The Role of Cd36 in Thrombospondin-1 Mediated

THE ROLE OF CD36 IN THROMBOSPONDIN-1 MEDIATED

ANTIANGIOGENESIS: A STUDY OF REGULATION OF CD36 ECTO-

PHOSPHORYLATION AND MECHANISMS OF VEGF INHIBITION

LING-YUN CHU

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Thesis Advisor: Dr. Roy L. Silverstein

Program of Cell Biology

CASE WESTERN RESERVE UNIVERSITY

May, 2012

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Ling-yun Chu

candidate for the Doctor of Philosophy degree *.

Gary Landreth

(Chair of the committee)

Roy L.Silverstein

Donald Jacobsen

Stanley Hazen

February 22, 2012

* We also certify that written approval has been obtained for any

proprietary material contained therein.

TABLE OF CONTENTS

List of Figures……………………………..…………………………………………….vi

Acknowledgements……………………………………………………………………..x

Abstract………………………………………...…………………………….…………xii

Introduction………………………………………………………………………………1

1. Angiogenesis and tumor growth……………………………………………1

2. Thrombospondin-1 and thrombospondin-1 type 1 repeats (TSR)………6

3. The role of endothelial CD36 in antiangiogenesis……………………....10

4. Ecto-domain phosphorylation of CD36…………………………………15

5. VEGF signaling and VEGF receptors…………………………………….18

Chapter 1*

CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding:

Structure - Function Relationships and Regulation by Protein Kinase C

Abstract…………………………………………………………………………………22

Materials and Methods……………………………………………………………..…24

iii

Results………………………………………………………………………………….30

CD36 CLESH domain phosphorylation blocks binding to recombinant TSR

domains…………………………………………………………………………30

Basal levels of cellular CD36 phosphorylation in vivo are low…………....37

Cellular CD36 phosphorylation can be increased by PMA………………..39

New protein synthesis and trafficking through Golgi are required for PMA-

induced CD36 phosphorylation………………………………………………47

PMA-induced CD36 phosphorylation blunts TSR-mediated recruitment of

Src family proteins to CD36…………………………………………………..51

Discussion of Chapter 1………………………………………………………………55

Chapter 2

Thrombospondin Signaling via CD36 Regulates SHP-1 Localization and

Phosphatase Activity: A Mechanism of VEGF Inhibition

Abstract…………………………………………………………………………………61

Materials and Methods………………………………………………………………..63

Results………………………………………………………………………………….68

Thrombospondin-1 and TSR inhibit VEGF-induced VEGFR2

phosphorylation in MVECs…………………….…………………….………..68

TSR induces SHP-1 association with CD36 and VEGFR2 in MVECs…..72

CD36 is required for TSR-induced SHP-1 association with VEGFR2 and

VEGFR2 dephosphorylation in MVECs……………………………………..77

TSR inhibition of MVECs tube formation is mediated by CD36 and SHP-

1………………………………………………………………………………....82

Cd36 null mice exhibit increased VEGFR2 phosphorylation and decreased

SHP-1 association with VEGFR2 after VEGF infusion…………………....86

Discussion of Chapter 2………………………………………………………………90

Discussion………………………………………………………………………………95

Bibliography…………………………………………………………………………..104

(Contents marked with * are e-published in Arteriosclerosis, Thrombosis, and Vascular Biology on January 12, 2012)

LIST OF FIGURES

Introduction

Figure 1. Mechanisms of angiogenesis...………………………………………..3

Figure 2. Angiogenesis is required for tumor growth and metastasis...………5

Figure 3. Structures of thrombospondin family……………………………….....9

Figure 4. Structure of CD36……………………………………………………...12

Figure 5. The model of CD36-mediated thrombospondin-1 antiangiogenic

responses…...... 14

Chapter 1

Figure 1. Extended CD36 CLESH domain can be phosphorylated on Thr92

by PKCα in vitro……………………………………………………...... 31

Figure 2. The maxima level of extended CLESH domain phosphorylation is

~25% by in vitro phosphorylation assay………………………...... 32

Figure 3. Extended CD36 CLESH domain binds thrombospondin-1………..34

Figure 4. Phosphorylation of CD36 extended CLESH domain inhibits binding

to thrombospondin type 1 repeats (TSR)……………………………35

Figure 5. Binding of TSR2/3 to CD36 extended CLESH domain inversely

correlates with level of phosphorylation…...... 36

Figure 6. Cellular CD36 phosphorylation levels are low……………………...38

Figure 7. Cellular CD36 phosphorylation is increased by treatment with

PMA……………………………………………………………………..41

Figure 8. CD36 phosphorylation in CD36 transfected Bowes cells is

increased by treatment with PMA…………………………………....42

Figure 9. CD36 phosphorylation in human platelets is not increased by

treatment with PMA and PKC expression is suppressed by

PMA……………………………………………………………………..43

Figure 10. CD36 phosphorylation in MVECs is not increased by treatment with

PMA and PKC expression is suppressed by PMA….…...... 44

Figure 11. Phosphorylation of endogenous CD36 in THP-1 cells is increased

by treatment with PMA………………………………………………..45

Figure 12. CD36 phosphorylation in CD36 transfected Bowes cells is

increased by treatment with PDK-1 activator……………………….46

Figure 13. No detectable ecto-kinase activity induced by PMA in CD36

transfected Bowes melanoma cells……………………………….…49

Figure 14. Protein synthesis and ER to Golgi trafficking are required for PMA-

induced CD36 phosphorylation………………………………………50

vii

Figure 15. PMA-induced CD36 phosphorylation inhibits TSR-mediated Src

recruitment to CD36…………………………………………………...53

Figure 16. TSR-mediated Src recruitment to CD36 can be recovered by

inhibitors of protein synthesis and ER-Golgi trafficking……………54

Figure 17. The summary of regulation of CD36 phosphorylation by PKC……59

Chapter 2

Figure 1. CD36 associates with VEGFR2 in endothelial cells……………….70

Figure 2. Thrombospondin-1 and TSR inhibit VEGF-induced VEGFR2

phosphorylation in MVECs……………………………………………71

Figure 3. TSR induces SHP-1 association with VEGFR2 in MVECs………..74

Figure 4. TSR induces SHP-1 association with CD36 in MVECs…...... 75

Figure 5. Phosphatase activity within the VEGFR2 complex is induced by

TSR and VEGF...... 76

Figure 6. CD36 is silenced by siRNA in MVECs after 48 hours……………..78

Figure 7. CD36 is required for TSR inhibition of VEGF-induced VEGFR2

phosphorylation in MVECs……………………………………………79

viii

Figure 8. Thrombospondin-1 and TSR does not inhibit VEGF-induced

VEGFR2 phosphorylation in human umbilical vein endothelial

cells…………………………………………...... 80

Figure 9. CD36 is required for TSR-induced SHP-1 association with VEGFR2

in MVECs……………………………………………………………….81

Figure 10. TSR inhibits formation of MVECs tube-like structures…………….83

Figure 11. TSR inhibits the number of branches and average branch length of

MVECs tube-like structures via CD36……………………………….84

Figure 12. TSR inhibits the number of branches and average branch length of

MVECs tube-like structures via SHP-1……………………………...85

Figure 13. CD36 associates with VEGFR2 in mice lung tissue……………….88

Figure 14. Genetic deletion of cd36 increases VEGFR2 phosphorylation and

decreases SHP-1 association with VEGFR2 after in vivo infusion of

VEGF……………………………………………………………………89

Figure 15. The summary of thrombospondin-1 inhibition of VEGF-signaling

mediated by CD36……………………………………………………..94

Discussion

Figure 1. The potential strategies to improve binding of TSR to CD36 and

inhibit angiogenesis…………………………………………………103

Acknowledgements

It was quite a long time in my life. When I left my home country for the Ph. D study, I was a 24 years old young boy. Now I am over 30 and get married. The life in a foreign country is not easy at first. The culture difference and language bothered me. I acknowledge my wife, my parents and my friends help me during this time. Chin-I Wu, my dear girl-friend and wife, is always a good listener to share my life in United States, even we were apart from the Pacific Ocean. It’s difficult to maintain a distant relationship and she never think of leaving me. My parents always care about me and give me confidence. My friends in Taiwan and US make my life interesting.

I graduated from the Department of Zoology of National Taiwan University in

Taipei, Taiwan. The education and training are solid and useful in my study. I appreciate Dr. Nin-nin Chung’s training in my graduate study. Dr. Hsinyu Lee’s teaching makes me interested in angiogenesis and vascular biology.

My advisor, Dr. Roy L. Silverstein, is always nice to me and helps me a lot in both research and paper publication. He is also helpful in my life allowing me to visit my country once a year to meet my family and girlfriend. I appreciate to have him to be my advisor.

I appreciate Dr. Maria Febbraio in Cleveland Clinic. She is a kind mentor and helps me to solve problems in my research. I also appreciate all the

x members of my thesis committee, Dr. Gary Landreth, Dr. Donald Jacobsen and

Dr. Stanley Hazen. They always give me good advices in my research.

I acknowledge Dr. Alan Tartakoff and Cell Biology Program of Case Western

Reserve University to give me the opportunity to start my research in angiogenesis and vascular biology and support my study with full scholarship.

At last, I appreciate all the lab members in Dr. Silverstein’s lab in Cleveland

Clinic. They helped me a lot. I would like to thank Dr. Phil Klenotic, Jennifer

Major, Dr. Wei Li, Dr. David Kennedy, Dr. Young-Mi Park, Dr. Wen-Xin Huang,

Dr. Wei-Fei Zhu, Dr. Bin Ren, Dr. Ohid Rahaman, Dr. Mette Johansen, Ken Chen,

James Hale, Sowmya Srikanthan, Prasadh Ramakrishnan, and Kristen Nowak.

The Role of CD36 in Thrombospondin-1 Mediated Antiangiogenesis: A Study of

Regulation of CD36 Ecto-phosphorylation and Mechanisms of VEGF Inhibition

Abstract

LING-YUN CHU

CD36 is a multiligand scavenger receptor expressed in many cell types, including microvascular endothelial cells (MVEC). Binding of thrombospondin-1 to CD36 inhibits angiogenesis by inducing apoptosis of growth factor stimulated MVEC and also by inhibiting growth factor signaling at the receptor level. CD36 phosphorylation on its extracellular domain inhibits binding of thrombospondin-

1. The mechanisms of cellular CD36 ectodomain phosphorylation and whether it can be regulated in cells are not known. Here we determined structure-function relationships of CD36 phosphorylation related to thrombospondin-1 peptide binding in vitro and explored mechanisms regulating phosphorylation by protein

xii kinase C (PKC) in CD36 transfected melanoma cells. Treatment of CD36- transfected melanoma cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, induced substantial CD36 phosphorylation and decreased ligand- mediated recruitment of Src-family proteins to CD36. PMA treatment did not induce detectable extracellular or cell surface-associated kinase activity, and both cycloheximide and brefeldin A blocked CD36 phosphorylation. New protein synthesis and trafficking through the Golgi were required for PMA-induced CD36 phosphorylation, suggesting that phosphorylation probably occurs intracellularly.

These studies suggest a novel in vivo pathway for CD36 phosphorylation that modulates cellular affinity for thrombospondin-related proteins to blunt vascular cell signaling. Thrombospondin-1 inhibits vascular endothelial growth factor

(VEGF) signaling at the receptor level in MVEC and CD36 has been suggested to be involved in this inhibition, however, the mechanisms are not known. Here we showed CD36 is required for the inhibition by silencing CD36 expression in

MVEC by siRNA or genetic deletion of cd36 in mice. Immunoprecipitation experiments showed that CD36 is required for thrombospondin-induced association of a phosphatase, SHP-1, with VEGFR2 in MVEC. Phosphatase activity was increased in precipitated VEGFR2 complexes from thrombospondin treated cells, consistent with thrombospondin-1-induced SHP-1 association with the VEGFR2 complex. By using an in vitro endothelial tube formation assay, we showed that thrombospondin-1 inhibited VEGF-induced tube formation of MVEC on matrigel, while CD36 silenced cells and SHP-1 inhibitor treated cells were less responsive to thrombospondin-1. These studies suggest a mechanism by which

xiii thrombospondin-1 inhibits VEGF signaling at the receptor level. Together the results, we provide new understanding of the role of CD36 in regulation of angiogenesis.

xiv

INTRODUCTION

1. Angiogenesis and tumor growth

The term angiogenesis refers to the formation of new vasculature from pre-existing capillary beds by progressive invasion and sprouting of endothelial cells 1. This process contrasts that of vasculogenesis, which is mediated by differentiation of endothelial progenitor cells (angioblasts) into de novo endothelial cells 2. New blood vessels in adult arise mainly through angiogenesis, while vasculogenesis mainly happens in embryonic development. In a healthy adult, quiescent endothelial cells are long-lived and form a monolayer. The endothelial monolayer is protected by pericytes, which suppress endothelial proliferation and release cell-survival signals. When a quiescent blood vessel senses an angiogenic signal, a series of reactions occurs. Vasculature invasion, endothelial cells migration, and lumen formation are involved in angiogenesis subsequently 3. Vasculature invasion is initiated by detach of pericytes from the vessel wall in response to angiopoietin-2 4 and secretion of proteolytic enzymes, such as matrix metalloproteinases, to degrade basement membrane 5-7.

Vascular permeability is then increased in response to vascular endothelial growth factor (VEGF) 8-10. Endothelial cells loosen their junctions due to the reducing of VE-cadherin and the vessel dilates 11. After invasion, endothelial cells proliferation and migration are induced by VEGF and integrins signaling. In

1 this process, one endothelial cell, known as tip cell, is selected to lead the tip in the presence of VEGF and Notch signaling, and the neighbor cells form stalk cells to divide and elongate the stalk and form the lumen 12-17. Once lumen formation occurs, it is covered by pericytes in response to signals, such as platelet-derived growth factor and angiopoietin-1 18-21, and basement membrane is re-established by protease inhibitors, such as tissue inhibitors of metalloproteinases and plasminogen activator inhibitor-1, and a functional blood vessel is formed 19-22. The size and density of newly formed vasculature depend on the demand and are coordinately regulated by angiogenic factors and antiangiogenic factors 23. For example, the oxygen-sensing mechanisms triggered by tissue hypoxia stimulate VEGF transcription and translation, thus promote angiogenesis 24-26. The mechanisms of angiogenesis are summarized in Figure 1.

Figure 1. Mechanisms of angiogenesis.

Endothelial cells are long-lived and stable in quiescent vessels. The vessel is embedded by basement membrane and covered by pericytes. Angiogenic signals, such as VEGF, angiopoietin-2, and cytokines, initiate angiogenesis by detaching pericytes, degrading basement membrane, and increasing capillary permeability. One endothelial cell is then selected to differentiate to tip cell and leads the sprouting of capillary. The neighbor cells of tip cell differentiate to stalk cells and the dividing of stalk cells elongate the lumen and a functional vessel is formed.

In the early stage of tumor growth, rapid dividing tumor cells cannot grow beyond 1-2 mm3 due to lacking nutrients and oxygen 27. Tumor cells at such hypoxia condition secret angiogenic growth factors, like VEGF, to induce angiogenesis from nearby capillary bed 27-28. The new vasculature support tumor growth by providing nutrients and oxygen, taking away metabolic wastes, and also support further metastasis (summarized in Figure 2). The strategy of antiangiogenesis to inhibit tumor growth has been suggested over 40 years 27 and several VEGF blockers and VEGF signaling inhibitors have been approved for clinical use in this decade 29. However, these drugs only prolong the survival of patient with some kinds of cancer for several months 3. The mechanisms of angiogenesis need to be further studied to find more effective therapies of cancer.

In this manuscript we studied the regulation of the binding of antiangiogenic factor, thrombospondin-1, to membrane receptor, CD36, by ecto-phosphorylation and also showed the mechanism by which thrombospondin-1 inhibits VEGF signaling in endothelial cells. The improvement of understanding of antiangiogenesis can be applied to future cancer therapies based on this strategy.

Figure 2. Angiogenesis is required for tumor growth and metastasis.

Tumor cannot grow beyond 1-2 mm3 due to the lack of nutrients and oxygen.

The hypoxia condition induces the secretion of growth factors, like VEGF, from tumor cells to stimulate angiogenesis from nearby capillary beds. The newly formed vasculature provides tumor nutrients and oxygen, takes away metabolic wastes and supports tumor growth. The vasculature also provides a route for further metastasis of tumor cells.

2. Thrombospondin-1 and thrombospondin-1 type 1 repeats (TSR)

Since angiogenesis is required for tumor growth and it has been suggested that tumor growth can be inhibited by inhibition of angiogenesis 27, people are interested in discovering antiangiogenic factors. Thrombospondin-1 is the first identified endogenous antiangiogenic factor from human platelets 30.

The thrombospondin family has five members named thrombospondin-1~5

(thrombospondin-5 is also called cartilage oligomeric matrix protein or COMP).

They are high molecular weight extracellular proteins and secreted by most cell types regulating cellular phenotype 31. The expression of thrombospondin-1 in adult tissue is limited for the most part in tissue remodeling sites 32.

Thrombospondin-1 shares similar structure with thrombospondin-2 and is distinct from the other three members 33-34. Thrombospondin-1 and 2 are composed of an N-terminal globule domain, a procollagen homology domain, three thrombospondin type 1 repeats (TSR), three type 2 repeats, a series of type 3 repeats and a C-terminal globule domain 34-35. The N-terminal domain has been shown to interact with heparin and also suggested to bind integrins and other proteins 36. The procollagen homology domain is involved in oligomerization of thrombospondin-1 to form trimers 37. The three type 1 repeats are exclusively presented in thrombospondin-1 and 2, but not in other members of thrombospondin family 35. TSR is the antiangiogenic domain of thrombospondin-

1 and 2 and has been shown to bind CD36 and β1 integrins 38-40. The type 2 and

3 repeats have plenty calcium binding sites and calcium is required for the stable of structure 41. The C-terminal domain has been shown to interact with CD47 42.

Thrombospondin-1 has been shown to inhibit migration and induce apoptosis of endothelial cells 30, 43-44. Downregulation of thrombospondin-1 is a frequent step in tumor cells 45-47. The molecular weight of thrombospondin-1 monomer is about 150 kDa. However, the large size with multiple functional domains limits the applications of full-length protein. For example, while the role of thrombospondin-1 as an inhibitor of tumor growth is well documented, in vitro studies showed thrombospondin-1 promotes the invasion of breast cancer cell lines through collagen gels and is suggested to be pro-metastatic 48. Therefore, to identify the therapeutic domain of thrombospondin-1 brings advantages for applications. Many of the roles attributed to thrombospondin-1 in regulating angiogenesis, wound healing, cell adhesion and migration have been shown to be localized to TSR repeats 35. TSR repeats are highly conserved domains in animals and protozoans and can be found in over 40 human proteins 49-50. The length of TSR repeats is about 60 amino acids and the crystal structure has been solved with a three-stranded anti-parallel design 50. An antiangiogenic therapeutic molecule ABT-510 has been developed based on the sequence

GVITRIR in the second β-strand of the second TSR repeat of thrombospondin-1

51-52. TSR has been suggested to bind CD36 with the positive charges of this sequence to the negative charges of CD36 50. ABT-510 has been tested clinically in renal cell carcinoma and soft tissue sarcoma, however, the effect is not significant and failed in phase II trials 53-55. Although ABT-510 failed, TSR still

7 has the potential to be a therapeutic agent and the mechanisms of TSR to inhibit angiogenesis are worth to be further studied. In this manuscript, we used prokaryotic expressed recombinant TSR protein which includes the second and third TSR repeat of thrombospondin-1 (TSR2/3) to study the effect of CD36 ecto- phosphorylation to TSR binding and the antiangiogenic effect of TSR to VEGF signaling. The improved understanding of TSR binding is helpful for future development of thrombospondin-1 based therapies. The structures of thrombospondin family are summarized in Figure 3.

Figure 3. Structures of thrombospondin family.

Thrombospondins are multidomain proteins. Thrombospondin-1 and 2 share similar structures and have TSR repeats, which is not presented on other members. The binding partners of each domain and the amino acid sequence of the second type 1 repeat of thrombospondin-1 are shown, and the sequence

GVITRIR for ABT-510 development is underlined.

3. The role of endothelial CD36 in antiangiogenesis

One of the receptors of thrombospondin-1 and TSR repeats on endothelial cells has been identified to be CD36 56. CD36 is a multiligand scavenger receptor expressed on the surface of platelets, microvascular endothelial cells

(MVECs), mononuclear phagocytes, erythroid precursors, adipocytes, hepatocytes, myocytes, and some epithelia 57. It was first identified as glycoprotein IV on platelets 58. CD36 is also a highly conserved protein in mammals, and orthologs of CD36 can been identified in insects, nematodes and sponges 59-61. Human CD36 is an 88 kDa transmembrane protein with a highly glycosylated extracellular domain and two short cytoplasmic domains with palmitoylation at terminals 62-63. CD36 has been localized in a specialized plasma membrane microdomain known as caveolae with caveolin-1 64. CD36 modulates cell signaling in some cases by interacting with other receptors on the cell surface, including VEGF receptor 2 (VEGFR2) 65, integrins 66-68, tetraspanins

66 and Toll-like receptors (TLRs) 69-70. The ligands of CD36 include oxidative modified LDL 71-72, components of bacterial cell wall 70, 73-74, Plasmodium falciparum-infected erythrocytes 75, apoptotic cells 76-78, photoreceptor outer segments 79-80, fibrillar β-amyloid 81, and extracellular matrix proteins containing

TSR 43, 82-83. The TSR binding domain of CD36, which spans amino acids 93 to

120, was identified by us and others and is named CLESH (CD36, LIMP-II, Emp sequence homology) domain 84-85. CLESH domain is also found in other proteins, such as HIV gp120 and histidine-rich glycoprotein, and these proteins are also

10 known to bind thrombospondin-1 86-87. A threonine residue besides CLESH domain of CD36 on amino acid 92 has been reported to be phosphorylated and block the binding of thrombospondin-1 to CD36 88. The intracellular signals of

CD36 are mediated by recruitment and activation of Src family non-receptor tyrosine kinases, such as Fyn, Lyn and Yes 43, 89-91. The structure of CD36 is summarized in Figure 4.

Figure 4. Structure of CD36.

CD36 is a transmembrane protein localized in caveolae with a large extracellular domain and two small intracellular domains. The extracellular domain is highly glycosylated and the intracellular domains are palmitoylated. Ligands of CD36 bind distinct domains. A threonine residue on amino acid 92 has been reported to be phosphorylated and block the binding of thrombospondin-1. CD36 associates with other receptors on the membrane and the intracellular signals are mediated by recruitment and activation of Src family non-receptor tyrosine kinases.

The role of CD36 is varied on different cell types with different ligands. On microvascular endothelial cells, CD36 is a receptor for thrombospondin-1 and related proteins, and it functions as a negative regulator of angiogenesis 56-57, 92-93 and therefore plays a role in tumor growth, inflammation, wound healing, and other pathological processes requiring neovascularizaton. CD36-mediated mechanisms by which thrombospondin-1 inhibits angiogenesis have been well studied 43, 57. In endothelial cells, binding of thrombospondin-1 to CD36 CLESH domain leads to recruitment of Fyn to a CD36 membrane complex with activation of the kinase and subsequent downstream activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase 43, 56. The signals then lead to cell apoptosis via caspase-3 pathway, expression of Fas ligand, and tumor necrosis factor-α 94-95. This antiangiogenic signaling of thrombospondin-1 inhibits growth factor-induced proangiogenic signals that mediate endothelial cell proliferation, migration, and tube formation 43, 96. The model of CD36-mediated thrombospondin-1 antiangiogenic responses are summarized in Figure 5.

Figure 5. The model of CD36-mediated thrombospondin-1 antiangiogenic responses.

During angiogenesis, growth factors, such as VEGF, bind receptors on endothelial cells and promote cell migration, proliferation and tube formation.

The binding of thrombospondin-1 to CD36 CLESH domain leads to recruitment and activation of Fyn and p38 MAP kinase. These signals switch the responses of endothelial cells to growth factor from proangiogenic to antiangiogenic, and lead to apoptosis via caspase-3 pathway.

4. Ecto-domain phosphorylation of CD36

CD36 has been reported to be phosphorylated on a canonical protein kinase C (PKC) target sequence in human platelets, microvascular endothelial cells, and CD36-transfected fibroblasts 88, 97, although the relative proportion of

CD36 that is phosphorylated under basal conditions has not been defined.

Unusually, the site of CD36 phosphorylation (Thr92) is on its extracellular domain

88, where it has been shown to influence extracellular ligand binding. For example, phosphorylated CD36 does not bind thrombospondin-1 and has significantly lower affinity for Plasmodium falciparum-infected erythrocytes 88, 97.

It has been hypothesized that constitutive phosphorylation of platelet CD36 explains the lack of demonstrable binding of thrombospondin-1 to resting platelets despite abundant levels of surface CD36 expression and that regulation of endothelial cell CD36 phosphorylation could influence angiogenesis and malaria cytoadhesion.

Importantly, intracellular or extracellular mechanisms by which CD36 can be phosphorylated on its ectodomain have not been identified, nor have molecular pathways by which phosphorylation can be regulated. Two groups have postulated that an extracellular protein kinase phosphorylates CD36 outside the cells 98-99, but convincing evidence for such an enzyme in vivo is lacking.

Asch et al. 88 have suggested that the key regulatory step in platelet CD36- thrombospondin binding is extracellular dephosphorylation by an unknown

15 ectophosphatase secreted from or activated by activated platelets. In the first part of this manuscript, we showed that CD36 is phosphorylated intracellularly and then transported to the plasma membrane.

The Thr92 extracellular phosphorylation site of CD36 is immediately adjacent to the CLESH domain. This domain, which spans from amino acid 93 to

120, was identified by us and others as the binding site for thrombospondin-1 and other antiangiogenic proteins containing TSR domains 10, 84. Although the mechanism by which Thr92 phosphorylation inhibits TSR-containing protein binding is not clear, it is reasonable to hypothesize that addition of a bulky phosphate group immediately upstream to the CLESH domain could disrupt the structure of the domain or block access to TSR-containing proteins by steric hindrance. In the first part of this manuscript, we used recombinant peptides to show that TSR domains bind to the CLESH domain and that phosphorylation of a small recombinant protein containing the consensus PKC target site and the

CLESH domain blocks TSR binding.

The observations that TSR proteins have robust CD36-dependent antiangiogenic activities in vitro and in vivo suggest that if indeed MVEC CD36 is phosphorylated under basal conditions, the level of phosphorylation must not be sufficient to prevent TSR-mediated responses. We thus propose that CD36 is only partially phosphorylated on MVECs and that upregulation of phosphorylation might be a mechanism by which MVECs lose responsiveness to an important endogenous antiangiogenic pathway triggered by TSR proteins.

In the first part of this manuscript, we showed that CD36 phosphorylation could be detected at low levels in cultured cells and that it can be further phosphorylated in vitro by exposure to active PKC. Furthermore, endogenous

CD36 phosphorylation could be increased by exposing cells to the PKC activator, phorbol 12-myristate 13-acetate (PMA). Induction of phosphorylation required new protein synthesis and trafficking through the Golgi, suggesting that only newly synthesized CD36 becomes phosphorylated and that phosphorylation occurs intracellularly. Finally, we showed that PMA induced phosphorylation inhibits CD36-mediated signaling downstream of TSR-proteins.

5. VEGF signaling and VEGF receptors

It was recently shown that thrombospondin-1 also inhibits growth factor signaling at the receptor level in endothelial cells and a mouse model 65, 67, 100. In these studies, VEGF-induced VEGFR2 phosphorylation and downstream Akt signaling was inhibited by pretreatment of cells with recombinant TSR. The studies showed that CD36 and VEGFR2 form a complex in MVECs and therefore suggest that inhibition of VEGFR2 phosphorylation was mediated by CD36. No direct evidence has demonstrated a CD36 requirement in this inhibition and the molecular mechanisms remain unclear.

VEGF (also known as VEGF-A) was first identified as vascular permeability factor (VAF) 101. It is the most potent permeability enhancing factor known and responsible for the vascular hyper-permeability in tumors 101. VEGF promotes survival 102, induces proliferation 103-104 and enhances migration and invasion 105 of endothelial cells. The VEGF family in mammals consists of

VEGF-A, B, C, D and placental growth factor (PLGF). They are secreted dimeric glycoproteins 106-107. VEGF-A is mainly involved in angiogenesis, and is the most studied factor in this family 108-110. There are at least five isoforms of human

111 VEGF-A with different biological activities and VEGF-A165 is the most predominant and also the most potent isoform to stimulate angiogenesis 108. In this manuscript we use VEGF to refer VEGF-A165 isoform and in the second part we used VEGF-A165 isoform to stimulate VEGF signaling in vitro and in vivo.

VEGF family binds three receptor tyrosine kinases on endothelial cells,

VEGFR1 (also known as flt-1), VEGFR2 (also known as Flk-1/KDR), VEGFR3

(also known as flt-4) and also receptors lacking the tyrosine kinase domain, such as Neuropilin-1 and 2 108, 112-113. Among these receptors, VEGFR2 is the primary mediator of angiogenic signaling 114. The role of VEGFR1 in angiogenesis is still elusive 115-116 and VEGFR3 is a key regulator in lymphangiogenesis 117.

Neuropilins associate with VEGFR2 as coreceptors and enhance the activity of

VEGFR2 113. The binding of ligands induces dimerization of VEGFR2 and activation of tyrosine kinase domain and leads to the autophosphorylation of the receptors. Phosphorylated VEGFR2 recruits downstream signaling proteins and activates signaling pathways. There are several tyrosine residues on VEGFR2 that become phosphorylated upon VEGF exposure. The kinase activity of receptor is regulated by Tyr1054 and Tyr1059 118. The others activate different signaling pathways. Among these, Tyr1175 is the most important in angiogenesis 119. Phospholipase C-γ (PLCγ) binds to phosphorylated Tyr1175 and activates the MAPK cascades and proliferation of endothelial cells 120-121.

Phosphorylation of Tyr1175 is also required for activation of phosphatidylinositol-

3 kinase (PI3K) and mediates survival of the endothelial cells 122-123.

Phosphorylation of Tyr951 is required for the binding of T-cell-specific adaptor

(TSAd) and Src-mediated endothelial cell migration 124-125. Phosphorylation of

Tyr1214 has been implicated in p38 MAPK mediated actin remodeling and regulation of vascular permeability 126-127. In the second part of this manuscript,

19 we detected VEGFR2 activity by a specific antibody to VEGFR2 phospho-

Tyr1175.

Phosphorylation of VEGFR2 is regulated by members of the SH2 domain- containing protein tyrosine phosphatase (SHP) family 128-130. There are two members in this cytoplasmic phosphatase family, SHP-1 and SHP-2; dephosphorylation of VEGFR2 Tyr1175 is mediated by SHP-1 131. SHP-1, also call as SHPTP-1, is a cytoplasmic phosphatase and has been shown to dephosphorylate the receptors which it binds 129, 132-133. Knockdown of SHP-1 by siRNA promotes VEGF-mediated DNA synthesis in human umbilical vein endothelial cells and accelerate angiogenesis in a rat model 128-129. Interestingly, our group recently showed that SHP-2 interacts with CD36 in macrophages and that its activity was modulated by binding of oxidized-LDL to CD36 134. Whether

SHP-1 interacts with CD36 is not known.

In the second part of this manuscript, we used in vitro and in vivo assays to define a mechanism by which thrombospondin-1 inhibits VEGF-induced

VEGFR2 phosphorylation in MVECs via CD36-dependent recruitment of SHP-1 phosphatase to the VEGFR2 complex. We found that CD36 binding to recombinant TSR peptides induced association of SHP-1 with both CD36 and

VEGFR2, and that CD36 was required for TSR-mediated inhibition of VEGFR2 phosphorylation by SHP-1 in vitro and in vivo.

CHAPTER 1

CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding:

Structure - Function Relationships and Regulation by Protein Kinase C

ABSTRACT

CD36 phosphorylation on its extracellular domain inhibits binding of thrombospondin-1. The Mechanisms of cellular CD36 ectodomain phosphorylation and whether it can be regulated in cells are not known. We determined structure-function relationships of CD36 phosphorylation related to thrombospondin-1 peptide binding in vitro and explored mechanisms regulating phosphorylation by protein kinase C (PKC) in melanoma cells. Phosphorylation of CD36 peptide on Thr92 by PKCα suppressed binding of thrombospondin-1 peptides in vitro, and the level of inhibition correlated with the level of phosphorylation. Basal phosphorylation levels of CD36 in vivo in platelets, endothelial cells, and melanoma cells were assessed by immunoprecipitation and immunoblot and were found to be very low. Treatment of CD36-transfected melanoma cells with phorbol-12-myristate-13-acetate (PMA), a PKC activator, induced substantial CD36 phosphorylation and decreased ligand-mediated recruitment of Src-family proteins to CD36. PMA treatment did not induce detectable extracellular or cell surface-associated kinase activity, and both cycloheximide and brefeldin A blocked CD36 phosphorylation. New protein synthesis and trafficking through the Golgi are required for PMA-induced CD36 phosphorylation, suggesting that phosphorylation probably occurs intracellularly.

These studies suggest a novel in vivo pathway for CD36 phosphorylation that

22 modulates cellular affinity for thrombospondin-related proteins to blunt vascular cell signaling.

MATERIALS AND METHODS

Reagents

Antibodies to phospho-PKCα and phosphothreonine were purchased from Cell

Signaling Technology. Rabbit anti-CD36 polyclonal antibody (ab36977) and monoclonal IgG FA6-152 were purchased from Abcam. Polyclonal rabbit anti-

PKCα antibody was from Gibco. Human platelet thrombospondin-1 was prepared as previously described 135 or purchased from CalBiochem.

Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgGs were purchased from GE Healthcare. Enhanced chemiluminescence (ECL) substrate was purchased from Thermal Scientific. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich.

Cells

Human dermal microvascular endothelial cells (MVECs) were purchased from

Lonza and maintained in microvascular endothelial cell growth medium (EGM-2

MV, Lonza) with full supplements (5% fetal bovine serum, 0.4 % human fibroblast growth factor-2, 0.1 % vascular endothelial growth factor, 0.1 % R3-insulin-like growth factor-1, 0.1 % human epidermal growth factor, 0.04 % hydrocortisone,

0.1 % ascorbic acid, 0.1 % GA-1000). Human umbilical vein endothelial cells

(HUVECs) were provided by Dr. P. DiCorleto and maintained in endothelial cell growth medium (EGM-2, Lonza) with full supplements (2 % fetal bovine serum,

0.4 % human fibroblast growth factor-2, 0.1 % vascular endothelial growth factor,

0.1 % R3-insulin-like growth factor-1, 0.1 % human epidermal growth factor, 0.04

% hydrocortisone, 0.1 % ascorbic acid, 0.1 % heparin, 0.1 % GA-1000). Bowes melanoma cells stably transfected with human CD36 cDNA or control plasmid were prepared and maintained as described previously 92. The human monocyte cell line THP-1 was obtained from American Type Culture Collection and maintained in RPMI-1640 medium containing 10 % fetal bovine serum. Before

PMA treatment, cells were placed in Dulbecco’s modified Eagle’s medium with 1

% fetal bovine serum for 16 hours. In some studies, cells were exposed to 100

µg/mL cycloheximide (Sigma-Aldrich) or 5 ng/mL brefeldin A (Biolegend) before

CD36 phosphorylation was assessed.

Recombinant proteins

A cDNA encoding an “extended” CD36 CLESH domain including the putative

PKC target site around Thr92 was cloned by polymerase chain reaction from human endothelial cell cDNA with the following primer pairs: 5'-

CACCAGCAACATTCAAGTTAAGCA-3' and 5'-TCAGGCACCATTGGGCTGCA

GGA-3'. Polymerase chain reaction was performed with high-fidelity DNA polymerase (Roche), and the products were gel-purified and cloned into the prokaryotic expression pET102 vectors (Invitrogen) by TA cloning to generate

His-patched, thioredoxin-tagged fusion proteins. The plasmids were then transformed to BL21-competent cells (Invitrogen), and recombinant protein expression was induced by 0.2 mmol/L isopropyl β-D-1-thiogalactopyranoside

(IPTG) for 4 hours at 37°C. Recombinant proteins were purified after cell sonication by incubation with Ni2+ beads and elution with imidazole. Dialyzed proteins were stored at -80°C. A Thr92Ala point-mutated CLESH domain was generated by overlap extension polymerase chain reaction with the following primer pairs: 5'-GGTCCTTATGCGTACAGAGTTCG-3' and 5'-CGAACTCTG

TACGCATAAGGACC-3'. The product was cloned and protein purified as described above. A plasmid encoding the second and third thrombospondin-1

TSR domains was obtained from Dr. J. Lawler (Beth Israel Deaconess Medical

Center) 50. The insert was recloned into the vector described above for purification of recombinant protein.

All constructs were sequenced by the dideoxynucleotide method to confirm that the recombinant protein sequences were correct and in frame. The recombinant proteins were also examined by Western blotting and ELISA to confirm appropriate size and immunoreactivity.

Immunoprecipitation and immunoblotting

Cells were scraped and lysed in 500 µL lysis buffer (20 mmol/L Tris-HCl, pH 7.5,

150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 % Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, and

26 protease inhibitors tablet [1 tablet in 10 mL of lysis buffer, Roche]) on ice for 5 minutes. Lysates were then centrifuged at 12,000 g for 5 minutes to remove cell debris, and supernatants containing 500 µg of protein were incubated with monoclonal anti-CD36 IgG (1 µg) or polyclonal anti-PKCα antibody at 4°C for 4 hours and then precipitated with 20 µL of protein G agarose beads (GE

Healthcare) for another 4 hours. Isotype matched mouse IgG or nonimmune rabbit Ig were used as controls. The beads were washed twice with lysis buffer at 4°C, resuspended in heated Laemmli sample buffer and boiled for 5 minutes before being subjected to electrophoresis in 7.5% sodium dodecyl sulfate- polyacrylamide gels and then transferred to polyvinylidene difluoride membranes

(Millipore). For detection, membranes were blocked with 5% nonfat milk in 25 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.05 % Tween 20 for 1 hour at 22°C and then incubated with 1:1000 diluted primary antibodies overnight at 4°C.

Membranes were incubated with 1:5000 diluted horseradish peroxidase-linked secondary antibodies for 1 hour at 22°C and then washed and developed by enhanced chemiluminescence. The blot was quantified by scanning and analyzed by ImageJ software (National Institute of Health).

In vitro PKCα phosphorylation

Purified CD36 recombinant proteins (25 µg) were diluted in 100 µL of kinase buffer (25 mmol/L Tris-HCl, pH 7.5, 5 mmol/L β-glycerophosphate, 2 mmol/L dithiothreitol, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2). Then, 2 mmol/L ATP and 1

27 ng/µL active PKCα (Cell Signaling Technology) were added and incubated overnight at 37°C. In other experiments, CD36 immunoprecipitates prepared as described above were resuspended in 150 µL of kinase buffer and divided into 3 aliquots. PKCα (1 ng/µL) was added to 2 aliquots and incubated at 37°C for 1 hour. At the end of the incubation, beads were washed twice and resuspended in 50 µL of phosphatase buffer (50 mmol/L Tris-HCL, pH 7.9, 100 mmol/L NaCl,

10 mmol/L MgCl2, 1 mmol/L dithiothreitol). In 1 tube, 10 units purified alkaline phosphatase (New England Biolabs) was added and incubated at 37°C for 1 hour. At the end of the incubation, beads were washed twice and processed for

Western blotting.

Solid phase protein binding assay

Thrombospondin-1 or recombinant TSR proteins (1 μg) were immobilized on 96- well polystyrene plates by overnight incubation in 0.5 mol/L NaHCO3, pH 9.6, at

4°C. Saturation coating conditions were first determined using antibodies against coated proteins. The wells were then washed 3 times with TBST (20 mmol/L

Tris-HCl, 150 mmol/L NaCl, pH 7.4, containing 0.1% Tween 20) and then blocked with 0.5 % bovine serum albumin in TBST at 22°C for 1 hour.

Recombinant CD36 peptides were then added in TBST containing 1 mmol/L

CaCl2 and incubated at 22°C for 2 hours. Wells were then washed 3 times with

TBST/CaCl2 before the addition of anti-thioredoxin or anti-CD36 polyclonal antibody (1:1000) for 1 hour at 22°C. Horseradish peroxidase-anti-rabbit IgG

28 was then added for 1 hour at 22°C. Bound antibody was detected using the

3,3′,5,5′-Tetramethylbenzidine-ELISA reagent (Pierce) reading absorbance at

450nm with a SpectraMax 190 Microplate Reader (Molecular Devices).

Mass Spectrometry

Protein bands were cut from SDS-PAGE gels; washed/destained in 50 % ethanol,

5 % acetic acid; and then dehydrated in acetonitrile. The proteins were then reduced with dithiothreitol and alkylated with iodoacetamide before the overnight in-gel digestion at 22°C with Lys-C (0.1 ng/μL) in 50 mmol/L ammonium bicarbonate. The peptides were then extracted from the polyacrylamide in 2 aliquots of 30 μL 50 % acetonitrile with 5 % formic acid. These extracts were combined, evaporated to <10 μL, and resuspended in 1 % acetic acid to a final volume of ~30 μL for liquid chromatography-mass spectrometry analysis using a self-packed 9 cm x 75 μm Phenomenex Jupiter C18 reversed-phase capillary chromatography column and a Finnigan LTQ linear ion trap mass spectrometer system operated at 2.5 kV. Chromatograms for each of these peptides were plotted and the peak areas determined manually with the program Qual Browser

(Thermal Scientific). The pThr92/Thr92 ratio was estimated by the observed peak area ratio for these peptides.

RESULTS

CD36 CLESH domain phosphorylation blocks binding to recombinant TSR domains

To confirm that CD36 ectodomain phosphorylation blocks thrombospondin-1 binding and determine whether phosphorylation of the CLESH domain by itself impacts binding of small TSR-containing peptides, we prepared a recombinant extended CLESH domain beginning at amino acid 81 that includes the Thr92-X-Arg PKC target motif (Figure 1A). As shown in the Western blot in Figure 1B the extended CLESH domain, but not a mutant substituting Ala for Thr at position 92, was readily phosphorylated by incubation with PKCα in vitro. Phosphorylation at Thr92 was confirmed and quantified by mass spectrometry. A dose response and time course study revealed that ~25 % of the total CD36 protein was phosphorylated at maximum (Figure 2A and 2B), but because phosphorylation reduces the efficiency of peptide ionization by at least half 136-137, this percentage is likely an underestimate by at least 2-fold.

(A) (B)

Figure 1. Extended CD36 CLESH domain can be phosphorylated on Thr92 by

PKCα in vitro.

(A) Schematic representations of CD36 showing the CLESH domain and PKC phosphorylation site (Thr92) and the recombinant thioredoxin/extended CLESH domain fusion protein. TM indicates transmembrane domains. (B) Wild-type and

Thr92Ala mutated fusion proteins were incubated with 1 ng/μL PKCα at 37°C for

16 hours, and then 5 µg were analyzed by Western blot with an anti- phosphothreonine (p-Thr) antibody. Blots were reprobed with anti-thioredoxin antibody as loading controls. The blot shows that the wild-type (WT) extended

CLESH domain was phosphorylated, whereas the Thr92Ala mutant or recombinant thioredoxin alone were not. Blots are representative of n=3.

(A) (B)

Figure 2. The maxima level of extended CLESH domain phosphorylation is

~25% by in vitro phosphorylation assay.

(A) CD36 extended CLESH proteins (100 μg, 50 μg and 25 μg) were incubated with 0.2, 2, and 8 mmol/L ATP in 100 μL in vitro PKC phosphorylation reactions overnight. The relative fold of phosphorylation level to 100μg protein with 0.2 mmol/L ATP is determined by Western blot with an anti-phospho-Threonine antibody. (B) CD36 extended CLESH proteins (25 μg) were incubated with 0.2 or 2 mmol/L ATP in 100 μL in vitro PKC phosphorylation reactions overnight.

Phosphorylated CD36 extended CLESH proteins were analyzed by Western blot with an anti-phospho-Threonine (p-Thr) antibody and also digested by Lys-C protease, and the phosphorylation ratio of pThr92/Thr92 is determined by mass spectrometry.

The extended CLESH domain peptide retained capacity to bind thrombospondin-1 as assessed in a solid phase binding assay (Figure 3A). The recombinant thioredoxin tag alone did not bind thrombospondin-1, demonstrating specificity. As with intact CD36, binding to the extended CLESH domain peptide was Ca2+-dependent. Figure 3B shows that soluble thrombospondin-1 also bound to the extended CD36 CLESH domain when the latter was immobilized.

To determine whether inhibition of thrombospondin-1 binding by CD36 phosphorylation was mediated by interactions outside of the TSR and CLESH domains, we studied binding of a recombinant peptide made up only of the second and third TSR domains to the recombinant extended CLESH domain. As shown in Figure 4A, the nonphosphorylated recombinant CD36 extended CLESH protein bound immobilized TSR2/3 in a concentration-dependent manner.

Binding of the phosphorylated protein was significantly reduced, but it was restored by pretreatment with alkaline phosphatase to remove most of the phosphorylation (Figure 4B). To determine whether inhibition of binding correlates with level of CD36 phosphorylation, we mixed nonphosphorylated and phosphorylated proteins at ratios of 1:0, 1:1, 1:2, and 0:1 to create mixtures containing 0, ~25 %, ~33 % and ~50 % phosphorylated CLESH (Figure 5A). As shown in Figure 5B, at the highest input concentration, binding of CD36 CLESH proteins was inversely proportional to the degree of phosphorylation.

(A) (B)

Figure 3. Extended CD36 CLESH domain binds thrombospondin-1.

(A) CLESH domain fusion protein (10 μg/mL) or thioredoxin control was incubated in TBS containing 1 mmol/L CaCl2 with or without 5 mmol/L EDTA in microtiter wells on which thrombospondin-1 had been adsorbed. After washing, bound proteins were detected by colorimetric assay using specific antibodies and

HRP-conjugated second antibodies. Significant calcium-dependent binding of the CLESH domain was seen. Thioredoxin did not bind, demonstrating specificity. (B) Thrombospondin-1 bound immobilized recombinant CD36 extended CLESH domain. Thrombospondin-1 (10 µg/mL) was incubated as in panel A in wells on which CLESH domain fusion proteins or thioredoxin control had been absorbed. (n=3, significance was determined by t-test.)

(A) (B)

Figure 4. Phosphorylation of CD36 extended CLESH domain inhibits binding to thrombospondin type 1 repeats (TSR).

(A) TSR2/3 protein was adsorbed on microtiter wells, and then untreated fusion proteins (♦ WT), proteins exposed to PKCα as in Figure 1 (■ pWT), or proteins exposed to PKCα followed by exposure to alkaline phosphatase (▲ AP) were added to the wells at concentrations of 0 – 10 µg/mL. Bound fusion proteins were detected as in Figure 3B. (n=3, significance was determined by t-test.) (B)

Fusion proteins were either exposed to PKCα as in Figure 1 or PKCα followed by purified alkaline phosphatase (AP) then analyzed by Western blot with an anti- phosphothreonine (p-Thr) antibody. Blots were reprobed with anti-thioredoxin as a loading control. NT indicates untreated fusion protein. Blots are representative of n=3.

(A) (B)

Figure 5. Binding of TSR2/3 to CD36 extended CLESH domain inversely correlates with level of phosphorylation.

(A) Nonphosphorylated and phosphorylated fusion proteins were mixed at 4 different ratios, 1:0, 1:1, 1:2, and 0:1. Mixtures were analyzed by Western blot as in Figure 4B. Blots are representative of n=3. (B) Mixtures of nonphosphorylated and phosphorylated extended CLESH domain prepared as in panel A were assayed for binding to immobilized TSR2/3 as in Figure 4A. (n=3, significance was determined by t-test.)

Basal levels of cellular CD36 phosphorylation in vivo are low

To determine basal phosphorylation levels in vivo, CD36 was immunoprecipitated from human MVECs and platelets, and the precipitates were analyzed by immunoblot with an anti-phosphothreonine antibody. Human umbilical vein endothelial cells were used as negative control because they do not express CD36. As shown in Figure 6A, phosphorylated CD36 was detected in both platelets and MVECs, but in both cases, the signal was very weak compared with that seen with recombinant CD36 peptides. Although the expression level of CD36 in human platelets is much higher than in MVECs, the signal of phosphorylated CD36 is similar, suggesting that the proportion of phosphorylated CD36 is higher in MVECs than in platelets. Because basal CD36 phosphorylation levels are low, we tested whether they could be increased by in vitro phosphorylation; i.e. whether full-length native CD36 retained the capacity to be phosphorylated. CD36 was immunoprecipitated from platelets and then incubated with PKCα in vitro for 1 hour. As shown in Figure 6B, this significantly increased the level of phosphorylation. The PKC-phosphorylated CD36 was then treated with alkaline phosphatase for 1 hour to show that phosphorylation could be reversed. These results indicate that the “default” phosphorylation level of

CD36 in cells is very low but that there is a capacity to increase phosphorylation.

(A) (B)

Figure 6. Cellular CD36 phosphorylation levels are low.

(A) CD36 was immunoprecipitated from human umbilical vein endothelial cells

(HUVEC), human microvascular endothelial cells (HMVEC), and platelets, and phosphorylation was detected by Western blot with an anti-phospho-Thr antibody

(pThr). Blots were also probed with antibodies to CD36 and IgG as loading controls. (B) Cellular CD36 was immunoprecipitated from human platelets and then incubated with vehicle control or PKCα at 1 ng/μL for 1 hour. An aliquot of

PKC-treated CD36 was then exposed to alkaline phosphatase (AP) at 0.2 U/μL for 1 hour. CD36 phosphorylation was then detected by Western blot as in panel

A. Blots are representative of n=3.

Cellular CD36 phosphorylation can be increased by PMA

To determine the impact of intracellular PKC activation on CD36 phosphorylation, we treated cells from a well-characterized Bowes melanoma cell line stably transfected with CD36 cDNA with a broadly active PKC activator,

PMA. We used melanoma cells rather than MVECs in these experiments because previous studies showed that PMA induced dramatic and rapid transcriptional suppression of CD36 expression in MVECs 138. Bowes cells were exposed to 0.01, 0.1 or 1 µg/mL PMA, and PKCα activity was confirmed by

Western blot using an antibody to phospho-PKCα (Figure 7A).

As shown in Figure 7B, before PMA treatment, levels of phosphorylated

CD36 were very low, and treatment with serum starvation or staurosporine, a

PKC inhibitor, had no significant impact. CD36 phosphorylation, however, increased in a dose- and time-dependent manner on exposure of the cells to

PMA (Figure 7B and Figure 8), with a maximum effect seen at 0.1 µg/mL and a modest reduction seen at higher concentration (1µg/mL). PMA-induced CD36 phosphorylation was first detected after 2 hours of exposure and increased further after 4 hours. Interestingly, PMA did not induced CD36 phosphorylation in platelets (Figure 9) or MVECs (Figure 10), but these results are difficult to interpret because prolonged PMA treatment suppressed PKC expression in these cells (Figure 9 and 10). On the other hand, PMA induced CD36

39 phosphorylation in THP-1 monocytic cells (Figure 11). In these cells PMA treatment was associated with prolonged PKC activation (rather than downregulation) and with upregulation of CD36 expression.

To test whether activation of other kinases could also induce CD36 phosphorylation, Bowes cells were exposed to cAMP and PS48, which are activators of protein kinase A and PDK-1, respectively. As shown in Figure 12, cAMP had did not influence either PKC activation or CD36 phosphorylation.

PS48, however, induced CD36 phosphorylation equivalently to PMA, presumably related to the ability of PDK-1 to activate PKC.

(A) (B)

Figure 7. Cellular CD36 phosphorylation is increased by treatment with PMA.

(A) CD36-transfected Bowes melanoma cells were incubated for 16 hours in low- serum media (1 % FBS) and then treated with PMA for 4 hours. PKCα was then immunoprecipitated with anti-PKCα IgG and analyzed by Western blot with an antibody specific for the PKCα auto-phosphorylation site (pPKCα). Blots were reprobed with antibodies to PKC as a loading control. (B) CD36 transfected

Bowes melanoma cells were treated with PMA as in panel A for 4 hours or with 1

μmol/L staurosporine. CD36 phosphorylation was then assessed by Western blot as in Figure 6. Blots are representative of n=3.

Figure 8. CD36 phosphorylation in CD36 transfected Bowes cells is increased by treatment with PMA.

CD36-transfected Bowes melanoma cells were incubated for 16 hours in low- serum media (1 % FBS) and then treated with 0.1 μg/mL PMA for 1 to 4 hours.

CD36 phosphorylation was then assessed by Western blot as in Figure 6. PKC activity in whole cell lysates was assessed by Western blot with an anti-phospho-

PKC (pPKC) antibody. Blots were reprobed with antibodies to PKC and tubulin as loading controls. Blots are representative of n=3.

Figure 9. CD36 phosphorylation in human platelets is not increased by treatment with PMA and PKC expression is suppressed by PMA.

Platelets-rich plasma (PRP) fraction was collected by centrifuge at 150 g for 10 minutes. Prostaglandin E1 (100 nmol/L) was added in PRP to rest platelets.

Platelets were collected by centrifuge at 500 g for 5 minutes and resuspend in modified Tyrodes buffer. 0.1 μg/mL PMA and 0.5 mmol/L levamisole were added and incubated for 0, 2, 4, 8 hours. CD36 phosphorylation was then assessed by

Western blot as in Figure 6. PKC activity in whole cell lysates was assessed by

Western blot with an anti-phospho-PKC (pPKC) antibody. Blots were reprobed with antibodies to PKC and tubulin as loading controls. Blots are representative of n=3.

Figure 10. CD36 phosphorylation in MVECs is not increased by treatment with

PMA and PKC expression is suppressed by PMA.

MVEC cells were incubated for 16 hours in low-serum medium (0.5% FBS) and then treated with 0.1 μg/mL PMA for 2 to 8 hours. CD36 phosphorylation was then assessed by Western blot as in Figure 6. PKC activity in whole cell lysates was assessed by Western blot with an anti-phospho-PKC (pPKC) antibody.

Blots were reprobed with antibodies to PKC and tubulin as loading controls.

Blots are representative of n=3.

Figure 11. Phosphorylation of endogenous CD36 in THP-1 cells is increased by treatment with PMA.

THP-1 cells were incubated for 16 hours in low-serum medium (1% FBS) and then treated with 0.1 μg/mL PMA for 1 to 4 hours. CD36 phosphorylation was then assessed by Western blot as in Figure 6. PKC activity in whole cell lysates was assessed by Western blot with an anti-phospho-PKC (pPKC) antibody.

Blots were reprobed with antibodies to PKC and tubulin as loading controls.

Blots are representative of n=3.

Figure 12. CD36 phosphorylation in CD36 transfected Bowes cells is increased by treatment with PDK-1 activator.

CD36-transfected Bowes melanoma cells were incubated for 16 hours in low- serum media (1% FBS) and then treated with 0.1 μg/mL PMA, 5 μmol/L cAMP, or

10 μM PS48 for 4 hours. CD36 phosphorylation was then assessed by Western blot as in Figure 6. PKC activity in whole cell lysates was assessed by Western blot with an anti-phospho-PKC (pPKC) antibody. Blots were reprobed with antibodies to PKC and tubulin as loading controls. Blots are representative of n=3.

New protein synthesis and trafficking through Golgi are required for PMA-induced

CD36 phosphorylation

It is not known whether CD36 phosphorylation occurs in an intracellular compartment or extracellularly. To test whether extracellular or cell-surface associated kinase activity was induced by PMA, Bowes cells were treated with

PMA for 4 hours and then the cells were incubated with recombinant CD36 extended CLESH domain peptide (1 µmol/L) with 0.2 mmol/L ATP for 1 hour.

Although the peptide was readily phosphorylated by addition of exogenous PKCα

(1 ng/µL), no phosphorylation was detected when the peptide was incubated with cells (Figure 13). These results suggest that CD36 phosphorylation is not the result of PMA-induced expression of a secreted or surface-associated kinase.

The prolonged time required to see an increase in CD36 phosphorylation after PMA treatment suggests that new protein synthesis could be involved in this process, and it may be newly synthesized CD36 that is phosphorylated in response to PMA-activated PKC. To test this hypothesis, we exposed Bowes cells to chemical inhibitors of protein synthesis (100 µg/mL cycloheximide) or endoplasmic reticulum-Golgi trafficking (5 µg/mL brefeldin A) concomitantly with

PMA. Whereas PMA induced robust CD36 phosphorylation (Figure 14A), both cycloheximide and brefeldin A inhibited the induction almost completely. The inhibitory effect was not due to a change in PKCα activity, as shown in Figure

14B. These data suggest that only newly synthesized CD36 is phosphorylated and that phosphorylation occurs after endoplasmic reticulum to Golgi transit.

Figure 13. No detectable ecto-kinase activity induced by PMA in CD36 transfected Bowes melanoma cells.

CD36-transfected Bowes melanoma cells were treated with PMA as shown in

Figure 7A for 4 hours and then incubated with 1 µmol/L recombinant CD36 extended CLESH domain and 0.2 mmol/L ATP for 1 hour. After incubation, the peptide was purified on 25 µL of Ni2+ beads and then analyzed by Western blot with an anti-phospho-Thr (pThr) antibody. Blots were reprobed with anti- thioredoxin antibody as loading controls. As a positive control (second lane from the right) a similar amount of CD36 peptide was incubated with exogenous PKCα.

The right lane (Total) shows the amount of recombinant peptide added before

Ni2+ bead pull-down and shows near quantitative recovery. Blots are representative of n=3.

(A) (B)

Figure 14. Protein synthesis and ER to Golgi trafficking are required for PMA- induced CD36 phosphorylation.

(A) CD36-transfected Bowes cells were incubated for 16 hours in low-serum medium and then treated for 4 hours with 0.1 μg/mL PMA along with either 100

µg/mL cycloheximide (CHX) or 5ng/mL brefeldin A (BFA). CD36 phosphorylation was then assessed by Western blot as shown in Figure 6. (B) Cells treated as in

Panel A were assessed for PKCα activity in whole cell lysates by Western blot with phospho-PKCα IgG. Blots were reprobed with an antibody to total PKCα as a loading control. Blots are representative of n=3.

PMA-induced CD36 phosphorylation blunts TSR-mediated recruitment of Src family proteins to CD36

A proximal event in CD36-mediated cell signaling is ligand-dependent recruitment of specific Src-family kinases to a CD36-containing multiprotein signaling complex 43. To determine whether PMA-induced CD36 phosphorylation influences CD36 signaling functions, we assessed TSR-induced recruitment of

Src family proteins to CD36. CD36 expressing Bowes melanoma cells were exposed to 0.1 µg/mL PMA for 4 hours and then treated with 100 nmol/L TSR or a control protein, thioredoxin, for 15 minutes before CD36 immunoprecipitation.

CD36-nonexpressing cells were also studied as a control. As shown in Figure 15,

Src family protein(s) were recruited to the CD36 signaling complex after exposure to TSR but not thioredoxin or PMA. The amount of Src protein recruited to the complex after TSR exposure was reduced by ~75 % in cells treated with PMA (p < 0.01). Exposure of the cells to alkaline phosphatase

(40u/mL) for 1 hour after PMA treatment restored TSR-induced Src protein association with CD36.

To test whether cycloheximide and brefeldin A also restored TSR-induced

Src protein association with CD36, Bowes cells were exposed to 0.1 µg/mL PMA along with cycloheximide or brefeldin A for 4 hours and then treated with 100 nmol/L TSR or a control protein, thioredoxin, for 15 minutes before CD36

51 immunoprecipitation. As shown in Figure 16, cycloheximide and brefeldin A both restored TSR-induced Src protein association with CD36. These data show that

PMA-induced CD36 phosphorylation changed the signaling potential of CD36 in response to TSR.

Figure 15. PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36.

CD36 transfected Bowes melanoma cells were incubated for 16 hours in low- serum medium and then treated for 4 hours with 0.1 µg/mL PMA or vehicle control. Some cells were then treated for 1 hour with 40 U/mL alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100 nmol/L recombinant TSR2/3 or thioredoxin control for 15 minutes. CD36 was then immunoprecipitated, and the precipitates were analyzed by Western blot with an antibody to Src family proteins. Blots were reprobed with anti-CD36 antibodies.

Blots are representative of n=3. Blots were scanned, and the amount of coprecipitated Src was normalized to total CD36. (n=3, significance was determined by t-test.)

Figure 16. TSR-mediated Src recruitment to CD36 can be recovered by inhibitors of protein synthesis and ER-Golgi trafficking.

CD36 transfected Bowes melanoma cells were incubated for 16 hours in low- serum medium and then treated for 4 hours with 0.1 µg/mL PMA along with either 100 µg/mL cycloheximide or 5 ng/mL brefeldin A. Cells were then exposed to 100 nmol/L recombinant TSR2/3 or thioredoxin control for 15 minutes. Src family association was then assessed by Western blot and analyzed as shown in

Figure 16. Blots are representative of n=3. Blots were scanned and the amount of coprecipitated Src was normalized to total CD36. (n=3, significance was determined by t-test.)

DISCUSSION OF CHAPTER 1

Although CD36 phosphorylation is potentially important as a modulator of ligand binding 88, 97, the mechanisms by which cells accomplish and regulate ectodomain phosphorylation as well as the mechanism by which phosphorylation influences thrombospondin binding are not known. In this chapter we shed light on all of these issues. We used recombinant peptides to show that inhibition of thrombospondin binding to phospho-CD36 is an inherent property of the two small peptide domains involved in the direct binding interaction – CLESH and

TSR. TSR domains are highly conserved structures found on many CD36- binding proteins 50, 139-140. Binding of these proteins to CD36 is mediated by interaction between the CLESH domain of CD36 with the TSR domain(s) 10, 82-84,

141-142. We found that phosphorylation of a recombinant CD36 CLESH domain extended by only a few amino acids to include the PKC substrate site blocked its ability to bind to recombinant TSR2/3 in a manner that was proportional to the degree of phosphorylation. Thrombospondin-1 is a large multi-domain protein and it is possible that the blocking effect of CD36 phosphorylation could be mediated by interactions between the phosphorylated residue and other domains of thrombospondin-1, or that phosphorylation disrupts CD36 structure in such a way to make the CLESH domain inaccessible. Recapitulating the inhibitory effect with small recombinant peptides strongly suggests that other domains are

55 not involved and that phosphorylation directly impeded TSR binding to CLESH, perhaps by steric or charge hindrance. These data also suggest that binding of other antiangiogenic TSR containing proteins to CD36 could also be modulated by CD36 phosphorylation.

The low basal levels of platelet and MVEC phosphorylation seen in our studies are consistent with the well-described robust CD36-dependent antiangiogenic and prothrombotic effects of TSR-proteins in vitro and in vivo 43, 56,

82, 92. Our findings that inhibition of TSR binding correlated with the level of phosphorylation of CD36 and that basal levels of cellular CD36 phosphorylation were very low indicates that cellular up-regulation of CD36 phosphorylation could be an important mechanism for dampening responses to TSR proteins. Indeed we identified a PKC-mediated pathway that significantly increased CD36 phosphorylation and decreased functional response to TSR in CD36 transfected melanoma cells. We speculate that growth factors or cytokines released from tumor cells or from non-malignant cells within the tumor microenvironment could activate this pathway in the tumor microvasculature and thus decrease the sensitivity of endothelial cells to endogenous anti-angiogenic factors, such as thrombospondin-1. Our studies also suggest that inhibiting this pathway in tumor endothelial cells could be a potential mechanism to overcome loss of responsiveness to TSR-mediated anti-angiogenesis.

While PMA is known to suppress CD36 expression in MVEC and induce

CD36 expression in monocytes and T-cells 138, 143-145, we did not see a significant change of CD36 expression in Bowes melanoma cells after 4h treatment and

56 thus the increase seen in CD36 phosphorylation was not due to a proportional increase in total CD36 expression. Our studies of PDK-1, which is an upstream activator of PKC, are consistent with studies on the role of PKC in platelet CD36 phosphorylation7, and support the hypothesis that PMA-induced CD36 phosphorylation is mediated by activation of PKC. PDK-1, like PMA activates

PKC family members broadly 146, thus whether CD36 phosphorylation is mediated by a specific PKC isoform remains to be determined. Although cAMP had been shown to induce CD36 phosphorylation in platelets by activating an ecto-PKA activity 98, it did not induce CD36 phosphorylation in Bowes cells.

Cellular mechanisms of CD36 ectodomain phosphorylation remain poorly understood. That significant upregulation of CD36 phosphorylation in Bowes cells was not seen until ~4 hours after exposure to PMA even though activation of PKC was seen in minutes suggests an indirect mechanism. Since there was no detectable kinase activity in either Bowes cell post culture media or on Bowes cell surfaces at the point where CD36 phosphorylation was detected, it is unlikely that PKC directly phosphorylates CD36 on the plasma membrane, or that the mechanism reported for platelets; i.e. induction of a surface-associated enzyme with protein kinase A-like activity is present in Bowes cells 98. In MVEC and platelets, where PMA did not enhance CD36 phosphorylation, PKC expression was dramatically suppressed by prolonged PMA exposure (not shown), suggesting that sustained PKC activity is necessary for CD36 phosphorylation.

In contrast to MVEC and platelets, the two cellular systems in which we observed up-regulation of phosphorylation - transfected Bowes cells and PMA-stimulated

THP-1 cells - are both characterized by high levels of CD36 gene transcription and protein synthesis. We thus hypothesized that CD36 phosphorylation occurs on newly synthesized protein and indeed found that phosphorylation was blocked by inhibiting new protein synthesis. Furthermore, inhibition by brefeldin suggests that phosphorylation occurs in an intracellular post-ER compartment.

In summary, we showed with in vitro assays that CD36 phosphorylation directly inhibits binding of TSR domains to CD36 CLESH domains and provide convincing in vivo evidence that low basal levels of cellular CD36 phosphorylation can be enhanced in the setting of robust CD36 synthesis by activating an intracellular PKC–mediated signaling pathway. CD36 phosphorylation occurs intracellularly on newly synthesized protein in a post ER compartment. By showing that PMA-induced CD36 phosphorylation blunted

TSR-dependent assembly of an intracellular CD36 signaling complex we suggest that upregulation of CD36 phosphorylation may be a mechanism to desensitize endothelial cell responses to TSR-mediated antiangiogenic factors. Regulation of CD36 phosphorylation by PKC is summarized in Figure 17.

Figure 17. The summary of regulation of CD36 phosphorylation by PKC.

PKC is activated upon PMA treatment. Activated PKC phosphorylates new synthetic CD36 in the cytoplasm either in the transported vesicles or in the Golgi apparatus. Phosphorylated CD36 is then transported to the plasma membrane and the phosphorylation of CD36 blocks the binding of TSR-containing protein.

Activation of PDK-1 by PS48 also activates PKC and induces CD36 phosphorylation. brefeldin A (BFA) blocks ER-Golgi transportation and blocks

PMA-induced CD36 phosphorylation. Cycloheximide (CHX) blocks protein translation and also blocks PMA-induced CD36 phosphorylation.

CHAPTER 2

Thrombospondin signaling via CD36 regulates SHP-1 localization and

phosphatase activity: A mechanism of VEGF inhibition

ABSTRACT

Thrombospondin-1 inhibits growth factor signaling at the receptor level in microvascular endothelial cells and CD36 has been suggested to be involved in this inhibition, however, the mechanisms are not known. We hypothesized that binding of thrombospondin-1 to CD36 induces association of Src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 with a vascular endothelial growth factor (VEGF) receptor 2 signaling complex, and attenuates

VEGF signaling. A recombinant protein containing the CD36 binding domain of thrombospondin-1 (known as thrombospondin type 1 structure homology repeats or TSR domains) inhibited VEGF-induced VEGF receptor 2 phosphorylation in endothelial cells at nanomolar concentrations. Silencing CD36 expression in endothelial cells by siRNA or genetic deletion of cd36 in mice showed that CD36 is required for the inhibition both in vitro and in vivo. Immunoprecipitation experiments showed that CD36 is required for TSR-induced association of SHP-

1 with VEGF receptor 2 in endothelial cells. Phosphatase activity was increased in precipitated VEGF receptor 2 complexes from TSR treated cells, consistent with TSR-induced SHP-1 association with the VEGF receptor 2 complex. By using an in vitro endothelial tube formation assay, we showed that TSR inhibited

VEGF-induced tube formation of endothelial cells on matrigel, while CD36 silenced cells and SHP-1 inhibitor treated cells were less responsive to TSR.

These studies suggest a mechanism by which thrombospondin-1 inhibits VEGF signaling at the receptor level, provide new understanding of the role of CD36 in angiogenesis regulation, and point to a novel target to modulate angiogenesis therapeutically.

MATERIALS AND METHODS

Reagents

Recombinant human VEGF and rabbit monoclonal antibodies to VEGFR2, phospho-VEGFR2 and phospho-SHP-2 were purchased from Cell Signaling

Technology. The Src homology 2 domain containing protein tyrosine phosphatase (SHP) inhibitor NSC-87877 was purchased from CalBiochem 147.

Rabbit anti-CD36 polyclonal antibody (ab36977) and monoclonal IgG FA6-152 were purchased from Abcam. Mouse anti-SHP-1 and anti-SHP-2 monoclonal antibodies were purchased from BD Biosciences. Human platelet thrombospondin-1 was prepared as previously described 135 or purchased from

CalBiochem. HRP-conjugated anti-mouse and anti-rabbit IgG were purchased from GE Healthcare. Enhanced chemiluminescence substrate (ECL) was purchased from Thermo Scientific.

Cells

Human dermal MVECs were purchased from Lonza and maintained in MVECs growth medium (EBM-2 MV, Lonza) with full supplements (5 % fetal bovine serum, 0.4 % human fibroblast growth factor-2, 0.1 % vascular endothelial growth factor, 0.1 % R3-insulin-like growth factor-1, 0.1 % human epidermal

63 growth factor, 0.04 % hydrocortisone, 0.1% ascorbic acid, 0.1% GA-1000).

Before VEGF treatment, cells were placed in EBM-2 MV medium with 0.5% fetal bovine serum for 16 hours.

Recombinant proteins

A plasmid encoding the second and third thrombospondin-1 TSR domains was obtained from Dr. J. Lawler (Beth Israel Deaconess Medical Center) 50. The plasmids were then transformed to BL21 competent cells (Invitrogen) and recombinant protein expression induced by 0.2 mM isopropyl β-D-1- thiogalactopyranoside for 4 hours at 37°C. Recombinant proteins were purified after cell sonication by incubation with Ni2+ beads and elution with imidazole.

Dialyzed proteins were stored at -80°C. All constructs were sequenced by the dideoxynucleotide method to confirm that the recombinant protein sequences were correct and in frame. The recombinant proteins were also examined by western blotting and ELISA to confirm appropriate size and immunoreactivity.

Immunoprecipitation and immunoblotting

Cells were scraped and lysed in 500 µl lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % CHAPS, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4 and protease inhibitors tablet (1 tablet in 10 ml lysis buffer, Roche) on ice for 5 minutes. Lysates were then centrifuged at 12,000g for 5 minutes to remove cell debris, and

64 supernatants containing 500 µg of protein were incubated with monoclonal anti-

CD36 IgG (1 µg) or polyclonal anti-VEGFR2 antibody at 4°C for 4 hours, and then precipitated with 20 µl protein G agarose beads (GE Healthcare) for another

4 hours. The beads were washed twice with lysis buffer at 4°C, resuspended in heated Laemmli sample buffer and boiled for 5 minutes before being subjected to electrophoresis in 7.5 % sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Millipore). For detection, membranes were blocked with 5 % nonfat milk in 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05 % Tween 20 for 1 hour at 22°C and then incubated with

1:1000 diluted primary antibodies overnight at 4°C. Membranes were incubated with 1:5000 diluted horseradish peroxidase-linked secondary antibodies for 1 hour at 22°C and then washed and developed with ECL. The blot was quantified by scanning and analyzed by ImageJ software (National Institute of Health).

In vitro phosphatase activity assay

MVECs were pretreated with 10 nM TSR before exposure to VEGF for 15 minutes. Cells were lysed with lysis buffer and VEGFR2 complex were immunoprecipitated as above and then resuspended in 1 M diethanolamine, pH

9.8. Phosphatase substrate, p-nitrophenyl phosphate disodium salt (PNPP 1 mg/ml, Thermo Scientific), was added and incubated for 1 hour at room temperature 129. The reaction was stopped by addition of 2 N NaOH.

Phosphatase activity was detected as absorbance at 405 nm using a

SpectraMax 190 Microplate Reader (Molecular Devices). 65

siRNA transfection

MVECs less than passage 6 were cultured to 60-80% confluent on 6-well plates.

Cells were incubated with 60 nM CD36 siRNA or control “scrambled” siRNA

(Santa Cruz) and 6 µl siRNA Transfection Reagent (Santa Cruz) in 1 ml serum- free medium (EBM-2 MV) at 37°C for 6 hour. After transfection, cells were cultured in EGM-2 MV medium for 42 hours.

In vitro endothelial tube formation assay

MVECs were transfected with CD36 or control siRNA as above and then incubated with 10 nM TSR in serum-free medium at 37°C for 4 hours. Cells were then trypsinized, resuspended in serum-free EBM-2 MV medium, and added to wells in a 24-well plate (105 cells/well) that were previously coated with Matrigel

(BD Biosciences). To some wells recombinant human VEGF (50 ng/ml) with or without the SHP inhibitor NSC-87877 (100 μM) was added. Cells were incubated at 37°C for 16 hours and then fixed with 4 % paraformaldehyde and stained with fluorescence-labeled phalloidin (Invitrogen). Fluorescence images were taken from 4 randomly-chosen areas and tube formation analyzed by ImageJ software

(National Institute of Health) as previously described 138.

In vivo VEGFR2 phosphorylation assay

C57Bl/6 wild type and cd36 null mice 148 were injected with 1 μg VEGF in 100 μl saline or saline alone via jugular vein. Lung tissue was harvested 5 minutes after injection, minced in liquid nitrogen, and then lysed in 1 ml buffer as for immunoprecipitation for 5 minutes. Lysates were centrifuged at 12,000g for 5 minutes to remove cell debris and then analyzed by western blot with antibodies to VEGFR2, pTyr1175 VEGFR2, CD36 and Tubulin. Lysates were also incubated with anti-VEGFR2 antibodies to precipitate VEGFR2 and the precipitates were analyzed by western blot with antibodies to VEGFR2, CD36 and SHP-1. All procedures on animals were approved by the Cleveland Clinic

Institutional Animal Care and Use Committees (IACUC). Mice were housed in a facility fully accredited by AALAC and in accordance with all federal and local regulations.

RESULTS

Thrombospondin-1 and TSR inhibit VEGF-induced VEGFR2 phosphorylation in

MVECs

To confirm the previously reported association of CD36 with VEGFR2,

MVEC lysates were precipitated with anti-CD36 IgG and VEGFR2 was detected as shown in Figure 1 by western blot. CD36 expression levels in MVECs decrease during sequential passage in culture, so as a negative control we repeated the studies in late passage MVECs with low CD36 expression and showed no VEGFR2 precipitated. As reported by others 65, 67, the association of

VEGFR2 and CD36 was sensitive to the conditions of cell lysis. Coprecipitation was not seen when cells were lysed with Brij97 or Triton X-100 (not shown), but was readily detected when the cells were lysed with Brij99, Brij96 or 3-[(3- cholamidopropyl) dimethylammonio]-1 propane sulfonate (CHAPS) in the presence of magnesium.

To test whether thrombospondin-1 inhibits VEGF signaling at the receptor level, MVECs were treated with thrombospondin-1 for 4 hours and then exposed to 50 ng/ml VEGF for 5 minutes. As shown in Figure 2, basal VEGFR2 Tyr1175 phosphorylation levels were very low in untreated cells, but increased

68 significantly in those exposed to VEGF. Pretreatment with thrombospondin-1 at nanomolar levels inhibited VEGF-induced VEGFR2 phosphorylation in a concentration dependent manner with 0.1 nM and 100 nM thrombospondin-1 inhibiting phosphorylation by 40 % and 80 %, respectively.

Thrombospondin-1 is a large multi-domain protein that interacts with many receptors on the cell membrane. To determine the specific role of CD36, we showed that a recombinant TSR peptide behaved similarly to intact thrombospondin-1 (Figure 2). The thioredoxin tag of the recombinant protein was used as a negative control and showed no effect on VEGFR2 phosphorylation.

Figure 1. CD36 associates with VEGFR2 in endothelial cells.

CD36 was precipitated from early (passage <6) or late (passage >9) passage

MVECs, and the precipitates were analyzed by western blot with an antibody to

VEGFR2. Blots were stripped and reprobed with anti-CD36 antibodies. Lower blot shows total VEGFR2 in whole cell lysates before immunoprecipitation. Blots were reprobed with anti-tubulin antibody as a loading control. Blots were representative of n=3.

Figure 2. Thrombospondin-1 and TSR inhibit VEGF-induced VEGFR2 phosphorylation in MVECs.

MVECs were cultured to confluence and then treated with thrombospondin-1, recombinant TSR or thioredoxin control for 4 hours before exposure to 50ng/ml

VEGF for 5 minutes. VEGFR2 phosphorylation was analyzed by western blot with an antibody to VEGFR2 pTyr1175. Blots were reprobed with anti-VEGFR2 as loading control. Blots shown are representative of n=3. VEGFR2 phosphorylation levels were expressed as the ratio of phosphorylated VEGFR2 to total VEGFR2. (n=3, significance was determined by t-test.)

TSR induces SHP-1 association with CD36 and VEGFR2 in MVECs

As shown in Figure 3, VEGF-induced VEGFR2 phosphorylation in MVEC was markedly diminished by TSR pretreatment. Phosphorylation of VEGFR2 is regulated by the SHP family of phosphatases. The two members of this family,

SHP-1 and SHP-2, dephosphorylate different tyrosine residues on the receptor.

To determine the role of these phosphatases in TSR-mediated inhibition, the presence of SHP phosphatases with VEGFR2 and CD36 was detected by western blot. SHP-2 was coprecipitated by both anti-VEGFR2 (Figure 3) and anti-CD36 (Figure 4) in untreated cells, but treatment with VEGF, TSR, or both had no effect. The activity of CD36-associated SHP-2, as determined by western blot assessment of the level of phospho-SHP-2 was low and was also not influenced by VEGF or TSR treatment. These results suggest that SHP-1, not

SHP-2 is likely to be the key regulator of VEGFR2 dephosphorylation in response to TSR and are consistent with previous reports that VEGFR2 Tyr1175 is dephosphorylated by SHP-1 but not SHP-2. In support of this hypothesis, we showed that SHP-1, in contrast to SHP-2, was precipitated from the cells by anti-

VEGFR2 (Figure 3) or anti-CD36 (Figure 4) only after TSR treatment. VEGF alone did not induce SHP-1 association with VEFGR2 or CD36.

Since an antibody against phospho-SHP-1 was not available, we performed in vitro phosphatase assays on the anti-VEGFR2 immunoprecipitates.

As shown in Figure 5, in the absence of VEGF, no phosphatase activity was

72 detected. VEGF alone induced a modest increase in phosphatase activity (P <

0.05), consistent with previous reports 129, 131, but pretreatment with TSR increased VEGF-induced phosphatase activity within the VEGFR2 complex by more than two fold (P < 0.01). These results are consistent with a model whereby TSR induces association of SHP-1 with VEGFR2, resulting in tyrosine dephosphorylation of the receptor upon VEGF treatment.

Figure 3. TSR induces SHP-1 association with VEGFR2 in MVECs.

MVECs were treated with TSR (10 nM) in low-serum medium for 4 hours before exposure to VEGF (50 ng/ml) for 15 minutes. Cell lysates were immunoprecipitated with anti-VEGFR2, and coprecipitated proteins were detected by western blot with antibodies to SHP-1, SHP-2, phospho-SHP-2, phospho-VEGFR2 and VEGFR2. Blots were representative of n=3.

Figure 4. TSR induces SHP-1 association with CD36 in MVECs.

MVECs were treated with TSR (10 nM) in low-serum medium for 4 hours before exposure to VEGF (50 ng/ml) for 15 minutes. Cell lysates were immunoprecipitated with anti-CD36, and coprecipitated proteins were detected by western blot with antibodies to SHP-1, SHP-2, phospho-SHP-2, and CD36.

Blots were representative of n=3.

Figure 5. Phosphatase activity within the VEGFR2 complex is induced by TSR and VEGF.

MVECs were treated with TSR (10 nM) in low-serum medium for 4 hours before exposure to VEGF (50 ng/ml) for 15 minutes. Anti-VEGFR2 or control immunoprecipitates were incubated with PNPP substrate for 1 hour at room temperature, and phosphatase activity was detected by absorbance at 405nm.

(n=3, significance was determined by t-test.)

CD36 is required for TSR-induced SHP-1 association with VEGFR2 and

VEGFR2 dephosphorylation in MVECs

To test whether the association of SHP-1 with VEGFR2 is mediated by

CD36, siRNA was used to silent CD36 expression in cultured MVECs. As shown in Figure 6, CD36 expression was dramatically decreased 48, 72 and 96 hours after siRNA transfection, while the scrambled siRNA control had no effect.

Phosphorylation of VEGFR2 was analyzed by western blot with an antibody to

VEGFR2 pTyr1175, and as shown in Figure 7, TSR dramatically inhibited VEGF- induced VEGFR2 phosphorylation in a concentration-dependent manner in cells transfected with the control siRNA. The inhibitory effect was completely lost in cells transfected with the specific CD36 siRNA, strongly suggesting that CD36 is required for TSR-mediated inhibition of VEGFR2 phosphorylation. As an additional control, we showed that human umbilical vein endothelial cells, which do not express CD36, showed no inhibitory effect of TSR (Figure 8). As shown by immunoprecipitation in Figure 9, TSR-induced SHP-1 association with

VEGFR2 (lane 2) was blocked in cells transfected with CD36 siRNA (lane 4).

Figure 6. CD36 is silenced by siRNA in MVECs after 48 hours.

MVECs were transfected with CD36 or control siRNA for 6 hours and then cultured for 48, 72, or 96 hours before analysis of CD36 expression by immunoprecipitation and western blot. Blots were representative of n=3.

Figure 7. CD36 is required for TSR inhibition of VEGF-induced VEGFR2 phosphorylation in MVECs.

MVECs transfected as in Figure 6 for 48 hours were treated with TSR in low- serum medium for 4 hours and then exposed to 50 ng/ml VEGF for 5 minutes.

VEGFR2 phosphorylation was analyzed by western blot with an antibody to pTyr1175. Blots were reprobed with an antibody to VEGFR2. The level of phosphorylation was shown as the ratio of phosphorylated to total VEGFR2.

(n=3, significance was determined by t-test.)

Figure 8. Thrombospondin-1 and TSR do not inhibit VEGF-induced VEGFR2 phosphorylation in human umbilical vein endothelial cells.

Human umbilical vein endothelial cells were treated with thrombospondin-1

(TSP-1) or TSR in low-serum medium for 4 hours and then exposed to 50 ng/mL

VEGF for 5 minutes. VEGFR2 phosphorylation was analyzed by Western blot with an antibody to pTyr1175. Blots were reprobed with an antibody to VEGFR2.

The level of phosphorylation was shown as the ratio of phosphorylated to total

VEGFR2. (n=3, significance was determined by t-test.)

Figure 9. CD36 is required for TSR-induced SHP-1 association with VEGFR2 in

MVECs.

MVECs transfected as in Figure 7 were treated with TSR in low-serum medium for 4 hours and then exposed to 50 ng/ml VEGF for 15 minutes. VEGFR2 was then immunoprecipitated and the precipitates were analyzed by western blot with anti-SHP-1 antibody. Blots were reprobed with an antibody to VEGFR2. Blots are representative of n=3.

TSR inhibition of MVECs tube formation is mediated by CD36 and SHP-1

To test the functional role of CD36 and SHP-1 on TSR inhibition of VEGF signaling, we performed in vitro endothelial cell tube formation assays using

MVECs transfected with CD36 or control siRNA. As shown in Figure 10, cells rarely formed tube-like structures in low-serum medium. Exposure to VEGF significantly induced tube formation in both cell types as expected. Pretreatment with TSR significantly inhibited both number of branches (P < 0.05) and the average branch length (P < 0.01) of VEGF-induced tube-like structures in control siRNA, but not in CD36 siRNA transfected cells (Figure 11). To elucidate the role of SHP-1 in TSR inhibition, control siRNA transfected cells were treated with

TSR and then exposed to VEGF in the presence of a pharmacological SHP inhibitor. The inhibitor significantly rescued the number of branches (P < 0.05) and average branch length (P < 0.01) of VEGF-induced tube-like structures in the presence of TSR (Figure 12). To exclude the possibility that the SHP inhibitor itself induced tube formation, cells were treated with VEGF plus the inhibitor without TSR and no significant increase in tube formation was seen.

Figure 10. TSR inhibits formation of MVECs tube-like structures.

MVECs were transfected with CD36 or control siRNA as in Figure 7 and then treated with 10 nM TSR in low-serum medium for 4 hours before being transferred onto matrigel-coated tissue culture wells. Cells were then exposed to

50 ng/ml VEGF for 6 hours and then fixed with 4 % paraformaldehyde and then stained with 30 nM fluorescence-labeled phalloidin for 1 hour. Images from 4 randomly-chosen areas were obtained with a fluorescence microscope. Scale bar equals 200μm.

Figure 11. TSR inhibits the number of branches and average branch length of

MVECs tube-like structures via CD36.

MVECs were treated as in Figure 10. The number of branches and the average branch length of tube-like structures were quantified using ImageJ software

(National Institute of Health). (n=4, significance was determined by t-test.)

Figure 12. TSR inhibits the number of branches and average branch length of

MVECs tube-like structures via SHP-1.

MVECs were transfected with control siRNA and treated with TSR as in Figure

10. Cells were then exposed to 50 ng/ml VEGF for 6 hours in the presence of

100 μM SHP inhibitor, NSC-87877, or vehicle control. The number of branches and the average branch length of tube-like structures were quantified using

ImageJ software (National Institute of Health). (n=4, significance was determined by t-test.)

Cd36 null mice exhibit increased VEGFR2 phosphorylation and decreased SHP-

1 association with VEGFR2 after VEGF infusion

To elucidate the role of CD36 in the inhibition of VEGF signaling in vivo, we assayed VEGFR2 phosphorylation in lung tissue of cd36 null mice treated with an intravenous infusion of VEGF. Endothelial cells are the most predominant cells in lung tissue. As shown by western blot in Figure 13, CD36 and VEGFR2 were expressed in lung tissue of wild type mice, and deletion of cd36 had no effect on the level of expression of VEGFR2. As with cultured

MVECs, CD36 was coprecipitated from wild type lung tissue by anti-VEGFR2, suggesting that CD36 and VEGFR2 form a complex in vivo. Lack of coprecipitation from cd36 null lung tissues demonstrated specificity. To induce

VEGFR2 phosphorylation in vivo, 1 μg VEGF was injected via the jugular vein 5 minutes before harvesting lung tissue. As shown in Figure 14, VEGF induced a

2 fold increase in the level of phosphorylation of VEGFR2 in tissue from wild type mice compared to the basal level seen in non-treated mice (P < 0.05). Lung tissue from cd36 null mice, however, showed a 4.7 fold increase in VEGFR2 phosphorylation after VEGF infusion (P<0.05 comparing wild type to cd36 null), suggesting that endogenous CD36 signaling suppressed VEGF-induced

VEGFR2 phosphorylation in vivo. Anti-VEGFR2 immunoprecipitates from these tissues revealed substantially less SHP-1 associated with VEGFR2 after VEGF

86 injection in cd36 null mice compared to wild type, suggesting that CD36 is required for VEGF-mediated recruitment of SHP-1 to the VEGFR2 complex.

Figure 13. CD36 associates with VEGFR2 in mice lung tissue.

Lysates of lung tissue removed from cd36 null or wild type mice were analyzed by western blot with anti-VEGFR2, anti-CD36, or anti-tubulin (top panel) or by immunoprecipitation with anti-VEGFR2 followed by western blot (lower panel).

Blots were representative of n=3.

Figure 14. Genetic deletion of cd36 increases VEGFR2 phosphorylation and decreases SHP-1 association with VEGFR2 after in vivo infusion of VEGF.

Lung tissue from wild type or cd36 null mice was harvested 5 minutes after 1 μg

VEGF injection via jugular vein. Top panel shows VEGFR2 Tyr1175 phosphorylation detected by western blot of tissue lysates. Blots were reprobed with antibodies to total VEGFR2, CD36, and tubulin. Bottom panel shows western blot of anti-VEFGR2 immunoprecipitates of the tissue lysates probed with antibodies to SHP-1 and VEGFR2. Blots are representative of n=3.

VEGFR2 phosphorylation levels were expressed as the ratio of phosphorylated

VEGFR2 to total VEGFR2. (n=3, significance was determined by t-test.)

DISCUSSION OF CHAPTER 2

TSP-1 was identified as an angiogenesis inhibitor more than 20 years ago

30, 149. The mechanisms underlying this activity were demonstrated to involve a specific MVEC receptor, CD36, which generates antiangiogenic signals that lead to apoptosis in the presence of angiogenic growth factors 43, 56. An inhibitory effect of TSP-1 on VEGF-induced VEGFR2 phosphorylation is a more recent observation 65, although molecular mechanisms have not been identified. In this chapter we defined two mechanistic components of the pathway. We used CD36 siRNA transfected MVEC and cd36 null mice to provide evidence that CD36 is required for TSR-mediated VEGFR2 inhibition in vitro and in vivo. Detection of the CD36-VEGFR2 interaction depended on the choice of detergent used to lyse the cells, suggesting perhaps that the interaction occurs in specific membrane microdomains 150-151. We also found that the suppression of VEGF-induced

VEGFR2 phosphorylation by TSP-1 or TSR peptide was mediated by CD36- dependent recruitment of a specific phosphatase, SHP-1 to the VEGFR2 signaling complex.

CD36 was previously shown by Zhang et al to interact with VEGFR2 in cultured MVEC 65. Here we confirmed that observation and report for the first time that CD36 and VEGFR2 form a complex in vivo in murine lung, and that the association has functional consequences with regard to VEGF response. The concentration of TSP-1 and TSR that inhibits VEGF-induced VEGFR2

90 phosphorylation in MVEC is at the nanomolar level, well within the range of reported plasma concentrations of TSP-1 152. Our in vivo finding that VEGF- induced VEGFR2 phosphorylation was enhanced by genetic deletion of cd36 suggests that physiological levels of TSP-1 or other endogenous CD36 ligands normally dampen VEGF signaling. This is consistent with the observation that endogenous levels of TSP-1 were sufficient to inhibit VEGF-induced Akt phosphorylation in the mouse retina 100.

SHP-1 was reported to associate with VEGFR2 constitutively in human umbilical vein endothelial cells 129, while in VEGFR2 transfected porcine aortic endothelial cells, the association required induction by VEGF 153. In murine lung we now show that the association was induced by VEGF. In human MVEC, however, the association was neither constitutive nor induced by VEGF, but rather was induced by TSR in a CD36-dependent manner. The association of

SHP-1 with target proteins is mediated by binding of its SH2 domain to phospho- tyrosine residues. It is possible that the multiple potential sites for tyrosine phosphorylation on VEGFR2 that can be SHP-1 targets are differentially phosphorylated under basal conditions in endothelial cells from different sites so that some cells require VEGF stimulation to create a docking site while others constitutively present a docking site. Our data are also compatible with a model in which SHP-1 is recruited to CD36, rather than VEGFR2, in MVEC. CD36 is not known to contain any phospho-tyrosine, but it does form a signaling complex with adaptor proteins, such as Vav that could function in this regard 154.

While others have shown using siRNA that SHP-1 is the critical phosphatase for VEGFR2 in endothelial cells 128-129, we now showed with an in vitro assay that phosphatase activity within the VEGFR2 complex is induced by

VEGF and increased by TSR treatment. Using a pharmacologic inhibitor in an in vitro endothelial tube formation assay we showed that SHP-1-mediated VEGFR2 dephosphorylation suppressed the angiogenic phenotype. Although the SHP inhibitor used blocks both SHP-1 and SHP-2 activity 147, 155, pTyr1175 of

VEGFR2 has been demonstrated to be dephosphorylated only by SHP-1 131 and our data also showed that the association and activity of SHP-2 were not affected by VEGF or TSR.

Previous work from our lab demonstrated that CD36 signaling in macrophages in response to oxidized phospholipids results in down-regulation of

SHP-2 activity 134. Mechanistically this was due to generation of intracellular oxidant stress and oxidative modification of a critical cysteine residue at the active site of the phosphatase. Endothelial cells signaling by TSR is fundamentally different in that SHP-2 was not affected, yet SHP-1 activity was increased. This difference could be due to the use of different CD36 ligands

(TSR peptide vs oxidized phospholipid) and/or differences in downstream src- family or mitogen-activated protein kinases in the different cell types. CD36 signaling in macrophages is mediated primarily by Lyn and c-Jun N-terminal kinase 156, while in endothelial cells Fyn and p38 mitogen-activated protein kinase are more relevant 43.

In summary, we showed that CD36 and VEGFR2 form a complex in human MVEC in vitro and mouse lung tissue in vivo. Binding of TSP-1 or TSR to

CD36 suppressed VEGF-induced VEGFR2 phosphorylation via CD36-dependent recruitment of SHP-1 phosphatase to the VEGFR2 signaling complex. These studies identify a novel mechanistic regulatory pathway that could potentially be targeted to enhance or inhibit angiogenesis in a therapeutic context.

Thrombospondin-1 inhibition on VEGF-signaling mediated by CD36 is summarized in Figure 15.

Figure 15. The summary of thrombospondin-1 inhibition of VEGF-signaling mediated by CD36.

CD36 and VEGFR2 form a complex in endothelial cells. The binding of thrombospondin-1 (TSP-1) to CD36 recruits phosphatase SHP-1 to this complex.

VEGFR2 is autophosphorylated upon binding of VEGF. SHP-1 associates with phosphorylated VEGFR2 by SH2 domain. VEGF-activated VEGFR2 phosphorylates SHP-1 and activates SHP-1 phosphatase activity. Activated

SHP-1 then dephosphorylates VEGFR2 and attenuates VEGF signaling in endothelial cells.

DISCUSSION

The idea of inhibiting tumor growth by antiangiogenesis has been proposed for over 40 years 27 and clinical cancer research has clearly shown the importance of angiogenesis in cancer growth. In this decade, several antiangiogenic drugs, such as anti-VEGF monoclonal antibody, bevacizumab, and small molecule VEGFR tyrosine kinase inhibitors (TKIs), sorafenib and sunitinib, have been approved by Food and Drug Administration for treatment of several cancers 3. However, while assessing the efficiency of these drugs in cancer patients, their response rate, progression-free survival, and overall survival rate were modest 3. Therefore the search for effective antiangiogenic molecules to treat cancer continues. TSR, the antiangiogenic domain of TSP-1, is a potential molecule to treat cancer by inhibiting angiogenesis. While the use of full-length TSP-1 is prohibitive due to its size and multiple functional domains, its small TSR domain is ideal to develop peptides to inhibit angiogenesis.

Although the TSR based peptide ABT-510 failed in phase II clinical trial in monotherapy 53-55, the use of TSR based peptides to treat cancer still has the potential to be improved with better understanding of the mechanisms involved in

TSR-mediated antiangiogenesis. In this manuscript, we studied two mechanisms related to TSR-mediated antiangiogenesis.

In the first part, we showed that the binding of TSR to CD36 can be blocked by phosphorylation of CD36 on Thr92 and that this phosphorylation can be enhanced in the setting of robust CD36 synthesis, such as in CD36

95 transfected Bowes melanoma cells and PMA-treated THP-1 cells, by activation of

PKC. This system may be more important in endothelial cells, the major cell type involved in angiogenesis. Our results suggest that CD36 phosphorylation in endothelial cells could be increased with activated PKC during a phase of rapid proliferation and new CD36 protein synthesis. PKC in endothelial cells is known to be activated by many growth factors, including VEGF, via activation of phospholipase C-γ 119, 157. During angiogenesis, a specialized group of endothelial cells adjacent to “tip” cells differentiate into highly proliferative “stalk” cells 13. Furthermore, CD36 expression in endothelial cells has been shown to be upregulated during hypoxia 158. Taking these results together, we speculate that CD36 could be highly phosphorylated by activating PKC in endothelial stalk cells. This suggests that upregulation of CD36 phosphorylation by activation of

PKC could be a mechanism to desensitize TSR-mediated antiangiogenesis in cancer therapy. It would be worthwhile to study the regulation of CD36 phosphorylation in endothelial cells by tumor-released growth factors and its effect on angiogenesis in vivo in future. An antibody specific for the phosphorylated form of CD36 would be extremely valuable for in situ studies to test this hypothesis. Unfortunately we were unable to generate such an antibody using synthetic peptides, but with such an antibody, one could assess the phosphorylation status of CD36 in specific tissue in vivo, for example, in stalk cells or around tumor cells. The development of transgenic “knock-in” mice expressing a point mutation on CD36 that substitutes a bulky charged amino acid residue (e.g. Asp) for Thr 92 to mimic phosphorylated CD36 would be also useful.

With such transgenic mice, we could study the effect of CD36 phosphorylation on angiogenesis in vivo.

Our results support that dephosphorylation of CD36 ecto-domain could be an effective way to improve TSR-mediated antiangiogenesis by increasing the binding of TSR to CD36. The dephosphorylation of CD36 ecto-domain could be achieved by two potential methods. The first method would be to inhibit PKC activity in endothelial cells. PKC isoforms are important regulatory proteins in both cancer and endothelial cells 159-160. Therefore, PKC isoforms are therapeutic targets in cancer therapy and many inhibitors of PKC isoforms have been developed 161-162. There is differential expression of PKC isoforms in different tissues and their function can differ based on the signals that activate them 163-165. It would be worthwhile to find out the specific PKC isoforms involved in CD36 phosphorylation in endothelial cells in the future. The second method would be to dephosphorylate CD36 extracellularly by ecto-phosphatases. As shown in our results and previous studies, CD36 can be dephosphorylated by alkaline phosphatase extracellularly 88. A tumor labeling method by using activatable cell-penetrating peptides has been developed in recent years 166. In this method, a fluorescent peptide coupled with a neutralizing peptide via a matrix metalloproteinases-cleavable linker is developed. This cell-penetrating fluorescent peptide is used to label the tumor upon exposure to matrix metalloproteinases secreted by tumor tissue. With a similar strategy, it is possible to develop an activatable phosphatase coupled with an inhibitory peptide via a matrix metalloproteinases-cleavable linker. Such a phosphatase

97 would be expected to be activated in and around tumor and dephosphorylate

CD36 on nearby endothelial cells. If combined with TSR treatment, the efficiency of antiangiogenesis could be improved. The same strategy could also be applied to increase the local concentration of TSR around tumor by developing an activatable TSR coupled with its inhibitor via a matrix metalloproteinases-cleavable linker. Such a protein is supposed to increase the local concentration of active TSR peptide and therefore inhibit angiogenesis as the inhibitory effect of TSR is dosage-dependent. The potential strategies to improve binding of TSR to CD36 and inhibit angiogenesis are summarized in

Figure 1.

The binding of thombospondin-1 to CD36 not only plays a role in endothelial cells, but also in platelets and tumor cells. Binding of thrombospondin-1 to CD36 induces platelets activation 167 and aggregation 168.

Our results and previous studies 88 have shown that in platelets, phosphorylation level of CD36 is very low. Since platelets are not proliferating cells and have no gene transcription, it is unlikely that CD36 phosphorylation can be induced in platelets by activation of PKC. However, the possibility of inducing CD36 phosphorylation in platelets cannot be completely excluded. In tumor cells, binding of thrombospondin-1 to CD36 induces tumor invasion 48 and cell migration 169. Our results suggest that phosphorylation of CD36 on tumor cells can block the binding of thrombospondin-1. It would be interesting to study the regulation of CD36 phosphorylation and its effect in tumor cells.

In the second part of this thesis, we showed that the mechanism of inhibition of VEGF signaling by TSR is mediated by recruitment and activation of

SHP-1. CD36 and VEGFR2 form a multi-protein complex in vivo. Many proteins, such as SHP-1 128, SHP-2 170, Fyn 43, Vav, PI3K, and spleen tyrosine kinase (Syk)

171, have been shown to be components of this complex. The relationship and the role of each component in this complex need to be studied further. SHP-1 and SHP-2 are small cytosolic tyrosine phosphatases. It has been shown that the targets of SHP-1 and SHP-2 include receptor tyrosine kinases, Src-family kinases, PI3K, and components of MAPK cascade 130, 172-173. Most of the targets of SHP-1 and SHP-2 are also downstream molecules of VEGFR2 signaling in endothelial cells 119. The colocalization of SHP-1 and SHP-2 with these molecules in the same signaling complex suggests that SHP-1 and SHP-2 may modulate these signals in endothelial cells. SHP-1 has been shown to negatively regulate many signaling pathways involving epidermal growth factor receptor 174, interleukin-3 receptor 175, and VEGFR2 176. On the other hand, SHP-2 has a positive effect on signaling through insulin receptor, platelet derived growth factor receptor, and fibroblast growth factor receptor 177. Since our results show that the association of SHP-1 with VEGFR2 is induced by TSR via CD36, it would be interesting to study whether the binding of TSR to CD36 can modulate SHP-1 mediated signaling.

The regulatory mechanism of SHP-2 activity is similar to SHP-1 biochemically 178. Basal phosphatase activity of SHP phosphatases is very low, because the N-terminal SH2 domain blocks the catalytic site 179. The activity of

SHP phosphatases can be induced by the binding of proteins containing phospho-tyrosine 180 or phospho-tyrosine residues on C-terminal of SHP phosphatases to N-terminal SH2 domain to release the catalytic site 130. SHP-1 has been shown to be activated via a tyrosine kinase, c-Src, in endothelial cells

129. The colocalization of tyrosine kinases, such as Fyn, Vav, and Syk, with SHP phosphatases in endothelial VEGFR2 complex suggests that the activity of SHP phosphatases may be modulated by these kinases. The regulation of SHP phosphatases in VEGFR2 complex needs to be further studied.

Activity of SHP phosphatases has also been suggested to be regulated by reactive oxygen species (ROS) 181. Previous studies from our lab show that oxidized LDL inhibits SHP-2 phosphatase activity by inducing ROS in macrophage in a CD36-dependent manner 134. Protein tyrosine phosphatases have a critical cysteine residue in their catalytic site that forms a transient intermediate with the phosphorus atom of the substrate 182. The oxidation of this cysteine residue by ROS forms sulfenic acid and prevents the formation of the intermediate with substrates 183. This oxidation is reversible 184 and it is a regulatory mechanism of protein tyrosine phosphatases in vivo 181. The production of ROS in endothelial cells is mostly mediated by NAD(P)H oxidases

185. VEGF has been shown to stimulate ROS production in endothelial cells via activation of endothelial NAD(P)H oxidases 186. It was proposed that VEGF induces ROS in endothelial cells and thereby enhances and sustains VEGFR2 phosphorylation 187. Our results and previous studies suggest that VEGF- induced ROS in endothelial cells might inhibit SHP-1 activity to enhance

100

VEGFR2 phosphorylation. It would be interesting to study the regulation of SHP-

1 activity by ROS in VEGF signaling in endothelial cells.

The role of the kinases in the VEGFR2 complex is not clear. While most of them are downstream signaling molecules of VEGFR2, some of them have been shown to regulate VEGFR2 phosphorylation. For example, Syk has been shown to promote VEGFR2 phosphorylation in vivo 171. A detailed mechanism of the regulation of VEGFR2 phosphorylation orchestrated by the components in the complex needs to be studied further.

Thrombospondin-1 has been shown to inhibit angiogenesis by both CD36 mediated cell apoptosis 43 and inhibition of VEGF signaling 65. Therefore, it would be interesting to study which pathway is predominant and/or whether it is specific to the prevailing conditions. Based on our observation, we speculate that TSR-induced cell apoptosis, but not the inhibition of VEGF signaling, is inhibited by cell-cell contact. The loss of cell-cell contact by disrupting VE- cadherin activates integrin α3β1 and increases the binding of thrombospondin-1 to integrin α3β1 in endothelial cells 188. While there is no direct interaction between VE-cadherin and integrin α3β1, this regulation is suggested to be mediated by intracellular signaling 188. Disruption of VE-cadherin in endothelial cells may also modulate the apoptotic signaling downstream of CD36 and promote the thrombospondin-1-mediated apoptosis. This raises the question if there is a crosstalk between VE-cadherin and CD36.

While the effects of antiangiogenic molecules in treating cancer patients are disappointing, it is still worthwhile to find ways to improve the current

101 practices, probably by finding new antiangiogenic molecules, optimizing the usage of current medicine, and using combinatorial therapeutic approaches. In this manuscript, we have explained the mechanisms of the antiangiogenic molecule, TSR, and have provided potential methods to improve the efficiency in antiangiogenesis and cancer therapy. These studies help us better understand

TSR-mediated antiangiogenesis and give new directions for improving cancer therapy.

102

Figure 1. The potential strategies to improve binding of TSR to CD36 and inhibit angiogenesis.

Phosphorylation of CD36 on Thr92 blocks TSR binding. The phosphorylation can be induced by PKC intracellularly. The inhibition of PKC can be a potential mechanism to inhibit CD36 phosphorylation, thus improve the responses of TSR mediated by CD36. The ecto-phosphorylation of CD36 can also be removed by phosphatase extracellularly. The improvement of TSR binding to CD36 leads to endothelial cells apoptosis and also attenuates VEGF signaling by recruitment of

SHP-1 to VEGFR2 complex and inhibits cell proliferation, survival and migration.

103

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