKP, such as IL-3 and c-KitL, while others, including IL-6 and mostly TPO impact MK maturation and plat elet release. One major component of MK maturation is the process of endomitosis, which generates MK that have an average DNA content of 32N, and ex hibit an increase in cell size, mRNA content, and protein production. 211,212 After fragmentation of polyploid MK, approximately 3000 platelets are generated, 213 all of which are small cells that lack a nucleus, but have a highly organized cytoskeleton, the major necessary components to control thrombosis; unique receptors and specialized secretory granules. 214,215 The level of platelet in an or ganism must be tightly regulated to prevent blood clot due to unwarranted production of platelet or excessive bleeding caused by a decrease in platelets levels. 70

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Figure 19. Megakaryocytopoiesis and cytokines that influence the process. The different steps of megakaryocytopoiesis are influenced by many cytokines in vitro and in vivo. 216,217 While the in vivo deletion of IL-3, IL-6, IL-11 and LIF does not affect megakaryocyte development, knockout mous e model for SF, TPO, and its receptor Mpl exhibit a megakaryocyte development defect. Interestingly, treatment of Tpo -/and Mpl -/mice with FGF-4 and SDF-1 induced platelet production to levels comparable to WT. 200 71

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The Involvement of SHIP in MK Signaling Pathway Early on many cytokines, including IL-3 IL-6, IL-11, LIF, c-KitL, Epo and G-CSF have been shown to impact megakaryocytopoiesis and thrombopoiesis. 193-197 It was shown that they can influence megakaryocyte progenitor (MKP) proliferation and differentiation, in vitro, in a synergistic manner, none of them being sufficient to promote megakaryocytopoiesis alone. However, it was always believed that megakaryocy topoiesis was controlled by a major growth factor that still had to be discovered. Even though the cytokine TPO was not physically isolated before 1994, the wo rd thrombopoietin was first used in 1958 to describe a factor that would stim ulate platelet production in mice and human. 201-203 In 1994, several groups identified and cl oned the humoral fa ctor primarily responsible for MKP and MK proliferation and maturation. 204,205,209 These groups identified TPO, as a molecule that can bind to c-mpl 204,205,209 a cellular protooncogene from a human erythr oid leukemia cell line wi th strong homology to vmpl, murine myeloproliferative leukemia virus oncogene. 218,219 Administration of TPO leads to a strong enhancement of M KP/MK proliferation, maturation and polyploidization, 205,207,210 as well as a dramatic in crease in platelet numbers. 208 TPO is constitutively produced by kidn ey and liver, and the control of its production appears not to be at the mRNA levels but to depend on platelet numbers. 220 One of the major mechanism by which TPO regulates 72

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megakaryocytopoiesis appears to be based on the level of free (unbound) TPO available in the blood, 221 such that free TPO levels in the blood are inversely proportional to the am ount of platelets. 222 If platelets levels are high, most of the TPO is bound to platelets, leading to a decrease in the megakaryocytopoiesis process. As levels of free TPO increase in the blood after injury or platelet insult, the megakaryocytopoiesis proc ess is induced in order to replace the lost platelets. TPO was initially thought to be a lineage specific cytokine that induces megakaryocytopoiesis in vivo and in vitro. However, TPO is now known to have an important role in the biology of seve ral other hematopoietic cell types such as erythrocytes, 223-225 granulocytes/macrophage, 223-225 neutrophils-colony forming cells (CFC), 225 and hematopoietic stem/progenitor cells. 184,225-227 Although TPO is the primary regulator for megakar yocytopoiesis, it has been shown that c-mpl and TPO deficient mice still have a reduced level of functional platelets, their numbers being 85% that of WT mice. 206,228 This suggests that other factors can impact megakaryocytopoiesis independently of TPO. It was demonstrated by several groups that SDF-1 can impact thrombopoiesis and in combination with other chemokines can com pensate for TPO deficiency. 198,199,205,229,230 In fact, SDF-1 and FGF-4 have recently been shown to have the capacity to palliate TPO deficiency. 200 Simultaneous administration of SDF-1 and FGF-4 to Tpo -/and cmpl -/mice, through an adenoviral system, led to a recovery of platelet numbers similar to the one observed in WT mice. 200 It appears that these chemokines are 73

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important for the interaction of MK with sinusoidal BM endothelial cells, promoting their maturati on and platelet shedding. 198,200 It was also shown that SDF-1 induces transendothelial MK mi gration and platelet production in vitro 199 and in vivo 230 Moreover, SDF-1 appears to enhance human thrombopoiesis in xenotransplanted NOD/SCID mice. 231 Interestingly, it was shown that SHIP might impact signaling pathways downstream of SDF-1. 27,152 In that study, the authors observed that CX CR4 engagement by its ligand SDF-1, led to phosphorylation and recruitment of Shp-2. In this cell context, Shp-2 was found in a protein complex including the proteins SHIP, cbl, and fyn. 27 Furthermore, SHIP-deficient myeloid progenitors exhibit enhanced chemotaxis towards SDF1. 152 When TPO binds to c-mpl it induces phosphorylati on and/or activation of an array of signaling molecules includi ng proteins in the JAK/STAT pathway, STAT3, STAT5, Shc, Lyn, SHIP and PI3K. 191,232-236 In that cell context, PI3K converts PI (,4,5) P2 to PI (3,4,5) P3, the latter can then recruit PH domain containing proteins, leading to Akt activation, increa sed cell survival and proliferation of MK. 235 Interestingly, TPO activation of PI 3K in mature platelets result in enhanced -granule secretion and thrombin induced aggregation activation. 237 SHIP has been shown to oppose PI3K signaling by removing the 5 phosphate of PI (3,4,5) P3, 2,3,5 and to be phosphorylated following c-mpl 74

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engagement by TPO in mature MK and c-mpl transfected BA/F3 cells. 235 Therefore, SHIP could block this ke y signaling pathway downstream of the c-mpl receptor 152,238 On the other hand, engagement of c-mpl by TPO leads to Shc phosphorylation and associati on with Grb2/SOS, resulting in the promotion of Ras activation of the MAPK pathways. 24,239,240 Knowing that SHIP can compete with Grb2 for binding to Shc, SHIP could negatively control the MAPK pathway activation. 1 Furthermore, other cytokines that have been shown to play a role in megakaryocytopoiesis are known to st imulate or to depend on SHIP for the dampening of their signaling pathways, including IL-3, c-KitL, and GCSF. 3,13,22,216,241,242 Interestingly, Lyn -/mice exhibit an increase in megakaryocytopoiesis. 243,244 Lyn kinase is a negative regulator of cell signaling pathways downstream of receptors for di fferent cytokines including M-CSF and TPO. 45,236 Lyn is known to phosphorylate and recruit SHIP to the membrane after cytokine stimulation of the cells. Since SHIP is important for the negative control of several signaling pathways do wnstream of cytokines implicated in megakaryocytopoiesis, we hypothesize that SHIP-deficient mice might show an increase in megakaryocytopoiesis and thrombopoiesis. 75

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Aims: 1) Assess the level of MK and MKP in SHIP -/mice hematopoietic organs by flow cytometry as compare to WT mice. 2) Attempt to identify the factors res ponsible for the perturbation of the MK compartment. Results MKP are Increased in the BM and Sp leen of SHIP-Deficient Mice We analyzed BM, spleen and PB from SHIP -/, SHIP IP/ IP and their respective WT littermate s to determine the size of the MKP compartment in vivo by flow cytometry (Figure 20A), usi ng an immunophenotype defined by Hodohara and colleagues, 245 Lin c-Kit + CD41 + which contains the majority of CFU-Mk activity. We observed an expansion of t he MKP compartment in the BM of SHIP /and SHIP IP/ IP mice as compared to their WT littermates (Figure 20BI). In fact, SHIP -/and SHIP IP/ IP BM showed a mean 18.1-fold and 50-fold increase, respectively, in the absolute number of MKP relative to WT controls (Figure 20BI). However, the numbers of MK, as defined by the immunophenotype Lin cKit CD41 + were decreased by a mean 2-fold in SHIP P -/and SHIP IP/ IP BM as compared to WT control (Figure 20CI). 76

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Figure 20. Increased number of MKP in SHIP-deficient BM. (A) Flow cytometry analysis of BM, spleen, and PB from SHIP -/(-/-) and SHIP IP/ IP ( IP/ IP) and WT littermates. Representative flow cytometry plot of c-Kit vs. CD41 after gating on live cells and Lin cells. MKP are in the upper right quadrant and MK in the lower right quadrant. Percentages for each population are indicated on the plot. (B) Bar graph showing the absolute numbers of MKP (Lin c-Kit + CD41 + ) cells in (I) BM (sum of 2 femurs + 2 tibias), (II) whole spleen and (III) per l of PB. (C) Bar graph showing the absolute numbers of MK (Lin c-Kit + ) cells in (I) BM (sum of 2 femurs + 2 tibias), (II) whole spleen and (III) per l of PB. Bar graphs showing the different SHIP-deficient models (black) and their respective WT littermates (gray). Data acquired using FACS Calibur, CellQuest software and analysis done with FlowJo. Significance was established using the unpaired student t test (Prism 4): ***p<0.0001, +++p<0.0005, ++p<0.005, and +p<0.05. (mean SEM, n 3). Collaboration with N. Parquet and LE Perez MD. 77

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The observation of splenocytes for t he presence of MKP and MK by flow cytometry (Figure 20A, Bii, Cii) revealed that SHIP -/and SHIP IP/ IP spleens exhibited higher percentages of MKP and MK as compared to WT littermates (data not shown). The absolute number of splenic MKP was increased by at least 30-fold in both, SHIP -/and SHIP IP/ IP mice, as compared to their respective WT littermates (Figure 20BII). Furthermore, the absolute number of splenic MK was increased by a mean 10.9-fold in SHIP -/spleen as compared to WT littermates (Figure 20CII). SHIP IP/ IP mice exhibited an even more dramatic increase in the absolute num ber of splenic MK as compared to WT control (Figure 20Cii). Analysis of PB for t he presence of MK and MKP by flow cytometry revealed that it contains very few MKP, with an average of 3 to 5 MKP/ l. Using this method, we observ ed that the number of MKP was not significantly increased in SHIP -/mice when compared to WT (Figure 20BIII). However, MKP numbers were slightly but significantly higher, in SHIP IP/ IP PB as compared to WT littermates (Figure 20BIII). Furthermore, in the PB of SHIP -/mice there was a mean 7.7-fold increas e in the absolute num ber of MK and the same trend was observed for SHIP IP/ IP mice (Figure 20CIII). We then combined the number of MKP and MK present in the BM and spleen in SHIP-deficient mice as compared to WT controls (Figure 21). In both, the SHIP -/and SHIP IP/ IP mice, the total MKP numbers were increased by a 78

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mean 3.5-fold as compared to their res pective WT littermates (Figure 21A). Despite an increase in total MKP numbers in SHIP-deficient mice, we observed that these mice have a comparable tota l number of MK as compared to WT littermates (Figure 21B). Thus, the decrease in MK numbers observed in SHIPdeficient BM (Figure 20Bi) is compensat ed for by the increase in splenic MK numbers (Figure 20Ci). This results in SHIP-deficient mice containing a comparable number of total MK as compared to WT littermates. Figure 21. Total MKP but not total MK numbers are increased in SHIP-deficient mice are compared to WT. The total number of MKP and MK was calculated by adding the absolute number MKP or MK in the spleen and whole BM of one mouse. To calculate the number of MKP and MK in the whole BM of a mouse, multiply the number of MKP or MK in one femur by a 16.6. 156 (A) Total absolute number of MKP in the spleen and whole BM of SHIPdeficient mice and their WT littermates. (B) Total absolute number of MK in the spleen and whole BM of SHIP-deficient mice and their WT littermates. Data acquired using FACS Calibur, CellQuest software and analysis done with FlowJo. Significance was established using the unpaired student t test (Prism 4): +++p<0.0005 and ++p<0.005. (mean SEM, n 3). 79

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MKP and MK are Increased in BM and Spleen of SHIP-Ablated Mice To observe if inhibition of SHIP dur ing adulthood could also result in an increase in MKP production, we used the MxCre model. This model is described in the previous section on SHIP and HSC. Briefly, the treatment of MxCRE+ fl/fl mice with polyIC will lead to Cre recombinase expression through Type interferon-inducible Mx1 promoter, and del etion of the gene section between two lox P sites. In this case, the promoter and the first exon of the SHIP gene will be deleted resulting in the abl ation of SHIP expression. As control, MxCre /SHIP fl/fl are treated with polyIC in t he same manner than the MxCRE+ fl/fl mice. Twentyone days after the last polyIC treatment, mice were euthanatized and the level of MKP was evaluated by flow cytometry (Fi gure 22Ai). As observed in Figure 19Bi we see an increase in the percentage of MKP in the BM and spleen of SHIPablated mice as compared to MxCre mice. Furthermore, we observe that SHIPablated BM contains approximately 4 times more MKP than their MxCre counterpart (Figure 22Bii). As for the germline SHIP -/, we also observe an increase in the percentage of MK present in the sple en (Figure 22Biii). This result suggests that mice that undergo normal development can also exhibit increased MKP numbers once SHIP is delet ed during adulthood. This has some therapeutic implication, w here SHIP inhibition could be used to increase in megakaryocytopoiesis in adult patients. 80

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Figure 22. Significant increase in the percentage of MKP cells in SHIP-ablated BM and spleen. MxCre + and MxCre mice with floxed SHIP alleles were treated with polyIC 3 times prior to being analyzed. (A) Representative FACS plots showing detection of and MK and MKP in the BM and spleen of MxCre + and MxCre mice after treatment. (B) (i) Percentage of MKP in BM (top), spleen (Spl) (bottom) and of SHIP-ablated (black) and WT (grey) mice. (ii) Absolute number of MKP cells in BM (per femur and tibia pair). (iii) Percentage of MK found in the spleen. Data acquired on a FACS Calibur with CellQuest software (BD Biosciences, San Jose, CA), analyzed with FlowJo. Significance was established using the unpaired student t test (Prism 4). +++p<0.0005, ++ p<0.005, and +p<0.05. (mean SEM, n 3). 81

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Platelet Levels are Lower in SHIP-Def icient Mice as Compared to WT Mice One striking observation is that despi te the profound expansion of the MKP in SHIP -/and SHIP IP/ IP mice, these mice do not exhibit increased platelet levels relative to WT controls, when measured by hematolyzer (Table 2). The analysis of SHIP -/and SHIP IP/ IP PB revealed that these mice have a significant reduction in their platelet as compared to WT littermates (Table 2). Furthermore, SHIP -/as well as SHIP IP/ IP mice PB have a significant ly lower percentage of hematocrit. SHIP heterozygous mice platelet and hematocrit levels were not significantly different than the WT le vels. The hematocrit percentages and platelet counts observed in PB isolated from SHIP heterozyg ous mice were not significantly different than the one observe d for the WT PB (Table 2) all p values being above 0.5. Except for SHIP +/PB hematocrit percentage (p=0.072), all heterozygous values shown in Table 2 we re significantly difference than the values observed for their res pective SHIP-deficient PB. Table 2. Platelet and Hematocrit counts in SHIP-deficient mice. Mice genotype Platelet levels (#x10 3 / l) Hematocrit (%) SHIP -/672.8 43.4 ++ 42.5 1.4 + SHIP +/848.9 35.7 45.2 0.5 SHIP +/+ (C57Bl6) 803.7 31.5 46.6 0.5 SHIP IP/ IP 455.4 81.1 ++ 43.1 1.4 ++ SHIP +/ IP 652.3 28.0 46.8 0.5 SHIP +/+ (129SvJ) 647.9 28.0 47.0 0.6 + p < 0.05 compared to their respective WT littermates ++ p < 0.05 compared to their respective WT and SHIP hete rozygous littermates 82

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SHIP-Deficient MK are Morpholog ically Different than WT MK BM histopathology rev ealed that MK in SHIP -/BM have a hypolobulated micromegakaryocytic morphology when co mpared to WT BM, which contains mature hyperlobulated MK (Figure 23A). The increase in MK numbers found by flow cytometry in the SHIP-deficient spleens was corroborated by morphology, where the spleens of SHIP-deficient mice have a qualitative increase in the number of MK (Figure 23B). These observations suggest a shift in the site of megakaryocytopoiesis from the BM to t he spleen. This is in agreement with other findings suggesting that the spleen of SHIP-deficie nt mice becomes the site of intense extramedullar hematopoiesis. 95,156,246 Figure 23. Hematoxylin-eosin staining of SHIP-deficient WT BM and spleen. (A) Hematoxylin-eosin staining of SHIP -/and WT BM section, photographed at 63x. (B) Hematoxylin-eosin staining of SHIP IP/ IP and WT spleen section, the images were photographed at 40x (spleen). Cells were visualized on a Leica DM LB microscope. Pictures were taken using a RTcolor Spot camera (Diagnostic Instrument Inc) with Spot Advance v3.0 software. Collaboration with N. Parquet and L.E. Perez MD. 83

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Comparable Ploidy Distribution in SHIP -/MK as Compared to WT The hypolobulated micromegakar yocytic morphology of SHIP -/BM MK cells observed by histology suggested that these cells did not mature properly. Therefore, we evaluated the ploidy distribution in BM and splenic SHIP -/MK, as endomitosis is an obligatory step toward s MK maturation. However, these results revealed that SHIP -/BM and splenic MK cells have a similar ploidy distribution than WT MK (Figure 24). Thus, SHIP -/Lin CD41 + cells do exhibit a similar ploidy distribution, thus have a comparable ability to undergo endoreplication as compared to WT Lin CD41 + cells (Figure 24A, B). 84

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Figure 24. SHIP -/MK undergo endocytosis with the same efficiency as WT MK. BM cells and splenocytes from SHIP P -/and WT mice were enriched for CD41+ on AutoMACS, stained with Lin-panel and CD41-biotin/SA-APC, fixed/permeated and stained with PI in the presence of RNase. (A) Representative gating strategy to obtain the final FACS plot used to calculate the DNA content of Lin CD41 + cells. (B) Representative FACS plot used to calculate the DNA content of BM and spleen Lin CD41 + cells for SHIP -/and WT. (Ci) Bar graph representing the percentage of BM Lin CD41 + cells in the different ploidy stage visible on the plot. (Cii) Bar graph with emphasis on the higher ploidy distribution for BM Lin CD41 + cells. (Di) Bar graph representing the percentage of spleen Lin CD41 + cells in the different ploidy stage visible on the plot. (Dii) Bar graph focusing on the higher ploidy distribution for spleen Lin CD41 + cells Data acquired using FACS LSR, DiVaII (BD Biosciences). Statistical analysis was done using the unpaired student t test (Prism 4). (mean SEM, n 3). 85

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TPO Levels are Increased in SHIP -/Plasma as Compared to WT It has been proposed that SHIP might c ontribute in the control of pathways downstream of c-mpl and the lack of SHIP could result in a higher response to TPO stimulation as compared to WT litterm ates. However, an increase in sera TPO levels could also result in increas ed megakaryocytopoiesis Therefore, we assessed the level of TPO in SHIP -/mice sera. Consistent with the decreased platelet levels (Table 2) and the incr eased megakaryocytopoiesis (Figure 20) observed in SHIP -/mice, we saw that SHIP -/sera contained a significantly higher concentration of TPO as compared to WT mice. ELISA performed on SHIP -/and WT sera revealed that SHIP -/sera had a concentration of 12.8 0.2 ng/ml of TPO while WT had 11.67 0.2 ng/ml (Figure 25A). Alt hough this is only a difference of 1.2 ng/ml, it is statistically significant according to an unpaired student t test (p<0.005) (Figure 25A). Furthermore, IL -6, a cytokine that has been shown to impact early megakaryocytopoiesis 216 and TPO production during inflammation, 247 was increased in SHIP -/mice sera as compared to WT (Figure 25B), this was also observed in another SHIP knock-out model at the mRNA level. However, not all factors show n to impact megakaryocytopoiesis were increased in SHIP -/mice as shown in Figure 25C, LIF levels in SHIP -/mice were not significantly different from WT mice The increase in IL-6 and TPO observed in SHIP -/sera could contribute to the increase in megakaryocytopoiesis observed in these mice. 86

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Figure 25. TPO levels are significantly increased in SHIP -/sera as compared to WT littermates. Mice were bled by sub-mandibular bleeding and the blood was collected in regular Eppendorf tube to allow coagulation. The sera was then collected and sent for analysis by ELISA (Charles River Laboratories Inc.). (A) Levels of TPO in SHIP -/(square) and WT (triangle) sera. (B) Levels of IL-6 in SHIP -/(square) and WT (triangle) sera. (C) Levels of LIF in SHIP -/(square) and WT (triangle) sera. Significance was established using the unpaired student t test (Prism 4). ++ p<0.005 (mean SEM, n 3). Collaboration with D. Woods and WG Kerr Ph.D. 87

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Discussion The ablation of SHIP results in an augmentation of MKP production. However, we observe that the total number of MK in SHIP-deficient mice is comparable to the one observed in WT. The decrease number of MK in the BM of SHIP-deficient mice appears to be comp ensated for by a dramatic increase in splenic MK. Therefore, there are some indications that in SHIP-deficient mice the mature MK leave the BM prematurel y to be found in the periphery, in the spleen and PB. The shift in MK localization from t he BM to the periphery might result from an increased responsivenes s to SDF-1, which can cause transendothelial migration of MK from the BM to the circulation. 198-200 Thus, SHIP may participate in the control of pathways downstream of CXCR4 mediating MK migration, as it does in myeloid progenitors. 152 Alternatively, the increase in peripheral MK might result from extramedullar hematopoiesis as it was shown that SHIP-deficient spleen contain elev ated levels of HSC 156,246 and hematopoietic progenit ors (Figure 21B). 95 We observe that MK cells in the BM of SHIP-deficient mice are hypolobulated and less mature than the o ne found in WT BM by looking at morphology (Figure 23). This would sugges t that SHIP-deficient BM MK cells do 88

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not mature properly. However, we observed that BM, like splenic, SHIP -/MK exhibited a comparable ploidy distribution as the one seen for WT MK cells. This result suggests that the SHIP-deficient BM MK mature as well as WT BM MK. Alternatively, SHIP -/MK might exit the BM prematur ely, before the shedding of platelets, as they might be more responsive to SDF-1 induced migration signaling. Platelet levels limit megakaryocytopoiesis by sequestering TPO. 216 These platelets have c-mpl receptors on their surface that can bind TPO molecules. 248 Therefore, it has been sugg ested that the level of fr ee-TPO in circulation is inversely proportional to platelet levels. 249 Thus, a decrease in platelet levels would cause an increase in free-TPO and stimulate megakaryocytopoiesis in order to replenish the platelet stock. In SHIP-deficient mice, we observed a significant decrease in platelet levels an d, consequently, a significant increase in TPO levels as compared to WT contro ls. The increase in free-TPO might stimulate the proliferation and/or survival of MKP in SHIP-deficient mice, leading to an expansion of the megakaryocytic compartmen t. Furthermore, SHIPdeficient MKP could be hyper-responsive to the free-TPO available since it was shown previously that SHIP is phosphorylated after c-mpl engagement with TPO in primary MK and c-mpl transfected Ba/F3 cells. 235 Administration of TPO to patients or test animals causes the increas e in platelet levels only a few days, 3 to 5, after treatment. 250 This suggests that TPO does not regulate directly release of platelet but mainly the pro liferation and maturation of MKP and MK 89

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respectively. Thus, the increase in MKP numbers but not in pl atelet levels in SHIP -/mice might further suggest that megakaryocytopoiesis and platelet shedding are regulated by different mechanisms. Furthermore, it is worth noting that SHIP has been shown to be present in human platelets and to be respons ible for the production of PI (3,4) P2 during aggregation dependent activati on of platelets. PI (3,4) P2 in human platelets was shown to mediate actin assemb ly for filopodial growth, 251 and sustained exposure of integrin GpIIB-IIa leading to irreversible aggregation. 251-253 This process is probably hindered in SHIP-defici ent platelet since it is responsible for removing the 5 phosphate of PI (3,4,5) P3, to create PI (3,4) P2. Thus, SHIP-deficient platelet might have compromised function, affect ing their activation status and causing premature elimination fr om the circulation. As mentioned earlier, we observe a significant decrease in the mean platelet numbers in SHIP -/mice as compared to WT mice. This could in part explain the increase in TPO levels observe d in these mice. However, researches have shown that inflammation can contribute to increasing TPO production. 247 Actually, cytokines produced by monocyt es, including macrophages, can induce a chain reaction, involvin g an increase in IL-6 le vels, promoting increased production of TPO by the liver. 191,247 SHIP -/mice have increased number of macrophages in their BM, spleen and periphery, 95 and we observe that these mice exhibit significantly higher IL-6 leve ls (Figure 25B). In fact, we observed 90

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that SHIP -/mice have 49 pg/ml of IL-6 in their sera as compared to 1 pg/ml for WT (Figure 25B). This increase IL-6 sera concentration could induce TPO production by the liver Furthermore, we observe that both SHIP-deficient models exhibit a decrease in hematocrits as compared to their respective WT (Table 2). Helgason et al reported that F2 SHIP -/mice (129 mice backcrossed twice to C57Bl6 mice) have no difference in hema tocrit levels compared to WT. However, a more recent publication reveals that F6-7 SHIP -/mice exhibit a significant decrease in hematocrit levels as compared to WT. 254 In this later paper, it was mentioned that the increased severity of the erythroid defect might result from backcrossing onto C57Bl6 strain. However, since the SHIP IP/ IP mice are on a mixed background, this explanation seems unlik ely. Looking back at the data published in 199 8, there was already a great difference in the percentage of hematocrits between WT and SHIP-defici ent mice with 50.7.0% for WT versus 42.4.7 for SHIP-deficient mice. However the standard error mean was large for SHIP-deficient mi ce, probably caused by a small sample population. 95 In the second study, they seem to have used a larger sample group, 254 which could have helped reduce the standard error mean, and show a significant difference between the two sample groups. 91

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Our results suggest that SHIP negativ ely controls pathways promoting early megakaryocytopoiesis. However, the fact that we observe a similar level of MK in SHIP -/and WT mice suggests that di fferent pathways control MKP proliferation, MK matura tion and platelet shedding. On the other hand, SHIPdeficient MK could leave the BM premat urely due to increased responsiveness to SDF-1, which could hamper MK matura tion and the platelet shedding process. These findings suggest t hat SHIP could be targeted in vivo to increase the pool of MKP, and subsequently enable this compartment to replenish platelets more rapidly following myeloablative chemotherapy and radi ation treatment. However, this assumes that SHIP def iciency does not adversely impact MK maturation, platelet shedding from MK or platelet function. Materials and Methods Mice Strains SHIP -/mice (F9 or F10 X C57BL6/J) produced in our laboratory have a deletion of the SHIP pr omoter and first exon. 43 A second SHIP-deficient mouse model was also analyzed, SHIP IP/ IP (129SvJ), 97 in which the inositol phosphatase domain is deleted was kindly provided by Dr. Jeffrey Ravetch, Rockefeller University, NY, USA. All studies described herein were conducted on six to eight week-old adult mice. For the inducible model, SH IP fl/fl mice were 92

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backcrossed to MxCre germline. The resulting MxCre+ fl/fl and MxCrefl/fl mice were treated 3 times with 625 g polyIC in the course of 7 days. Mice were analyzed at least 21 days after the last injection. Experiment s were performed in compliance with institutional guidelines of the University of South Florida. Cell Isolation Isolation of BM cells and splenocyt es was as described previously. 246 Following red blood cell (RBC) lysis, t he cells were resuspended in staining media.. 246 PB was obtained from the retro-orbital sinus, sub-mandibular bleed or heart puncture. For MKP analysis of PB, RBC were lysed twice in 1x RBC lysis buffer (eBioscience). Cells were then resuspended for antibody staining. Flow Cytometry Analysis and Antibodies Staining of MKP and MK wa s performed as per Hodohara et al 245 All antibodies were from BD Biosciences except when mentioned otherwise. The cells were treated with anti-CD16/CD32 (2 .4G2) to block Fc receptors and then stained with a Lin panelPE, CD41-FITC(MWReg30), and c-Kit-APC(2B8). The Linpanel was CD3 (17A2), CD4(GK1.5), CD8 (53-6.7), B220(RA3-6B2), Gr1(RB6-8C5), Mac-1 (M1/70) (Caltag, Burlingame, CA) and Ter119(TER-119). Dead cells were excluded usi ng 7-AAD (BD Biociences). Data was acquired on 93

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a FACS Calibur using the FACS Ques t software and analysis was done using FlowJo 4.5. Platelet Analysis Blood was collected by heart punctu re or sub-mandibular bleed and platelets were quantified using the CellDyn 3700 hematology analyzer (Abbott Diagnostic, Dallas, Texas USA). Histopathology Bone and spleen were fixed in 10% buffered paraformaldehyde. Bone were first decalcified using Routine Acid Decalcification (RDO; Apex engineering Products, Plainfield, IL, USA), followed by sectioning. The spleen and bone sections were then stained with hematoxylin-eosin afte r being deparaffinized with xylene. Briefly, slides we re stained using the modifi ed Meyers hematoxylin and eosin phloxine B solution (Fisher Scientific, Suwan ee, GA, USA) according to a modified Armed Forces Institute of Path ology (AFIP) protoc ol. Cells were visualized on a Leica DM LB microscope. Pictures we re taken using a RTcolor Spot camera (Diagnostic Instrument In c) with Spot Advance v3.0 software. Ploidy assay 94

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BM cells were isolated from hind/fore limb and vertebral column. Briefly, cells were incubated with CD41-biotin anti body (Serotec, Raleigh, NC, USA) on ice and then with anti-biotin be ads (Miltenyi Biotec) at 4 o C. CD41 + cells were then selected using the autoMACS (M iltenyi Biotec). Collected CD41 + cells were stained with Lin panel-FITC as menti oned above and with strept avidin-APC (BD Biosciences). The cells were fixed in 1% formaldehyde (Sigma-Aldrich) for 15 minutes on ice and then incubated overnight in a 70% ethanol solution at -20 o C. The day after, the cells were washed carefully of any residual ethanol with PBS and incubated for at least 3 hours in a prop idium iodine (PI) solution composed of 50 g/ml PI, 0.1% Triton X-100, 2.5U/ml RNa se in PBS without Ca or MG (all from Sigma-Aldrich except for the PBS which was from Gibco BRL/Invitrogen). Measurement of Cytokines and Growth Factors Levels in the Sera of Experimental Mice Blood was obtained by sub-mandibula r bleed, collected in a regular Eppendorf tube and left at RT for 4 hours to allow coagulation. The blood was then stored at 4 o C overnight. The following day, blood clots were removed using a wooden stick and the remaining blood was centrifuged at 4000 RPM for 10 minutes at 4 o C. The sera was then isolated by taking the supernatant and sent for analysis at a custom based service at Charles Rivers Laboratories Inc., where ELISA for multiple cytokines and growth factors was performed. 95

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Section III: Natural Ki ller Cells and SHIP Introduction to Natural Killer Cells NK cells were first defined as a cell fraction residing in the spleen, with the ability to spontaneously kill tumo r cells without prior immunization. 255-257 However, it is now known that NK cells are found in the BM, liver and PB. NK cells are derived from the same progeni tor cell as T-lymphocytes, either the common lymphocyte progenitor in BM or th e NK-T cell precursor in fetal liver. 258 Although common in origin key difference do exist between the two cell types. One primordial distinction is that NK ce lls do not undergo the maturation process that T-cells go through in the thymus and are unable to accomplish gene rearrangement for the production of antigen specific receptors. 259 Furthermore, NK cells are part of innate immunity, and the first line of defense of an organism against viral infection and tumorigen ic transformation of host cells. 255-257 These cells can kill target cells either by direct cell-mediated cytotoxic ity or by secretion of soluble factors. To this effect they can produce diverse cytokines, such as IL5, IL-10, IL-13 and GM-CSF. 260 However, NK cells secrete predominantly interferon(IFN, which helps control tumor cell proliferation and kill virally infected cells. 261,262 In fact, it was shown that NK cells store IFNtranscripts, 263 96

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which can be rapidly releas ed upon stimulation. They also express molecules member of the Tumor necrosis fact or (TNF) family such as, TNF Fas ligand (FasL) and TNF related apoptosis inducing ligand (TRAIL), wh ich bind to death domain-containing receptors on tar get cells causing their apoptosis. 264,265 Most importantly, NK cells contain granules stori ng perforin and granzyme, 266-268 all of which can be secreted shortly after stimulat ion. For example, perforin dependent cytotoxicity can be mediated within 20 mi nutes after activation of NK cells. 269 Thus, as soon as these cells encounter a potential target, they are ready for action. It is therefore su ited that NK cells be tightly regulated by an array of receptors that trigger activation and inhibitory response depending on the target cell and cytokines present. 270 This fine balance between inhibition and activation prevents inadvertent killing of normal healthy host cells. Activation of NK cells by engagement of activating receptors leads to the stimulation of kinases that triggers cytolysis of target cell. The whole process remains to be further elucidated, but to date we know that several molecule become phosphorylated or recr uited during NK cells activation, including Syk, 271 SLP-76, 272 ZAP-70, 273 Linker for activation of T cell (LAT), 274 Shc, 275 PI3K, 276,277 PLC 1 and 2. 278-280 Interestingly, it was observed that NK cells ablated for Syk and ZAP-70 can develop normally and lyse tumor cells in vitro and in vivo. 281 Thus, these molecules are used for activati on of NK cells but are not essential. On the other hand, simultaneous inhibition of PI3K and Src kinases prevented 97

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the cytolytic activity of these mutant NK cells and strongly reduced that of WT NK cells, suggesting that distinct and r edundant signaling pathways act in a synergistic manner to trigger cytotoxicity. 281 To this end, PI3K is known to mediate the production of PI (3,4,5) P3, which lead to calcium increase and activation of PLC molecules. PLC -2 ablated NK cells were shown to be greatly reduced in their ability to lyse target cells and secrete IFNin response to stimulation of signaling pathways mediated by DN AX adapter protein-10 (DAP10), which contains an ITAM motif. 279 Furthermore, PLC -2 ablated NK cells have a disrupted receptor repertoire, particularly for Ly49 receptors. 279 NK Cell Receptors As mentioned above NK cell cytotoxic activity is regulated by a fine balance between activating and inhibitory re ceptors. The inhibitory receptors expressed by NK cells are many and diffe r in structure and lig and specificity. These receptors are encoded in a defined region on chromosome 6 that was named the NK cell gene complex. 282 This chromosomal region encodes for series of receptor molecules that can form disulfidebound homodimers, characterized by being type II integral membrane proteins with C-type lectin domains. The NK cell gene complex encode for the activation receptor such as CD69 283 and for members of the Nkrp1 (N KR-P1A, B, C) multigene family. 282,284 Nkrp1-C, also called NK1.1, and part the of the Nkrp1 family 285 is expressed on 98

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NK cell and a subset of T cells, and even though it s ligand is unknown it is believed that NK1.1 is an activation mole cule since antibody cross-linking leads to activation of NK cells and lysis of target cells. 286 Most importantly for this work, the NK cell gene co mplex encodes for members of the Ly49 receptor family, which is composed of at leas t 23 members (Ly49A through W) (Table 3). 287 The ligands for 14 of these recept ors are known so far (Table 3). 287 Table 3. Functions and ligands of Ly49 NK cell receptors Receptor Function Cellular Viral ligand name Ligands Ly49A Inhibitory D b D d D p D k Ly49B Inhibitory ? Ly49C Inhibitory K b K d K k D d D b Ly49D Activating D d Ly49E Inhibitory ? Ly49F Inhibitory D d Ly49G Inhibitory D d L d Ly49H Activating D b MCMV-m157 Ly49I Inhibitory K d MCMV-m158 Ly49J Inhibitory K b Ly49K* Activating ? Ly49L Activating K k Ly49M Activating ? Ly49N* Activating ? Ly49O Inhibitory D b D d D k L d Ly49P Activating D d Ly49Q Inhibitory ? Ly49R Activating D d D k L d Ly49S Inhibitory ? Ly49T Inhibitory ? Ly49U Activating ? Ly49V Inhibitory D b D d K k Ly49W Activating D d K k To date no full length transcript have been found 99

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These receptors are di sulfidebound homodimers, type II trans-membrane proteins with C-type lectin-like domains. Ten of these appear to be activating receptors, of which some bind MCH cl ass I molecules while others recognize MHClike molecules produced by viruses. For example Ly49H recognizes m157 molecules produced by murine cytomegalovirus infected cells. 288,289 Ly49 activating receptors contain no ITIM motif and do not seem to become phosphorylated upon engagement with ligand. Instead they necessitate the interaction with ITAM-containi ng adapter molecule to exer t their effect, such as death adapter prot ein 12 (DAP12), 290 CD3 291 DAP10, 292,293 and Fc RI 294 the latter being responsible for binding to CD16 and stimulate antibody-dependent cellular cytotoxicity (ADCC). 295 DAP12 is a 16kDa prot ein with an aspartic acid residue in its transmembrane region, whic h can interact with the asparagine on Ly49D transmembrane r egion upon its engagement. 296 After this interaction, tyrosine in the ITAM of DAP12 becomes phosphorylated, 297 which leads to a activation of signaling event s involving the phosphorylat ion of Syk, PLCy-1 and calcium mobilization. 290,296 It was also shown that the SH2 domain of Syk can interact with DAP12 phosphorylated tyrosine. 296,297 The activation and/or phosphorylation of these effect or molecules will lead to the release of perforin, granzyme and cytokines to kill the target cell and stimulate a complete immune response. 100

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The activation of NK cells has to be counterbalanced by inhibitory signaling in order to prevent inadvertent attack of hos t cells. To this effect, NK cells rely on the presence of inhibitory receptors that recognize MHC class I molecules on hosts cells such as recept ors of the Ly49 receptors family. The inhibitory receptor Ly49A was the firs t one to be cloned from the NK cell gene complex. 298 The human counter part of the Ly49 inhibitory receptors are the killer cell inhibitory receptors (KIR s), which belong to the Ig superfamily. 299 KIRs and Ly49 receptors evolved separately into totally different molecules that both have the ability to negatively control NK cell activation through recognition of MHC Class I molecules. Furthermore, they both exhibit a cytoplasmic ITIM sequence, and both were observed to asso ciate with Shp-1 and/or Shp-2 after engagement, leading to inhibitory signaling. 300,301 Like human KIRs, murine Ly49 recept ors such as Ly49 A, C, G2 and I, have a cytoplasmic ITIM motif that becomes tyrosine-phosphorylated upon engagement by self MHC class I molecules. 302-304 The murine ITIM sequence was first identified as a 13 amino acid sequence on Fc RIIB receptor necessary for inhibitory function. 305,306 ITIMs are now know to be present in several receptors involved in immunological pr ocesses and are usually composed of 6 amino acids (I/V/L /S)-X-Y-X-X-(L/V). 307 Upon engagement of inhibitory receptors, such as Ly49A and G2, the tyrosine residue in the ITIM becomes phosphorylated and can then recruit molecu les that contain a SH2 domain such 101

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as Shp-1 308 and possibly Shp-2. 309 Once activated, these molecules can then dephosphorylate and therefore deactivate Syk, PLC -1/2, ZAP70, and Shc, which would participate in the stimul ation of cytotoxic response. 310 These inhibitory receptors are responsible for prevent ing unwanted attack against host cells, however, once an infected or cancerous cells down-regulate expression of their MHC class I molecule, Ly49 inhibitory receptors can not be engaged and NK cells are then free to kill. This process was first described by Karre and his colleagues, who observed that an H-2B deficient lymphoma was rejected by the host, while a lymphoma that expressed MHC molecules would engraft in the host. They named this concept the mi ssing-self hypothesis (Figure 26). 311 It was later confirmed by other groups who noticed that 2-microglobulin deficient cells would get rejected by irradiated MHC-matched mice while 2-microglobulin expressing cells could engraft successfully. 312,313 These studies relate that NK cells will be inhibited from killing a possible target cell when this one expresses the right MHC molecules. The MHC molecu les bind inhibitory receptors on NK cells, which then block activation pathway. 102

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Figure 26. Missing-self hypothesis. Graphic representation of the possible outcomes when a NK cell encounters a possible target cell. MHC class 1 or non-MHC class I molecules expressed by the host can be recognized by inhibitory receptors on NK cells, dampening the possible response of NK cells. The level of activation and inhibitory signal received by the NK cell, and the qualitative difference in the signal transduced will determine the NK cell response. Loosely adapted from Lanier review on NK cells published in 2005. 314 103

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SHIP and NK Cell Development The transcriptional regulation of Ly 49 receptors remains to be fully elucidated. Why is their expression variegated? Why do certain subset of NK cells in the same individual express different but overlapping Ly49 receptors on their surface? Right before the prolifer ation phase, immature NK cells, which already express NK1.1 and the CD122 (IL-2 and IL-15 receptor ) receptor, acquire CD94-NKG2 and Ly49 receptors. 315 Thus, it is possible that upon acquisition of the Ly49 rec eptors, the cell subset t hat bear Ly49 engaged by the host MHC Class I molecules would be prompt to survive or proliferate. To this end, the Ly49 receptors specific for t he host MHC molecule would guide the proliferation and survival of the NK cell subset that expresses it. Consistent with this hypothesis, the p85 subunit of PI3K can be recruited to the membrane by inhibitory Ly49 receptors to promote formation of PI (3,4,5) P3. Even though that molecule has been implicated in the simula tion of NK cells through activation of PLCy, it is possible that PI (3,4,5) P3 recruit the PH containing protein AKT, which will promote survival and proliferation of the cell. Since SHIP contains an SH2 domain, it could be recruit ed to the membrane by Ly49 receptor phosphorylated ITIM motifs, where it would negatively co ntrol the survival an d/or proliferation signal received by NK cells through dephosphorylation of PI (3,4,5) P3. This event would prevent over-represent ation of a certain subset of receptors, thus, maintaining a proper repertoire of Ly49 receptor on NK cells. 104

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Aims:: 1) Define the NK cell compartment in SHIP -/mice. 2) Identify factors contribut ing to the disruption of NK cell receptor repertoire. Results Spleen of SHIP -/Have Increased Number of NK Cells Analysis of the NK compartment in the spleen at different stages of ontogeny indicated that NK cells devel op normally in juvenile SHIP -/mice (Figure 27Ai, B). However, in SHIP -/adult mice an abnormal population of NK cells appears that exhibit a 10-fold increase in the surface expression level of the NK receptor, NK1.1 (NK1.1 hi ), as compared to WT contro ls (Figure 27Aii, Aiii). The NK1.1 hi population lacks CD3 and thus is not an NK-T cell population. The appearance of the NK1.1 hi population coupled wit h an increase in NK cells with a normal 2B4 + NK1.1 + staining profile (NK1.1 + cells) leads to a 2 to 3-fold increase in peripheral NK cells in SHIP -/adult mice ( 8 weeks), relative to wild-type littermates (Figure 27B). 105

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Figure 27. Increased NK cell numbers in SHIP -/. (A) FACS analysis of splenic NK cells in SHIP +/+ and SHIP -/littermates at different stage of ontogeny, at (i) 3, (ii) 5, and (iii) 8 weeks. Genotype and age of the mice at the time of sacrifice and analysis are indicated.. (B) Absolute splenic NK cell numbers in SHIP+/+ and SHIP-/mice at different ages. Significance was established using the unpaired student t test (Prism 4). (mean SEM, n 3). The values determined for SHIP -/mice that are significantly different from that of their age-matched SHIP +/+ counterparts are indicated by the following symbols: +p<0.05 and,* p<0.01. Collaboration with J. Howsen, T. Ghansah Ph.D., S. May and K.HT Paraiso MS. 106

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Murine NK cells detect MHC class I molecules using receptors encoded by the Ly49 or CD94/NKG2 genes. 316 Expression of these MHC receptors is distributed among different NK subsets during the transition from neonate to adult. Because the number of peripheral NK cells increases in SHIP -/mice during this period, we asked whet her the relative representation of NK subsets expressing certain Ly49 and CD94 receptors might account for this increase. Indeed, the relative representation of several Ly49 receptors and CD94 was significantly altered in the SHIP -/NK compartment of older mice when compared to SHIP +/+ littermates (Figure 28). However, SHIP -/weanlings, at 3 weeks of age, showed no skewing of their NK repertoire relative to wild-type littermates (Figure 28). The NK cell receptor repertoire di stortion was most pronounced in mice 8 weeks of age and was found in both the NK1.1 + and the NK1.1 hi populations. We found that Ly49A + and C/I + NK cells were overrepresented in adult SHIP -/mice, while Ly49D + G2 + and CD94 + were underrepresented (Figure 28). Because the overwhelming majority of the NK1.1 + and NK1.1 hi cell populations lacked Ly49I in adult SHIP -/mice, the majority of the Ly49C/I + NK cells express only Ly49C. Thus, the repertoire distortion in adult SHIP -/mice leads to an NK compartment dominated by a subset of cells expre ssing Ly49A and/or Ly49C, where Ly49D, G2, I can CD94 are underrepresented (Figure 28). In vitro and in vivo studies indicated that Ly49A and Ly49C can bind ligands in the H2b haplotype of SHIP -/107

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mice; however, these two receptors also bind and transmit inhibitory signals from ligands in most or all H2 haplotypes. 317-320 Therefore, SHIP deficiency leads to an NK inhibitory repertoire that is both, self-specific and promiscuous for other ligands. A potential explanation for the r epertoire disruption seen in SHIP -/NK cells is that SHIP is recruited to certain inhibitory receptors expressed by NK cells to oppose intracellular signals that mediate survival of specific NK subsets expressing these receptors. Indeed, SHIP binds the phosphorylated ITIM motif of Ly49A in vitro. 42 These findings prompted us to examine whether SHIP associates in vivo with Ly49 receptors expressed by NK cells. 108

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Figure 28. MHC class I receptors on peripheral NK cells in SHIP -/mice. The mean percentage of peripheral NK cells expressing the indicated Ly49 or CD94 molecule after gating on 2B4 + NK1.1 + cells. The age and genotype of the mice are indicated. Significance was established using the unpaired student t test (Prism 4). (mean SEM, n 3). Values determined for SHIP -/mice that are significantly different from their agematched SHIP +/+ littermates are indicated: +p<0.05; p<0.01. Collaboration with J. Howsen, T. Ghansah Ph.D., S. May and K.HT Paraiso MS. 109

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SHIP is found associated with Ly49 receptor s where it may control the level of PI (3,4,5) P3 generated by PI3K and negativ ely regulate Akt phosphorylation Analysis of potential SHIP interact ion with different Ly49 receptors, revealed that SHIP is associated with Ly49A and Ly49C under physiological conditions (Figure 29A), but not with Ly49G2, Ly49F, or Ly49I (Figure 29B). As further confirmation that the protein co-precipita ting with Ly49A and Ly49C is SHIP, we analyzed NK lysates from SHIP +/+ and SHIP -/mice (Figure 29C). This analysis detected co-precipitation of SHIP with Ly49A and Ly49C only in the SHIP +/+ NK lysates, confirming the spec ificity of the Western Blot. Because SHIP limits the in vivo survival of myeloid cells by opposing the PI3K/Akt pathway, 2,3 we examined whether Akt is activated in SHIP -/NK cells in vivo based on its phosphor ylation at Thr308. 321,322 We found that both Akt phosphorylation and total Akt protein levels were sign ificantly increased in SHIP -/NK cells relative to those in SHIP +/+ NK cells (Figure 29D). The increase in total Akt levels is surprising; however primary B cell activation leads to increased Btk levels in a PI 3K-dependent manner. 323 This additional level of regulation may further amplify signals from PH domain-containing ki nases, such as Akt or Btk, which are recruited to PI (3,4,5) P 3 Taken together, these findings suggest the interplay of SHIP and PI3K may influence the relative survival of NK subsets expressing Ly49 receptors capable of recruiting these enzymes. Interestingly, 110

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PI3K is recruited to human KIR and can activate Akt in human NK cells. 324 Thus, despite their evolutionary divergence in how they bind MHC class I molecules, murine Ly49 receptors and human KIR may recruit SHIP to limit the in vivo survival of NK subsets, just as both receptors recruit Shp-1 to limit NK effector functions. 310,325 Figure 29. SHIP is recruited to NK inhibitory receptors in vivo to oppose activation of Akt. (A) Western blot detection of SHIP in Ly49A and Ly49C immunoprecipitates. Immunoprecipitation with a murine IgG2a antibody (IgG2a) was analyzed as a negative control and SHIP was immunoprecipitated as a positive control (SHIP). (B) Western blotting for SHIP in other Ly49 immunoprec ipitates (Ly49G2, Ly49F, and Ly49I). (C) Western blot analysis of SHIP in Ly49A and Ly49C immunoprecipitates prepared from lysates of SHIP ( ) and SHIP ( ) NK cells. (D) Western blot analysis of Akt phosphorylation at Thr 308 and total Akt protein in SHIP and SHIP NK cell lysates. To control for equal loading the blot was re-probed with antibodies specific for -actin, GAPDH, and -tubulin. Collaboration with T. Ghansah Ph.D., and K.H.T Paraiso MS. 111

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DAP12 is Expressed by SHIP -/BM Cells and NK Cells According to our data, the disruption in NK cell receptor repertoire in SHIP -/mice appears to result from a bias towards selection for Ly49A and C receptors, due to their increased survival Consequently, the level of Ly49D + NK cells in SHIP -/mice is decreased was compared to WT mice. However, we wanted to determine if the decrease in Ly49D + NK cells representation did not arise from a lack of DAP12 expression. It has been s hown that Ly49D and other activation receptors require the presenc e of DAP12 to migrate to the cell surface. 326 Thus, we considered the possibility that Ly49D was not found on the surface of SHIP -/NK cells due to the absence of its adaptor molecule DAP12. However, using reverse-transcription P CR (RT-PCR), we observed that DAP12 is expressed in SHIP -/BM (Figure 30A) and SHIP -/NK cells as well as WT (Figure 30B). Another molecule known to be involved in transduction of extracellular signaling by NK ce ll receptors is DAP10. Like for DAP12, we also observe that SHIP -/and WT BM cells express t he DAP10 adaptor molecule (Figure 31). Since we can detect DAP1 2 by RT-PCR it suggests that Ly49D receptors should reach the SHIP -/NK cell if they were to be expressed. 112

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Figure 30. DAP12 is expressed in SHIP -/and WT BM cells and NK cells. RNA from (A) BM and (B) NK cells was isolated and RT-PCR was performed to assess the presence of DAP12. Figure 31. DAP10 is expressed in SHIP -/and WT BM cells. RNA from BM cells was isolated and RT-PCR was performed to assess the presence of DAP10. 113

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SHIP but not Shp-1 is Found Associ ated with Ly49A under Physiological Conditions Signal transduction studies on NK cells have relied for a long time on the study of lymphokine-activated killer (LAK) cells. These cells are splenocytes that are grown for approximately 7 days in a media containing a combination of cytokines that promote NK cells proliferation. After th is 7-day culture, the cells are collected and activation experiments ar e performed. Several group observed that when LAK cells were treated with pervanadate, Shp-1 was recruited to the tyrosine-phosphorylated ITIM sequence of Ly49A molecule. 308 Once to the membrane, Shp-1 can dephosphorylat e molecules such as Syk, PLC -1/2, ZAP70, and Shc, 42,310 leading to their deactivation, preventing NK ce ll from killing target cells. Instead of culturing the cells for 7 days, we studied the biology of NK cells right after isolation, which would be clos er to physiological condition. In this experiment we observe that SHIP does co-immunoprecipitate with Ly49A, while Shp-1 does not (Figure 32A, B). This c onfirms that the roles of SHIP and Shp-1 in NK cell biology are very different (Figure 33). While Shp-1 appears to be important for the control of activati on signaling after Ly49 engagement, SHIP seems to play a role in NK cell homeo stasis and maintenance of receptor repertoire. 114

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Figure 32. SHIP but not Shp-1 is recruited to NK inhibitory receptor Ly49A in vivo. (A) Western blot detection of SHIP in Ly49A IP, Ly49G, a murine IgG2a antibody (IgG2a) IP used as a negative control. WCL from 70Z/3 and NK cells (same lysate than the one used for IP) were used as positive control. (B) Western blot detection of Shp-1 in Ly49A IP, Ly49G, a murine IgG2a antibody (IgG2a) IP used as a negative control. WCL from A431 and NK cells (same lysate than the one used for IP) were used as positive control. 115

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Figure 33. Signaling pathways downstream of Ly49A and C that can be influenced by SHIP in NK cells. As shown on this schematic representation of NK cells signaling pathways. SHIP, by being recruited to Ly49A and C can negatively control the survival and proliferation of NK cells in response to encounter with self H2b encounter. Once NK cell activation receptors are also engaged, Shp-1 will be recruited to the membrane to Ly49 receptors, where it will dephosphorylate kinases that, when activated, trigger an attack on target cells. 116

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Discussion Our study shows that F4 SHIP -/mice have an increased number of peripheral NK cells, and that their receptor repertoire is disrupted. This suggests that NK cells might undergo some sort of selection process once they acquire their Ly49 receptors during developmen t. The interaction of these NK cell inhibitory receptors with self MHC ligands may elicit si gnals that promote the survival or proliferation of these cells in vivo. 327 Thus, NK cells could be selected for their ability to be engaged by host MHC molecule (Table 3), which would ensure that the NK cells in an organism express the proper inhibitory receptors to prevent unwanted attack on self. SHIP, once recruited to the membrane by some of these Ly49 rec eptors could negatively regulate their survival and proliferation by cont rolling signaling pathways downstream of PI3K. Alternatively, SHIP could be recruit ed to the membrane by Ly49 receptors to participate in the control of NK cell activation, as it has been shown to negatively control calcium influx in mast cells. 99,241 In addition, PI3K 276,277 and PLCy molecules 278-280 have been shown to play a role in NK cell activation. Since SHIP has the potential to dephosphorylate PI (3,4,5) P3, it could downregulate these signaling pathways. 10,56-59,62,63 However, this scenario seems highly unlikely since SHIP -/NK cells are unable to kill allogeneic transplant 43 and are not as efficient as WT NK ce lls in lysing target cell in an in vitro assay. 43 117

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On the other hand, SHIP is know n to control signaling pathways downstream of cytokine receptors that can lead to activation of transcription factors, which could potent ially impact Ly49 expression. 328,329 Up to this date the transcription regulation of NK cells recept ors, including Ly49 receptors, appears to be highly stochastic with each recept or gene containing up to 3 separate promoters. 330 Thus, more studies are nece ssary to understand the mechanism for Ly49 variegated gene expression. Interestingly, NF B has been implicated in the regulation of the probabilistic switch regulating Ly 49 receptors expression. 331 Knowing that SHIP can impact NF B activation, 332 SHIP-deficiency could perturb Ly49 transcription regulation. Interestingly, PLC -/mice also have a disrupted Ly49 receptor repertoire. 279 This suggests that pat hways downstream of PI3K are important for the establishment of a proper Ly49 receptor repertoire. Materials and Methods FACS Analysis of the NK Cell Compartment and their Receptors To analyze the peripheral NK cell co mpartment spleens were collected from mice at various ages. A single cell suspension was prepared by crushing of the spleen with a syringe plunger, and NH 4 Cl lysis (Red blood cell lysis buffer, eBioscience) of erythrocytes. The cells were then stained with an antibody against the Fc receptor to prevent unspecif ic binding. All anti bodies were from 118

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BD Biosciences, except where mentio ned. For the NK cell compartment assessment, splenocytes were stained wit h fluorescently c onjugated antibody against the NK-associated markers 2B4PE, NK1.1-FITC and CD3-APC. The cells were then incubated into a solution of 7AAD (5 l per 1 million cells) for dead cell exclusion. Data acquired on FACS Calibur with CellQuest software, and analyzed with FlowJo. For the recept or repertoire analysis, isolated and Fc blocked splenocytes were stained with 2B4, NK1.1 and anti-Ly 49A (A1), -Ly49C/I (5E6), -Ly49D (4E5), -Ly49G2 (4D11) or -CD94 (eBioscience). To distinguish Ly49C staining from Ly49I, cells were stained with NK1.1, Ly49C/I and Ly49I (YLI90). All biotin conjugates were revealed by Streptavidin-APC (eBioscience). Cells were then exposed to 7AAD for dead cell exclusion and data was acquired on FACS Calibur with CellQuest so ftware, and analyzed with FlowJo. Protein Lysis Buffers Radioimmunoprecipitat ion (RIPA) buffer. The modified RIPA buffer protocol was obtained from the Upstate catalog. To prepare 100 ml of RIPA buffer, add 790 mg of Tris base (Fisher) to 75 ml of distilled water, add 900 mg of NaCl (Fisher), adjust the pH to 7.4 wit h HCl, then add 10 ml of 10% NP-40, 2.5 ml of 10% Na-Deoxycholat e and 1 ml of 100 mM EDTA, adjust the final volume to 100 ml with distilled water and store at 4 o C until use. Right before using the buffer add protease and phosphates inhibitors to it. A cocktail of protease 119

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inhibitors from Sigma-Aldrich was us ed at a concentration of 0.1%, or 10 l of protease inhibitor cocktail in 1 ml of RIPA. For phosphates inhibitor, we use pervanadate (NaVO 3 ) to a final concentration of 1mM, thus add 10 l of 100mM NaVO 3 to 1 ml of RIPA buffer. Digitonin cell lysis buffer. First, weigh 0.5 g di gitonin (CalBiochem, EMD Biosciences, Inc., San Diego, CA, U SA) into a 50 ml tube add 25 ml ddH 2 O and boil for about 2 h. Allow to cool on the bench and if a precipitate forms, boil again for longer period of time, until no more precipitates are found after cooling. Then, add the following reagent s: 0.6 ml of 10 % Triton X-100, 1.5 ml of 5 M sodium chloride (NaCl), 10 ml of 100 mM Triethanolamine, pH 7.8, 125 l of 1 M calcium chloride (CaCl 2 ), 50 l of 1 M magnesium sulfate (MgSO 4 ), 25 l of 20 % Sodium Azide an d top with ddH 2 O to 50 ml. As for the RIPA buffer, the protease and phosphatases inhibitors were added right before us e. Immunoprecipitation was carried as mentioned in the next sect ion. After immunopr ecipitation, the precipitated beads were washed 4 times with a solution that prevents (or diminishes) the detection of unspecific interaction. This washing buffer surnamed FROPS buffer contains 30 ml of 5 M NaCl, 5 ml of 1M CHAPS, 50 ml of 1 M Tris pH8 brought up to 1 li ter using deionized distilled water. 120

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Biochemical Analysis of SHIP and Akt NK-enriched C57BL6/J splenocytes we re prepared by depletion of B cells and macrophages by adherence to nylon wool followed by T cell depletion using anti-CD3 plus complement. NK cells were then lysed in modified RIPA buffer or digitonin buffer. Prior to immunoprecip itation the NK cell lysates were precleared twice by incubation with 0.25 g of an murine or rat IgG2a antibody (BD Biosciences) and 50 l of Protein A-Agarose or Protein G-Sepharose beads (Upstate, Charlottesville, VA, USA). The beads were pelleted at 15,000 RPM for 15 minutes at 4C. The pre-cleared super natants were immunoprecipitated with 2 g of anti-Ly49A (A1), -Ly49C/I (5E6), -Ly49F (HBF-719), -Ly49G2 (4D11), Ly49I (YLI-90) or IgG2a (BD Biosciences ). To pre-clear the lysate and as a negative control for immunoprecipitation, mouse IgG2a was used for Ly49A and Ly49C/I immune precipitates, rat Ig G2a was used for Ly49G2 immune precipitates, and mouse IgG1 was us ed to for Ly49F and Ly49I immune precipitates. Immune complexes were precipitated by addition of 50 l of Protein A-Agarose (Ly49A, Ly49C/I) or Protei n G-Sepharose (Ly49F, Ly49G2, Ly49I) beads. The immunoprecipitates were resolved on a 4-12% Tris-Bis polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Pharmacia, Piscataway, NJ). The filters were then probed wit h a 1:1000 dilution of anti-SHIP (P2C6, a kind gift of Larry Rohrschneider) and a horseradish peroxidase (HRP)-conjugated anti-mous e IgG secondary antibody (Amersham 121

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Pharmacia) at a 1:80,000 dilution. The presence of SHIP was revealed using the SuperSignal West Femto reagent (Pierce Bi otechnology Inc., Rockford, IL, USA). The results of the Ly49 immunoprecip itations in Figure 29 and 32 are representative of three in dependent analyses of NKenriched splenocytes. For analysis of Akt activation lysates of purif ied NK cells from the spleens of SHIP -/and SHIP +/+ were prepared as above. Equal quantities of protein from cell lysates prepared from SHIP +/+ and SHIP -/NK cells were resolved on a 4-12% Tris-Bis polyacrylamide gel (Novex/Invi trogen, Carlsbad, CA USA), transferred to a nitrocellulose membrane (Amersham Pharmacia) and the filters probed with an anti-Phospho-Akt(Thr308) antibody (Cell Signaling Technolog y, Danvers, MA, USA) at a 1:1000 dilution. The pres ence of Akt was detected by a HRPconjugated donkey anti-rabbit IgG secondary antibody (Amersham Pharmacia) at a 1:2000 dilution and revealed using ECL substrate (Amersham Pharmacia). The blot was then stripped and reprobed in a similar manner using anti-actin (Cell Signaling Technology), anti-GAPDH (Research & Diagnostics Antibodies, Benicia, CA) and anti-tubulin (Oncogene Research, Cambridge, MA) as internal controls for protein loading. The detection of increased Akt levels and its activation is representative of three s eparate analyses of NK cell lysates from SHIP -/and SHIP +/+ mice. 122

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RT-PCR for DAP10 and DAP12 Cells were isolated as mentioned above. For the NK cells RNA extraction, splenocytes were first treated with Fc block (CD16/CD32) for 15 minutes to prevent unspecific binding. The cells were then expo sed to NK1.1-FITC (NK1.1) and CD3 -PE (145-2C11). The FACS Vant age with the DiVa software was then used to sort CD3 NK1.1 + or CD3 NK1.1 ++ cells in the presence of 7AAD to exclude to dead cells. All antibodies were from BD Biosciences. RNA was isolated following t he protocol for the RNAqueous-4PC R kit (Ambion, Austin, TX, USA). RT-PCR was performed using MultiS cribe reverse transcriptase (Applied Biosystems, Foster City, CA, USA) protocol. Brie fly, 5xRT-PCR buffer (4 l), 25 mM MgCl 2 (2 l) 10 mM dNTP (2 l), RNase inhibitor (0.5 l), 100 mM DTT (2 l) and random hexamers (0.5 l), were mixed with RNase free water (up to 20 l) and RNA isolated from tested samples (equ ivalent of 10 000 cells). The enzyme, 0.3 l of MultiScribe RTase was then added to the reaction right before starting the reverse transcription on a Px2 therma l cycler PCR machine (Thermo Electron Corporation, Waltham, MA) using t he following program; 10 minutes at 25 o C and 12 minutes at 42 o C. The RT-PCR products were then placed at 4 o C for 1-2 hours. 10 l of the RT-PCR reaction was then used for a PCR reaction using the primers for specific genes. Briefly, cDNA (10 l), RNase free water (25 l), 5x AmpliTaq buffer, (8 l) 25 mM MgCl 2 (2.5 l), 10nM dNTP (3 l), 20 M DAP12 forward and reverse primers (.5 l each) and 0.5 l of AmpliTaq (Applied 123

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Biosystems) were mixed then PCR reaction was performed. PCR reaction was done on Px2 thermal cycler PCR machi ne using the following program; 10 minute polymerase activation at 95 o C, 43 cycles of 25 second at 94 o C and 1 minute at 60 o C, and then a final incubation at 72 o C for 7 minutes. The expected PCR product for DAP12 was 120 nucleot ides and the band on th e 1.2% agarose gel appears to be 120. When the P CR was performed for DAP10, DAP 10 forward and reverse primers were used for the PCR reaction, otherwise every thing else was as mentioned earlier, a 126 base pairs sequence was expected, as seen on the 1.2% agarose gel. Primers used for D AP10: Forward: 5-caa gtc aga cat cgg cag gtt c-3 and Reverse; 5 -gca tac ata caa aca cca ccc cta-3. Primer used for DAP12 Forward: 5cct t cc tgt cct cct gac tg t g and Reverse 5tca ccc aga aca atc cca gc-3. Western Blot for Shp-1 Lysis and immunoprecipitation was ca rried as mentioned above. The same immunoprecipitate wa s then loaded on two differe nt gels, one for Western blot with SHIP and the other one with Sh p-1. The Western Blot and probing for SHIP was performed as mentioned above. The membrane to be probed for Shp1 was then blocked in 5% non fat m ilk (NFM) in TBS (TBS-5%NFM) for 20 minutes at RT with constant agitation. The membrane was then incubated overnight at 4 o C in TBS-5%NFM containing 1 g/ml of anti Shp-1 antibody 124

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(Upstate). The morning after the membr ane was washed twice in distilled water and incubated for 1.5 hour at RT in TBS-5%NFM containing HRP conjugated anti-rabbit antibody (Cell Signaling Techno logy) at a concentration of 1:80,000. The membrane was then washed twice in distilled water, and once in TBS-0.05% Tween 20 for 5 minutes. The membrane was washed another 5 x in distilled water and revealed using the SuperSignal West Femto for 5 minutes (Pierce Biotechnology Inc.). 125

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Section IV: Murine ES Cells and s-SHIP Introduction to ES Cells Murine ES (mES) cells are pl uripotent cell lines derived from the culture of preimplantation embryos or epiblasts. 333-335 These cells have the potential to differentiate into any cells of an organism ES cells, contrarily to cells from 2cells-stage embryo, can not form t he trophoblast, and ther efore, should be referred to as pluripotent and not totipotent. 336 ES cells can be derived and grown for several passages, without any in tervention or immortalization agent. Throughout this process ES cells do not undergo senescence and retain their diploid karyotype. They can multiply in the absence of serum, if in the presence of feeder layer, and are not subject to contact inhibition or anchorage dependence. 337 Once ES cells are injected into a developing blastocyst, they have the potential to int egrate that blastocyst and change parts of its genetic background. Successful int egration of ES cells into blastocyst results in the colonization of germ cells. 337 These mice are then considered chimeras and can produce functional gametes containing t he genetic background of the injected ES cells. Thus, ES cells have a tremendous po tential to be used as a vehicle for introducing genetic modification in mi ce and many other organisms. It is 126

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important to note that inje ction of ES cells in adult mice has been associated with the formation of teratocarcinoma. 334 The maintenance of ES cells in vitro is paramount for the developm ent of genetic manipulati on strategies, therefore understanding the process of self-renewal vers us differentiation is very important. The first mES cell lines were derived by growing these cells on fibroblast feeder layers. However, the presence of these feeder cells made the study of mES cell signaling very difficult. Then, Smith and Hoopers f ound that mES cells could be grown, undifferentiated, in the presence of FBS and LIF. 338-340 Furthermore, it was established recently that signaling by bone morphogenetic proteins (BMP) works toget her with LIF to maintain the pluripotency of mES cells. 341,342 ES Cells Signaling Pathways LIF and ES cells. In mES cells, LIF, coming from the feeder layer or given in the culture media as a recombinant protein, appears to be essential for the maintenance of ES cell pluripotency. 338-340 LIF is a factor that belongs to the IL-6 cytokine family, which also includes IL11, ciliary neutropic factor, oncostatin M and cadiotrophin-1. 343 LIF engages the heterodimeri zation of receptor complex composed of LIFr and gp130, gp130 being a common recept or subunit of the IL6 cytokine family receptor. 344,345 The heterodimerization of this complex leads to 127

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activation of janus associated kinases (JAKs) that phosphorylate the receptor chain (Figure 34). Figure 34. LIFR/gp130 receptor complex signal transduction pathways and how s-SHIP may impact them in pluripotent stem cells. Based on research from several groups, it was found that the LIFR/gp130 receptor complex can impact ES cell self-renewal and differentiation. 337,346-348 Although s-SHIP does not become phosphorylated after LIF stimulation, s-SHIP is constitutively present at the membrane and thus can impact the signaling pathways downstream of ES cell receptors. As illustrated above, LIFR/gp130 has the potential to trigger either differentiation or self-renewal depending on which downstream signaling components are activated. 128

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The phosphorylated tyrosine on LIFr /gp130 complex can then serve as anchor site for SH2 containing domains. Some of the molecules recruited to the activated LIF/gp130 receptor complex cons ist of signal transducer and activator of transcription 3 (STAT3), 349 PI3K, 350 Erk, 337,351 and Shp-2. 352,353 These proteins can then be activated by surrounding kinases 354-356 and exert their effect. Interestingly, the activation and formati on of protein comple xes between these different proteins create a very fragile balance between self-renewal vs. differentiation during pr oliferation signaling. 337 STAT3 stimulates ES cell self-renewal. In mES cells, LIF predominantly activates STAT3, 349 and it appears to be essentia l for self-renewal (Figure 34). 357 Consequently, down regulat ion of STAT3 through ex pression of a dominant negative protein causes ES cell to differ entiate. Studies using a chimeric STAT3 molecule directly activated by estradiol showed that STAT3 is not only necessary but might even be sufficient to block differentiation of mES cells. 358 ERKs antagonize ES cell self-renewal. While recruitment of STAT3 to the activated LIFr/gp130 leads to self-renewal of ES cell, recruitment of Shp-2 and Grb2 will lead to stimulation of the Erk1/2 (p42/44) and differentiation of ES cells (Figure 34). 346 Attenuation of ERK and Shp-2 signaling (by pharmacological inhibition of MAPK or by overexpression of Erk phosphatase) result in an 129

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increase in self-renewal by reduction of differentiation. 347 However, inhibition of ERK does not replace the St at3 requirement but enhances its action (direct or indirectly) in the promoti on of ES cells self-renewal. 347 The balance between these two major signaling pathways is impo rtant for ES cells maintenance. Once phosphorylated, LIFr/gp130 rec eptor can recruit SH2 domain containing protein such as Shp-2. 352,353,356 Grb2 will then bind Shp-2, which will result in the recruitment of SOS to the membrane. On ce SOS is at the membrane, it can then activate RAS/RAF/MAPK/Erk and downstr eam transcriptional factors such as, Myc, Ets, Elk, and SRF. 359 Furthermore, Shp-2 also associates with Gab1, which can recruit PI3K. 360 The resulting phospholipids, PI (3,4,5) P3, can then provide a bi nding site for the PH domain of Gab1, stabilizing the association of Shp-2/Gab1/Grb2/Sos complex at the membrane and facilitating coupling to RAS signaling pathway (Figure 34). 361 However, the production of PI (3,4,5) P3 can also promote the recruitment of molecules that can increase su rvival or proliferation of the cells, such as Akt, which has been shown to stim ulate ES cell self-renewal. 362 Since SHIP, has been implicated in the control of several of these signaling pathways, s-SHIP mi ght also impact some of these pathways. Even if s-SHIP lacks the SH2 domain and does not appear to bind Shc, it does bind Grb2 and might be involved in the control of protein complex formation of Grb2/Shp-2/Gab1/Sos, which leads to differentiation stimulation. 49 Furthermore, 130

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s-SHIP has been shown to be constitutively located at the membrane 49 where it could hydrolyze the 5 phosphate of PI (3,4,5) P3 363 and dampen signaling downstream of PI3K, such as Akt or Gab1 activation. PI3K signaling in ES cells. Inhibition of PI3K using drug inhibitor or dominant negative del ta85, led to a decrease in Akt, GSK3alpha/beta, and S6 proteins activity and an increase in Erk phosphorylation, result ing in a reduction of self-renewal. 350 One of the lipid phosphatases responsible for degradation of PI (3,4) P2 and PI (3,4,5) P3 levels, is PTEN. In fact, PTEN -/ES cells have enhanced viability and rate of cell proliferation, correlated with increased level of PI (3,4,5) P3 and phosphorylation of Akt, and inactivati on of Bad (a pro-apoptotic protein). 364 The absence of PTEN results in an increa sed cell division rate, which is much more important in ES cells than in fibr oblasts, suggesting that PI3K signaling is more important in ES cells than fibroblast. 337 Even though PTEN appears to be primarily responsible for PI (3,4,5) P3 degradation and down modulation of PI3K effector signaling pathways in ES cells, sSHIP could also have a role in this process. 131

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s-SHIP s-SHIP is a predicted 104/97k Da protein that is expressed by ES cells and HSC but not normal lineage differentiated cells. 43 s-SHIP can be amplified by RT-PCR using primers that span the car boxyl terminal end of SHIP but not by primers that bind the amino terminal region. 49 This is explained by the fact that sSHIP sequence lacks the SH2 doma in found in full-length SHIP. 49 Studies revealed that s-SHIP is t he murine homologue of human SIP-110. Similar to s-SHIP, SIP-110 is a 109kDa SHIP isoform protein that lacks the SH2 domain. 4 We observed that the first ex on of SIP-110 and s-SHIP share 82% homology. Furthermore, s-SHIP has a 44 nucleotides sequence in its amino terminus that is not present in SHIP cDNA, this region was named stem-SHIP region (SSR). 49 In SIP-110, a similar 42 nucleotides SSR-like sequence is observed. 49 In our paper, it wa s proposed that s-SHIP, like SIP-110, resulted from the utilization of a di fferent transcriptional start site than full-length SHIP, this start site would be em bedded within the intron 5/6 of SHIP 49 It was recently shown that the intron 5/6 contains a promoter region that can drive the expression of a green fluorescent prot ein (GFP) construct in knock-in mice stem/progenitor cells, including regions where breast mammary stem cells reside. 55 Interestingly, others observed that SHIP-deficient ES cells express sSHIP at higher levels and for a longer perio d of time when primed to differentiate 132

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than WT ES cells. 365 Since s-SHIP appears to be selectively expressed in ES cells and HSC, but not in lineage differentiated cells, 49 we were interested in studying the signaling pathways in whic h this protein might be involved. s-SHIP and ES Cell ES cells maintain pluripotency thr ough stimulation of several signaling pathways. 366 In vitro culture of murine ES (mES) ce lls requires the presence of fibroblast feeder layer cells and/or LIF when grown on gelatin-coated paltes. 339,340,367 LIF engages the heterodimeric receptor complex composed of gp130 and LIF receptor (LIF R) and supports the cultur e of mES cells in the absence of a feeder layer. 368 Several signal transduction molecules have been shown to play a role in the survival, self -renewal, and/or prolif eration of mES cells via the LIFR/gp130 axis, such as Stat3, 349 PI3K, 350 Erk, 337 and Shp-2. 347 Interestingly, the activation and forma tion of protein complexes among these different proteins establishes the delicate balance between self-renewal and differentiation pathways in pluripotent stem cells. 337 In different cell models, severa l signaling pathways important for proliferation and survival do wnstream of PI3K can be dow nregulated by inositol phosphatases such as PTEN, 364,369 SHIP1, 3 and SHIP2. 77,370,371 In particular, SHIP1 can be recruited to the membra ne, where it can hydrolyze the 5phosphate of PI (3,4,5) P3 or I (1,3,4,5) P4. 2 In this manner, these inositol 133

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phosphatases oppose the activity of PI3K by preventing the recruitment of pleckstrin homology (PH) containing kinase s such as Akt and Btk. Previously, we found that ES cells and, to a lesser extent, HSC, express an isoform of SHIP1, s-SHIP. This isoform is not ex pressed in normal mature hematopoietic cells, making this molecule an excellent candidate as a key factor in the regulation of stem cell si gnal transduction pathways. In previous studies, we showed that s-SHIP interacts with Grb2 and does not with Shc. 49 In this present study, we show that s-SHIP is expressed by mES cells, but not by the mouse embryonic fibroblasts (MEF) that support their undifferentiated growth. MEF cells, howe ver, do express t he full-length SHIP isoform. In addition, we demonstrate t hat murine BM cells la cking the promoter and first exon of the full-length SHIP ge ne still express s-SHIP. This result confirms that expression of s-SHIP is not dependent on the upstream promoter active in differentiated cells. Knowing t hat the major growth factor for ES cell self-renewal and prolifer ation is LIF (Figure 34), we used this growth factor to stimulate ES cells to determine if it c an stimulate s-SHIP phosphorylation. We observed that s-SHIP does not bec ome tyrosine phosphorylated following stimulation of ES cells with LIF. Finally we show that s-SHIP associates with gp130 in vivo in ES cells. These findings implic ate s-SHIP in signaling pathways that control the self-renewal and differentiation of plurip otent stem cells. 134

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Aims: 1) Define if s-SHIP is expressed in MEF and SHIP -/BM cells. 2) Study what pathway s-SHIP might impact Results MEF Cells Express Full-Length SHIP, but Only ES Cells Express the s-SHIP Isoforms In our previous study, we analyzed SHIP and s-SHIP protein expression in ES cells after transfer fr om MEF feeder layer to gelatin-coated dishes. 49 Occasionally, we would find by Western blot analysis that some ES cell cultures grown on gelatin would express some le vels of the full length SHIP isoform. 49 We attributed this to residual contaminat ion of the ES cell cult ure with MEF. To resolve this issue, we established a cult ure of MEF cells alone, ES cells on MEF cells, and ES cells on gelatin. Our suspic ion that MEF cells express SHIP was confirmed as we found that MEF express the 145/135kDa full-length SHIP doublet, but not the s-SHIP 104/97kDa double t (Figure 35). In a culture of ES cells growing on MEF cells, SHIP and s-SHIP are present (Figure 35). The protein lysate used in t he third lane was produced us ing ES cells propagated on gelatin-coated dishes in the presence of LIF for several passages. This was done to reduce as much as possible the contribution of MEF cells to the analysis. The protein lysate of these ES cells, grown on gelatin, revealed that these cells express only the s-SH IP doublet (Figure 35). 135

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Figure 35. MEF cells express full-length SHIP, while ES cells express only s-SHIP. Protein lysates from MEF cells grown alone, ES cells grown on MEF cells, and ES cells grown on gelatin were analyzed for the expression of the different SHIP isoforms. Western blot was performed using the Novex/Invitrogen system. SHIP and s-SHIP were revealed using P2C6 anti-SHIP antibody and anti-mouse-HRP. Chemoluminescence was detected using SuperSignal Femto (Pierce Biotechnology Inc.). 136

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SHIP -/Murine BM Cells Express s-SHIP In our laboratory, we established a SHIP -/mouse model by deletion of the promoter and ex on 1 of the SHIP gene using the Crelox P technology. 43 This deletion led to the ablati on of SHIP production. 43 As previously published, bioinformatic analysis predict ed that the s-SHIP and SI P-110 promoter is in the intron 5/6 of the SHIP gene. 49 Therefore, we theorized that SHIP -/HSC might retain s-SHIP expression, since the internal promoter in intron 5/6 is intact in this SHIP -/model. As we antic ipated, s-SHIP mRNA is detected in SHIP -/BM cells by nested RT-PCR (Figure 36). This re sult is consistent with a study by Rohrschneider et al (2005), in which the s-SHIP promoter region in SHIP intron 5/6 promotes transcription of a reporter gene in stem and or early progenitor cells of a transgenic mouse model. 55 Figure 36. Nested RT-PCR detection of s-SHIP expression in SHIP -/BM. Nested RTPCR was performed, using sets of primers that span the SSR (1-44) region, unique to sSHIP. Initial round of amplification (43 cycles) with primers F1-25 and R459-478, secondary round of amplification (43 cycles) with primers F21-41 and R439-463. Amplified DNA was then resolved on a 2% agarose gel. Legend: 1) Hela cells, 2) WT WBM C57BL6, 3) SHIP -/WBM C57BL6, 4)WT WBM BalbC, 5) no RNA, 6) No RTase with RNA from WT C57BL6 BM cells, 7) No polymerase with RNA from WT C57BL6 BM cells). 137

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s-SHIP Does Not Become Phosp horylated Following LIF Stimulation The SHIP isoform expressed in mature hematopoietic cells is tyrosine phosphorylated upon stimulation by growth factor, 1,21,372 immune complexes, 5,34,372,373 or BCR engagement. 33,372,374 Phosphorylated SHIP is found associated with t he adapter protein Shc, which facilitates its recruitment to the plasma membrane 33 where it can then act on phosphat idylinositol substrates. However, we found that s-SHIP is not tyrosine phosphorylated in either ES cells grown in LIF at steady state or ES cells deprived of LIF for 5 hours and subsequently stimulated with 2 LIF (Figur e 37). It was also observed that sSHIP does not associate with Shc either at steady-state or after LIF stimulation. 49 Instead, s-SHIP co-immunoprecipitates with Grb2. 49 Furthermore, it has been observed that SHIP or SIP110 do not need to be phosphorylated to exert their catalytic effect. 54 Thus, it is highly probabl e that s-SHIP, can control PI (3,4,5) P3 accumulation in ES cells, despi te not being phosphorylated. 138

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Figure 37. ES cells express the s-SHIP protein isoform that does not become phosphorylated following growth factor stimulation. Immunoprecipitation and immunoblot detection of s-SHIP in ES cell lysates. Lysate from ES-TL1 cells cultured in LIF was immunoprecipitated with the P2C6 anti-SHIP monoclonal antibody, separated on gels, transferred to membranes, and probed with P2C6, revealing 104-kd and 97-kd proteins (lane 5). No tyrosine phosphorylation of these proteins was detected when they were probed with the 4G10 anti-phosphotyrosine antibody. For comparison, lysates from 293T cells transfected with SHIP cDNA (lane 1) and s-SHIP cDNA (lane 2) were included in the blots. To further assess the tyrosine phosphorylation status of s-SHIP, timed LIF stimulation studies were performed. ES-TL1 cells incubated for 5 hours without LIF were stimulated with 2000 U/ml LIF for 0, 2, 5, or 10 minutes and were rapidly lysed. Equal amounts of total protein were immunoprecipitated with the P2C6 antibody and probed separately with the P2C6 and 4G10 antibodies (lanes 6-9). No tyrosine phosphorylation of s-SHIP was detected at any time point with the 4G10 antibody. For comparison, A20 B-lymphoid cells were stimulated with anti-IgG antibody for 0 or 5 minutes, lysed, immunoprecipitated with P2C6, and probed with P2C6 and 4G10 (lanes 3,4), showing prominent tyrosine phosphorylation of SHIP at 5 minutes. Molecular mass standards are indicated on the right. Collaboration with J. Ninos MD. 139

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s-SHIP Associates with gp130 In Vivo Grb2 has been shown to be present in protein complexes formed after gp130 engagement. Since Grb2 can associate with s-SHIP, we assessed the potential of s-SHIP to associate with gp130 in ES cells. As demonstrated in Figure 38A and B, s-SHIP is present in gp130 immunopr ecipitates from protein lysates of ES cells at steady state. This result strongly suggests that s-SHIP is present in protein complexes th at interact with gp130 in ES cells. Inhibition of the s-SHIP/gp130 association by preincubat ion of gp130 antibody with specific blocking peptides establishes the specificity of the interaction (Figure 38B). This result provides a direct link betw een s-SHIP and an important signaling component of the pathway infl uencing ES cell self-renewal. 140

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Figure 38. s-SHIP is associated with gp130 in ES cells. A) Lysates of ES cells grown on gelatin were immunoprecipitated with gp130 antibody, s-SHIP antibody (positive control) and rabbit antibody (negative control). (B) Lysates of ES cells grown on gelatin. Whole cell lysate (positive control), immunoprecipitate with gp130 antibody, immunoprecipitation with gp130 antibody previously blocked with specific peptides, immunoprecipitate with Grb2 antibody, immunoprecipitation with Grb2 antibody previously blocked with specific peptides, and immunoprecipitate with rabbit antibody (negative control). 141

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Discussion In this study, we show that s-SHIP co-immunoprecipitates with gp130. This further suggests that s-SHIP can be found intimately associated with protein complexes that impact self-renewal and proliferation pat hways of mES cells. sSHIP may interact with gp130 directly, or indirectly, through an intermediate such as Grb2. The exact role that s-SHIP plays in ES cell biology remains to be defined. As shown previously, s-SHIP is found constitutively at the plasma membrane in ES cells. 49 Furthermore, it has been shown previously that SIP110 phosphorylation is not required for enzymatic activity. Thus, s-SHIP could downregulate basal activation of Akt in these cells th rough degradation of PI (3,4,5) P3. 363 In this context, the role of sSHIP would be to limit cell survival (Figure 34). Interestingly, PI3K inhibition leads to a decrease in PI (3,4,5) P3, resulting in Erk activation and a reduction in self-renewal. Therefore, s-SHIP may negatively control ES cell self-r enewal. Since PTEN appears to be necessary for the control of pathway downstream of PI3K in ES cells, 364 s-SHIP and PTEN might have a redundant role in controlling this pathway. Alternatively, s-SHIP w ould associate with Grb2 to prevent the formation of the protein complex com posed of Shp-2, Grb2, and SOS, and thereby limit the activation of MAPK and the downstream diff erentiation of ES cells (Figure 34). In previous studies, it was shown that s-SHIP expression declines as the stem cell differentiates into specific lineages. 49,365 Furthermore, SHIP -/ES cells express 142

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higher levels of s-SHIP and for a longer period of than WT ES cells. 365 Thus, ES cells might express the s-SHIP isoform to inhibit differentiation stimuli, since it binds Grb2 constitutively, potentially preventing the association of Shp2/Grb2/SOS protein complexes (Figure 34). Interestingly, the SHIP IP/ IP mice, produced by targeting the inositol phosphatase domain, are viable, 97 despite the likelihood t hat they do not express an enzymatically active form of s-SHIP. However, analysis of s-SHIP expression in primitive cell types or BM cells has not been demonstrated for this SHIP KO model. To date, there does not exist an s-SHIP -/model, which maintains WT levels of full length SHIP, t herefore it will be difficult to truly assess the role of sSHIP in stem cell biology until such a mouse model is dev eloped. In the meantime, studies in ES ce lls using RNA interference techniques to target sSHIP expression will be useful in understanding the contribution of this protein in the control of self-renewal and differentia tion in pluripotent stem cells. Most importantly, more studies are needed to completely define the relationship between s-SHIP, Grb2, and gp130 and their combined role in ES cell function. 143

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Materials and methods Propagation of mES Cells For our studies, we used TL1 strain mES cells, which were a kind gift from Patricia A. Labosky, University of P ennsylvania, Philadelphia, Pennsylvania, USA. mES cells were first thawed on MEF cells and then transferred and passaged on gelatin-coated dishes. Petri dishes (100mm plates, Nunc, Fisher Scientific Inc. Suwanee, GA) were cove red with 0.1% gelatin in ultrapure water (Chemicon International/Specialty Media, Phillipsburg, NJ, USA) for at least 30 minutes. The extra gelatin was then remov ed. Cells were cultured in KO DMEM media (Gibco BRL/Invitrogen), 15% ES cell qualified FBS (Invitrogen/Gibco BRL), 2 mM L-Alanyl-L-Glutamine (ATC C, Manassas, VA, USA), 0.1 mM Nonessential amino acids (Gib co BRL/Invitrogen), 0.1 mM -mercaptoethanol ( -ME) (Sigma-Aldrich) with 1000 U/ml LIF (Chemicon International). ES cells were split every other day using a 0.05% trypsi n-EDTA treatment for 3-5 minutes. Preparation of mES Cell Lysates for Biochemical Analysis ES cells were grown in the presence of 1000 U/ml LIF. In preparation for cell lysis, ES cells cell cult ure dish was placed on ice, the culture media was then removed and the cells were washed twice with PBS containing 1 mM sodium pervanadate (NaVO 3 ). ES cells were then lyse d with modified RIPA buffer (Upstate) containing 1 mM NaVO 3 and protease inhibitor (S igma-Aldrich). Lysis 144

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was performed at 4 o C for 45 to 60 minutes. Protein concentration was established using the BCA protein a ssay kit (Pierce Biotechnology Inc.). Nested Reverse-Transcription Polymerase Chain Reaction Assay for Detection of s-SHIP Expression Murine BM cells were isolated as mentioned previously. RNA was isolated from 1x10 6 WT and SHIP -/BM cells following the protocol for the RNAqueous-4PCR kit (Ambion). RT-PCR was performed using MultiScribe reverse transcriptase (Applied Biosystems) protocol. Briefly, 5xRT-PCR buffer (4 l), 25 mM MgCl 2 (2 l), 10 mM dNTP (2 l), RNase inhibitor (0.5 l), 100 mM DTT (2 l) and random hexamers (0.5 l) were mixed with RNase free water (up to 20 l) and RNA isolated from tested samples (equivalent of 1x10 4 cells). MultiScribe RTase enzyme, 0.3 l, was then added to the reaction, for a total volume of 20 l. The RT reaction was performed in a Px2 thermal cycler PCR machine (Thermo Electron Corporati on, Waltham, MA) using the following program: 10 minutes at 25 o C and then 12 minutes at 42 o C. The RT products were placed at 4 o C for 1-2 hours. Ten l of the RT reaction product was then used for PCR amplification us ing a forward primer spec ific for the s-SHIP SSR region and a reverse primer specific for a region approximately 460 bp downstream of the s-SHIP SSR region. 49 Briefly, cDNA (10 l), RNase free water (25 l), 5x Amplitaq buffer (8 l), 25 mM MgCl 2 (2.5 l), 10nM dNTP (3 l), 145

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20 M s-ship forward (1-25) and reverse (459-478) primers (0.5 l each) and 0.5 l of AmpliTaq (Applied Biosystems) were mixed prior to PCR amplification. Amplification was performed in a Px2 thermal cycler PCR machine using the following program: 10 minut es denaturation at 95 o C followed by 43 cycles of 25 seconds at 94 o C and 1 minute at 55 o C, with a final extension phase at 72 o C for 7 minutes. Twenty l of the PCR reaction was then placed in a second PCR using the same conditions, except that the pr imers were internal to the first round amplification primers and targeted the s-SHIP nucl eotides 21-41 (forward primer), within the SSR regi on, and 463-478 (reverse prim er). All primers were obtained from Integrated DNA Technologies Inc. (Coralville, IA, USA). The expected PCR product for the s-ship nested PCR product is 442 base pairs, which coincides with the product seen on a 1.2% agarose gel. Primers used for s-SHIP nested PCR: Initia l amplification: forward(125), 5-gtt ccc act agt tgt tga act tta c-3 and reverse(478-459), 5-tct cct tct ccg tct cca cc-3; secondary amplification: forward(21-41), 5-ttt acc ttg aac ctc tgc tcc-3 and reverse(463439) 5-cca cca aaa tca cca act tgt tta g-3. Western blot antibodies and techniques The Western blots apparatus consist ed of the Novex Xcell II system with Bis-Tris gel system (Novex/Invitrogen). Typically 4 to 12% gels were used (Novex/Invitrogen). Gels were run at RT fo r 2 hours at 150 Volts. Multi-Mark or 146

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BenchMark (Novex/Invitrogen) protein ladders were used to correlate molecular weight of target proteins. Proteins were electrophoretically transferred to nitrocellulose membranes (Millipore, Billeri ca, MA, USA) for i mmunoblotting. The blots were initially washed twice for 2 minutes in a solution of PBS 0.05% Tween (PBS-T), then blocked for 1 hour at RT in 5% NFM in PBS with 0.05% Tween (5% NFM/PBS-T). The blots were wash ed 3 x 5 minutes in PBS-T and then incubated overnight at 4 o C in anti-SHIP monoclonal antibody solution of P2C6 antibody (a kind gift from Dr. Larry Rohrschneider) at a concentration of 1:1000, or the P1C1 monoclonal ant ibody (Santa Cruz Biotec hnology Inc., Santa Cruz, CA) at a concentration of 1 g/ml in 5%NFM/PBS-T. The following day, blots were washed at least three times for 10 minutes and incubated with anti-mouseHRP conjugated antibody (Pie rce Biotechnology Inc.) at concentration 1:80,000 for 2 hours at RT. The blot was then wa shed four times 5 minutes in PBS-T and exposed with SuperSignal West Femto for 5 minutes (Pierce Biotechnology Inc.). Cell stimulation ES-TL1 cells were split into 4 di fferent 100-mm TC plates and grown overnight at 37 o C in ES media suppl emented with 1000 U/ml LIF to approximate 80% confluence. LIF-supplemented media was removed, cells were washed twice with PBS, medi a without LIF was added, and cells were incubated for 5 hours. Cells were washed once with PBS and twice with Hanks balanced salt solution (HBSS, Mediatech). Then they were pre-incubated in HBSS at 37 o C for 147

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10 minutes. Cells were stimulated by the addition of HBSS containing 2000 U/ml LIF and by incubation at 37 o C for either 2, 5, or 10 minutes. A control ES plate (0 minute) containing HBSS without LIF was incubated at 37 o C in parallel for 10 minutes. Stimulations were stopped by the removal of buffer and the addition of 10 ml ice-cold PBS/1 mM Na 3 VO 4 PBS/Na 3 VO 4 was removed, and 1mL ice-cold modified RIPA buffer was added i mmediately. Cells were scraped, and cell lysates were processed as described above. A20 cells were grown in RPMI and were washed once with PBS and twice with HBSS. Then 2 7 cells were placed in separate 15-mL conicals and were pre-incubated in HBSS at 37 o C for 10 minutes. Cells were spun down, and HBSS was removed. After that, cells were stimulated with 1 ml HBSS containing 20 g/ml goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) and were incubated at 37 o C for 5 minutes. Control A20 cells (0 minute) containing HBSS without anti-mouse IgG were incubated in parallel. Stim ulations were stopped by placing the conicals on ice and adding 2 ml ice-cold PBS/Na 3 VO 4 Cells were pelleted, supernatant was removed, and 1 ml ice-cold modified RIPA buffer was added. One mg in 1 ml of buffer was used for subsequent immunoprecipitation experiments. Western blot analysis of gp130 immunoprecipitates Equivalent amounts of prot ein lysate were first pr e-cleared twice with 0.25 g of appropriate nonspecific IgG isotype control followed by 50 l of appropriate 148

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50% agarose bead slurry (Protein A or G) (Upstate). The beads had previously been washed three times in co ld PBS. They were then resuspended in PBS containing 1mM NaVO 3 to obtain a 50% slurry. The pre-cleared lysates were then exposed to specific polyclonal ant ibodies against gp130 (M-20) (Santa Cruz Biotechnology) and Grb2 (Santa Cruz Biotechnology) for 3 hours at 4 o C on a rotator. When peptide bl ocked antibodies were used for immunoprecipitation, gp130 and Grb2 antibodies were incubated with their respective blocking peptides (Santa Cruz Biotechnology) for at least 1 hour on a rotator at 4C prior to being used for immunoprecipitation. Once incubation with antibody was completed, 50 l of Protein A or G agarose beads 50% slurry was then added to the immunoprecipitates and put on a rotator overnight at 4 o C. The following day, the supernatant was removed and beads we re washed four times with FROP buffer (150 mM NaCl, 5 mM CHAPS, 50 mM Tr is pH 8). LDS sample buffer with reducer (Novex/Invitrogen) was t hen added to the beads, and the bead/buffer mixture was heated at 100 o C for 5 minutes. Western blot analysis was then performed as described above. 149

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FINAL DISCUSSION One might be surprised to notice that th rough out this dissertation, SHIP is given multiple roles. For example, in HSC we attributed to SHIP the ability to impact proliferation, survival, homing and retention to the BM. In addition, a similar scenario was observed when we studied the role of SHIP in MKP. However, when we truly look at each of the signaling pathw ays that can be influenced by SHIP, each of them can be lin ked to a function attributed to SHIP. This protein can control proliferation through the r egulation of the MAPK by sequestration of Shc. 1 Alternatively, it can contribu te in the negative regulation of the Akt signaling pathway, which promotes cell survival and proliferation. 61 Furthermore, SHIP was shown to negativ ely control myeloid progenitor cell response to SDF-1, SHIP -/myeloid progenitors being more responsive to SDF-1 stimulation than WT. 152 Additionally, SHIP by being a major regulator of the PI3K downstream effector pathway can impact hematopoietic cell response to diverse cytokines. Interestingly, PI3K is also known to bind to focal adhesion kinase (FAK), 375 and in this pathway, PI3K activa tion leads to the production of PI (3,4,5) P3 which contributes to the control of cell motility by FAK. Thus, SHIP could contribute to the negative r egulation of this pathway. Moreover, we observed that SHIP appears to contribute to the shapi ng of the NK cell receptor repertoire. 150

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Our results suggest that SHIP regulates the NK cell receptor repertoire diversity through promotion of survival of certain NK cell subset. However, we can not exclude that SHIP could also participat e in NK cell receptors transcription regulation. SHIP has been shown to influence IB activity, which regulates NF B function. Most interestingly, NFB was shown to contribute to the regulation of Ly49 receptor expression. 331 More studies are needed to truly define the role of SHIP in all these di fferent pathways. Furthermore, it is necessary to establish which isoform or isoforms are involved in each of these pathways in every cell type, since each is oform contains different combination of functional domains. After studying SHIP for 5 years, it is cl ear to me that it plays different roles depending on the hematopoietic cells type, the cell context, and the cell environment. Another testament to its vari egated role in the control of cellular function is the fact that SHIP has seve ral isoforms and that each of them appear to be expressed differently in diverse cell types. 4,49,53 In theory, every SHIP isoform still contains a func tional 5 inositol phosph atase enzymatic domain. Phosphatidylinositols are used to transmit signal from different external stimuli to activate cellular response that can l ead to calcium mobilization, protein phosphorylation, cell pr oliferation, activation and surv ival. Therefore, SHIP has the potential to influence each of these pat hways. However, th e fact that these SHIP isoforms lack some of the protein interaction domain, will modify the 151

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cellular context in which these isoforms will be recruited to the membrane or site of action. For example sSHIP, which has no SH2 domain, will be restricted in its ability to interact with phosphorylated tyrosine. Another isoform, SHIP 183, lacks a region between the two NPXY leading to a di sruption of a consensus sequence necessary for the binding of PI 3K p85 subunit. Interestingly, the different isoform appear to be differentially expressed by diverse cell type. This can provide the cell with an isoform t hat will correspond to the need of that particular cell. For example, ES cells express only s-SHIP and not full length SHIP, while HSC express both isoforms. 49 It was also shown that untreated human BM cells express a 110kDa isofor m, but when the same cells were treated in vitro with IL-3 and M-CSF to promot e differentiation towards the myeloid lineage, full length SHIP was observed. 53 Noteworthy are the observations lin king SHIP-deficiency to premature aging. For example SHIP-def icient mice suffer from osteoporosis due to hyperresponsive osteoclasts. 101 Furthermore, SHIP -/BM have an in creased number of HSC, which are not as efficient as WT at reconstituting an irradiated host. 156,246 Moreover, the SHIP -/hematopoietic system is ske wed towards myeloid cell differentiation. 95 All of these are characteristi cs that have been associated with aging. 376-383 It would be interesting to addr ess the hypothesis that SHIP deficiency causes accelerated aging in mice. 152

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Although SHIP is known to influence many signaling pathways in different cell types, its deficiency is not associat ed with many diseases. SHIP appears to be absent in some forms of acute leukemias. 384 It was also associated with myeloproliferative disorder 95 and with osteoporosis. 101 Nevertheless, the study of the germline SHIP -/mouse model did not allow co nfirming that SHIP mutation might be associated with any other disease, yet. It will be important to study the conditional knock-out system to establish more precisely if SHIP deletion or mutation during adulthood might participate in the development of cancers, allergic, auto-immune or inflammatory condition. 153

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End Page ABOUT THE AUTHOR Caroline Desponts received a Bachelor of Science in Agriculture with a Major in Microbiology from McGill University. There, she developed her interest for research under the guidance of Drs. Brian T Driscoll and David Zadworny. She then joined an internship program at Merck Frosst Center for Therapeutic Research. There, she worked under the guidance of Dr. Axel Ducret at the development of 2-D gel electrophoresis met hod for the study of the effect of drug treatment on protein phosphoryl ation. She then worked with Dr. Ernest AsanteAppiah at the discovery of dr ug binding site to ensure spec ific inhibition of target proteins. Her contribution to different studies at this center led to four publications. Caroline then joined the Inte rdisciplinary PhD program in Cellular and Molecular Biology at the University of South Florida. T here, she developed an interest for stem cell research and decided to pursue her graduate studies under the guidance of William G. Kerr Ph.D. While working at the H. Lee Moffitt Cancer Center and Research Institute, Caroline contri buted to several studies, which led to the publication of five papers.