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Kaposi's sarcoma associated herpes-virus induces cellular proxi expression to modulate host gene expression that benefits viral infection and oncogenesis
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Kaposi's sarcoma associated herpes-virus induces cellular proxi expression to modulate host gene expression that benefits viral infection and oncogenesis
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KAPOSI'S SARCOMA ASSOCIATED HERPES-VIRUS INDUCES CELLULAR PROX1 EXPRESSION TO MODULATE HOST GENE EXPRESSION THAT BENEFITS VIRAL INFECTION AND ONCOGENESIS by Berenice Aguilar ________________________________________________________________________ A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY (GENETICS, MOLECULAR AND CELLULAR BIOLOGY) May 2012 Copyright 2012 Berenice Aguilar ii DEDICATION I dedicate my doctoral thesis to my wonderful family. They all mean the world to me. They are my strength, my inspiration and the reason I strive to be a better person … iii ACKNOWLEDGEMENTS The writing of this dissertation has been an incredible journey and a monumental milestone in my academic life. I could not have embarked on this journey and traveled this far without the continued support of my incredibly patient and kind advisors, colleagues, friends and family. It was a true group effort. When I embarked on this journey, I had no idea the extent of the commitment I was making not only for myself but for those who became part of my life as a graduate student. It all started with the person who took a chance on me, unaware of the challenges this commitment had in store for him. To my Ph.D. advisor Dr. Young-Kwon Hong, I would like to express my heartfelt gratitude. Dr. Young-Kwon Hong has been a major pillar of my professional development at USC during my doctoral training. Dr. Hong took it upon himself to transform my curiosity and interests into learning opportunities and skills that have prepared me well for the next challenge in my professional career. Dr. Hong has been extremely patient with me not only during the time it took me to develop the fundamental skills needed to effectively execute my experimental procedures, but also during those times my personal failings compromised my work in the lab. For his commitment to me, patience, support and kindness during my doctoral training, I'm deeply grateful to my advisor Dr. Young-Kwon Hong. My thesis committee also played a critical role in my development as a doctoral candidate and as a professional. These wonderful advisors are Dr. Michael R. Stallcup, Dr. J.H. James Ou, and Dr. Yves A. DeClerck. I could not have done it without them. Their generous support and guidance has been instrumental in strengthening the objectives of my projects and in unveiling new perspectives that have only improved my iv understanding and the purpose and reach of my thesis projects. They helped to enable and shape my work while continuing to provide firm support and constructive feedback along the way. For that, I will always be grateful. Dr. Stallcup has been a true inspiration for me, he has not only been supportive of my work but has also been a helping hand during those critical times I doubted myself. His wisdom and kindness steered me through some very challenging times during my formative years as a Ph.D. candidate and has generously extended his counsel and warm friendship to the development of professional career. Dr. J.H. James Ou and Dr. Yves A. DeClerck enlightened me in so many aspects throughout my journey, I cannot even begin to list all the valuable lessons I learned from them. They provided the expertise I needed to help me develop new perspectives regarding all aspects of my thesis projects. They have been wonderful advisors and I cannot thank them enough for the time and advice they provided for my development as a research professional. Although Dr. Debbie Johnson is not in my thesis committee, she has also been a wonderful mentor to me. I have been extremely fortunate to have had Dr. Johnson's support and guidance during my doctoral training. Debbie has been an inspiration and a role model whom I hope to emulate in my future career endeavors. I have no words to describe how important to me the aid all of these wonderful people gave me was; all I can say is that because of them, I have a brighter and more fulfilling future ahead of me. I will forever cherish the advice and the time I shared with them. Equally important to my development as a doctoral candidate are my wonderful and supportive lab mates. They have all been very generous with their time and assistance. I would not have finished important experiments on time without their help and advice. Dr. Inho Choi has been an inspiration and a person I will try my best to v imitate during my postdoctoral training. The commitment to his work and self- development is a true inspiration. Dr. Sun Ju Lee, Dr. Dongwon Choi, Dr. Yong Sun Maeng, Dr. Yong Suk Lee, Ha Neul Lee, Hee Kyong Chung, Eun-Kyung Parker, and Kyu Eui Kim have all been very generous with their time, knowledge and help. I'm very grateful to all since during my training, regardless of how busy they all were they always availed themselves to help me in any way they could. I have deep respect for all of them and I'm truly grateful for all they have done for me. I feel very fortunate to have met them all and will never forget their kindness and help. Finally, the support and friendship I found in my PIBBS sisters was incredibly important for my survival during my doctoral training. In Helty, Vivian, Jennifer, Kim and Nikki, I found the support and strength to keep trying my best despite the fact that at times my best was not enough. They gave me the courage to endure and believe that I could finish the journey we all started together. In them, I have found lifelong friends that I will forever cherish. vi TABLE OF CONTENTS DEDICATION………………………………………………………….………………..ii ACKNOWLEDGEMENTS.............................................................................................iii LIST OF FIGURES.........................................................................................................vii LIST OF TABLES..........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER I: DEVELOPMENT OF EMBRYONIC CIRCULATORY SYSTEM....1 Vascular Development............................................................................................1 Lymphatic Development.........................................................................................8 Molecular Interplay in Cell Reprogramming…………………............................17 CHAPTER II: HISTORY OF KAPOSI'S SARCOMA...............................................19 Kaposi's Sarcoma..................................................................................................19 Kaposi's Sarcoma-Associated Herpesvirus...........................................................23 Origin of KS Spindle Cell.....................................................................................26 CHAPTER III: PROX1'S ROLE IN KSHV-MEDIATED ONCOGENESIS….......33 KSHV Selectively Represses the Expression of RGS4........................................33 RGS4 Is Predominantly Expressed In BECs…………………............................37 PROX1 Down-Regulates RGS4 Promoter Activity……..………………….......39 PROX1 Represses RGS4 at the Promoter Level..................................................41 KSHV Down-Regulates RGS4 Through the PROX1-LRH1 Complex…….......48 RGS4 Attenuates vGPCR-Mediated Activation of Akt………………………...51 RGS4 Antagonizes vGPCR Tumor Formation………………………….….......54 CHAPTER IV: CONCLUSION....................................................................................57 CHAPTER V: MATERIALS AND METHODS.........................................................62 REFERENCES…...........................................................................................................66 ALPHABETIZED BIBLIOGRAPHY..........................................................................81 APPENDICES.................................................................................................................96 Appendix A: Supplementary Table 1...................................................................96 Appendix B: Supplementary Table 2...................................................................98 vii LIST OF FIGURES Figure 1: Schematic representation of lymphangiogenesis.......................................................11 Figure 2: Schematic representation of the blood vascular system and lymphatic system.........16 Figure 3: Kaposi's Sarcoma nodular lesions of the skin and oral cavity...................................20 Figure 4: Kaposi's Sarcoma tumor histology.............................................................................22 Figure 5: Schematic representation of KSHV infection and reprogramming of BECs.............30 Figure 6: RGS4 is down-regulated in BECs by KSHV infection..............................................36 Figure 7: RGS4 is predominantly expressed in BECs over LECs.............................................38 Figure 8: Regulation of RGS4 by PROX1.................................................................................40 Figure 9: RGS4 promoter analysis ............................................................................................43 Figure 10: RGS4 promoter analysis with wild type PROX1.....................................................44 Figure 11: RGS4 promoter analysis with PROX1-NR1/NR2 binding mutant .........................45 Figure 12: RGS4 promoter analysis with PROX1-DNA binding mutant.................................46 Figure 13: PROX1 down-regulates the RGS4 expression by binding to it promoter...............47 Figure 14: Cooperative regulation of RGS4 by KSHV-upregulated PROX1 and LRH1.........51 Figure 15: RGS4 inhibits cell proliferation, migration, VEGF secretion and Akt activation...53 Figure 16: Inhibition of vGPCR-mediated tumor formation by RGS4.....................................55 Figure 17: Working hypothesis of KSHV-mediated inhibition of RGS4 via PROX1..............60 viii LIST OF TABLES Supplementary Table 1: KSHV regulation of RGS genes in infected BECs............................79 Supplementary Table 2: KSHV regulation of nuclear receptors in infected BECs...................80 ix ABSTRACT Kaposi Sarcoma (KS) is the most prevalent neoplasm within HIV-infected patients and transplant recipients. Kaposi's Sarcoma-Associated Herpesvirus (KSHV) causes the disease by using a novel mechanism that reprograms endothelial cells making them susceptible targets for viral infection and dissemination. We and others reported that KSHV induces lymphatic differentiation of blood vascular endothelial cells (BECs), by inducing PROX1 up-regulation. Importantly, KSHV G-protein coupled receptor (vGPCR) has been identified as the major viral gene responsible for cellular transformation and disease maintenance. Given that PROX1 is an important mediator of KSHV-induced cell reprogramming, we set out to determine if it had other functional implications in KS pathogenesis. In this study, we report that the regulator of G-protein signaling (RGS)-4 is selectively expressed in BECs and not in LECs, and acts as a cellular agonist against the transformation function of vGPCR. In effect, we found that RGS4 is able to suppress cell proliferation, migration, VEGF-expression and activation of vGPCR-expressing cells in vitro. In accordance, RGS4 significantly antagonized vGPCR-induced tumor growth in two models of immune-deficient mice with reduced tumor-associated angiogenesis. Finally, we demonstrate that KSHV-up-regulated PROX1 and LRH1 cooperate to repress RGS4 expression in KSHV-infected BECs. Together, our study identifies a novel viral strategy that hinders a major host-GPCR regulatory mechanism that may function as an inhibitor for vGPCR activity. Based on our data, we propose a novel hypothesis that KSHV obstructs RGS4-mediated host inhibition of vGPCR transforming activity by up-regulating its transcriptional repressors, PROX1 and LRH1, in order to facilitate KS tumorigenesis. 1 CHAPTER I: DEVELOPMENT OF EMBRYONIC CIRCULATORY SYSTEM Vascular Development Organogenesis describes a sequence of events that transforms a formless cell mass into extremely sophisticated and complex organ systems within the developing embryo. The generation of entire organ systems from an ill-defined cell mass is an amazing miracle of nature that becomes more astounding as we gain a better understanding of the process. It is staggering to realize that this life giving process is set off by a single signaling event that upon initiation triggers a multitude of signaling cascades that take off like a set of falling dominos after the first domino falls into place. Once initiated, this remarkable process faithfully triggers the induction of precise and intricate consecutive molecular programs designed to communicate instructions resulting in cell proliferation, migration, differentiation, organization and integration of complex and highly developed organ systems. However, this process cannot take place without the existence of the now especially controversial embryonic stem cell. Embryonic stem cells are greatly priced due to their pluripotency and self-renewing capacity and highly controversial because they are of embryonic origin with the capacity to give life and form complete organisms. In effect, these remarkable cells are the essential component of the inner cell mass of the embryonic blastocyst (10, 12). Due to its pluripotent nature, these cells can differentiate into any and all of the specialized cell types found in a complete organ system. In vertebrate embryos, the cardiovascular system is the first functional organ system to develop (23, 44). The drive for the early onset of embryonic vasculogenesis is the necessity of a conduit system to supply nutrients and eliminate waste byproducts from the rapidly 2 developing embryo. However, more than a mere transportation network, the vasculature serves a more fundamental role; it facilitates essential signal exchange between distant developing tissues resulting in organ formation and integration of the various organ systems within the organism (44). In essence, vascular development relies on the meticulous orchestration of extremely sophisticated series of molecular programs that are initiated in a systematic, sequential and highly reliable manner (8, 13, 23). The fundamental building block of vascular structures is the mesoderm-derived endothelial cell. Once proper instructions are received, endothelial cells accurately erect the walls of vascular vessels and faithfully safeguard vessel-barrier integrity (6, 13). Although formation of the vascular walls is imperative for proper organ function, another critical function of endothelial cells within the vasculature is to detect and relay environmental signals in order to effectively communicate with neighboring or distant cells groups (20, 25, 32). Notwithstanding, far from being the inert building blocks that were once believed to be, endothelial cells are clearly interactive receivers, integrators, and transducers of information that is fundamental for organ development and function (33). Furthermore, essential for their effectiveness is their ability to dramatically alter their shape and behavior to adapt to new and challenging environmental conditions. To this end, endothelial cells meticulously safeguard all of their responses including cell proliferation, migration and morphogenesis (23, 33). Vasculogenesis, angiogenesis and arteriogenesis are the three processes that give rise and shape to the vascular system. The assembly of the vasculature begins with vasculogenesis involving de novo organization of blood vessels by in situ differentiation of endothelial cells from the mesoderm, making this process uniquely embryonic (9, 11). 3 This rudimentary blood vessel development occurs in the absence of hematopoiesis and it begins by orchestrating the aggregation of de novo forming angioblast into a network of simple endothelial pipelines (11, 23, 32). Despite being the simpler of the two processes, organs such as the lungs and spleen are known to be generated by vasculogenesis. Angiogenesis, on the other hand, takes advantage of pre-existing blood vessels and extensively remodels and refines them into mature vascular vessels (33, 36). In comparison to vasculogenesis, angiogenesis is a more elaborate process involving cell proliferation, polarization, sprouting, and branching of pre-existing vessels, and the process known to generate the brain among other organ systems (32, 36). Unlike vasculogenesis, angiogenesis occurs throughout the life of the organism as the main source of vessel formation and repair system during injury or pathogenesis. The final touch of vessel maturation is provided by the process of arteriogenesis. Arteriogenesis further remodels arterial vessel diameter in response to physiological stimulation such as blood flow increase or shear stress. Although we have accumulated a wealth of knowledge on the molecular mechanisms engaged in vascular development, we still lack a complete picture of all the molecular players and events involved in this vital and highly sophisticated process (33, 36, 42). Admittedly not all of the molecular details are known of how vasculogenesis begins, nevertheless, substantial evidence points to the Indian Hedgehog signaling activation as the signaling event that initiates vascular and hematopoietic cell-fate commitment from the primitive mesoderm at mouse embryonic day E8.5 (8, 11, 34). While the nature of upstream signaling is species-specific, the immediate downstream effect appears to be highly conserved (12, 15). All signaling converge to induce the 4 expression of tyrosine kinase receptors for the vascular endothelial growth factor (VEGF), VEGFR2/KDR (Flk1) and VEGFR1 (Flt1). During this process, VEGFR2 (Flk1) appears to be the earliest detectable marker of the developing endothelial cell lineage (32, 42). In effect, VEGFR2 (Flk1) expression appears to highlight the distinction between the outer-endothelial cells and the enclosed hematopoietic cells (42), establishing the hemangioblast as the progenitor of the hematopoietic and endothelial cells (20, 23). The significance of VEGFR2 expression during early embryonic development has been surmised by gene knockout studies which show that VEGFR2-/- mice fail to develop blood islands throughout the mouse embryo and yolk-sac, which is the primary task of vasculogenesis. Not surprisingly, failure to initiate vasculogenesis results in mouse lethality between embryonic day E8.5 and E9.5 (23, 45). These findings demonstrate the critical function VEGFR2 and its ligands have on the onset of vasculogenesis. To date, here are four ligands known to bind VERGFR2, namely VEGF-A, VEGF-C, and VEGF- D. However, it appears that from these four ligands, VEGF-A is the main ligand responsible for initiating vessel formation and for setting in motion the chain of molecular and cellular events that culminate in the development of a mature vascular network (20, 23, 44, 45). The significance of VEGFR1 is mostly seen during angiogenesis. VEGFR1 seems to be essential for the proper remodeling of the primary vascular plexus during angiogenesis. In effect, a single allele mutation in VEGFR1+/− mice results in large, dilated vessels in the yolk sac and throughout the embryo instead of a plexus of smaller and more refined vessels (45). Not surprisingly, complete ablation of VEGFR1-/- results in embryonic lethality between E8.5 and E9.5 (44, 45). Although these VEGFR1 5 deficient mice are able to develop blood islands, they fail to properly organize them and as a consequence endothelial cells end up in inappropriate locations such as the lumen of blood vessels (45). Nevertheless, the most critical effect of VEGFR1 ablation is the lack of vessel remodeling and consequently organization of the secondary vascular plexus required for angiogenesis fails to occur (46, 47). Admittedly, angiogenesis is by far better understood than vasculogenesis. In effect, several angiogenic factors have been identified to date. These factors include VEGF- ligands A, B and C; Transforming Growth Factor-Alpha (TGF- α); Interleukin-8 (IL-8); Fibroblast Growth Factor-1 and -2 (FGF-1 and FGF-2); PDGF; and Hepatocyte Growth Factor (HGF) (20, 44, 45, 47). The function of VEGF-A in angiogenesis appears to be similar to the role it plays in vasculogenesis, namely to initiate and regulate the entire angiogenic process. Furthermore, in vitro studies show that VEGFs main functions are to promote migration, proliferation and tube formation in endothelial cells (45, 47). However, it seems that each VEGF family member has differing capabilities in promoting these functions. For instance, a single allele deletion of VEGF-C results in mouse embryonic lethality between E11.5 and E12.5 from ubiquitous vascular defects (47). These defects include abnormally large vessels in the yolk sac and within the embryo, lack of vitelline vein fusion with yolk sac vessels, failure to promote vessel growth from the perineural plexus to the brain neuro-epithelium, and tissue necrosis (47, 48). Furthermore, complete ablation of VEGF-C-/- causes embryos to exhibit a number of developmental anomalies, among them malformations of the heart, rudimentary dorsal aorta and a reduced number of nucleated red blood cells in the yolk sac (20, 23, 47). These observations indicate not only the importance of VEGF-C during angiogenesis but 6 that a specific threshold level of VEGF-C is required to maintain angioblast differentiation. Interestingly, it has been document that organs developed by the process of vasculogenesis exhibit higher VEGF-C expression levels that those organs developed through angiogenesis (20, 23, 44, 47). Other important angiogenic molecular modulators are the tyrosine kinase receptorsTie-1 and Tie-2 and their ligands Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Like VEGF receptors, Tie receptors are also specifically expressed on endothelial cells and similarly, Tie-1 and Tie-2 receptor mutant mice undergo severe vascular malformations and embryonic lethality or death soon after birth (44, 47, 51). In effect, Tie-1 receptor deficient mice die between embryonic day E13.5 and post-partum P0 while Tie-2 receptor mutants perish between embryonic day E9.5 and E10.5 (23, 34, 47). Furthermore, a detailed analysis of Tie-2 receptor deficient embryos shows that the heart malformations are a consequence of the lack of interaction between the endocardium and the myocardium (48). Furthermore, these embryos present abnormally large vessels in the extra-embryonic yolk sac and in the vasculature throughout the embryo. More importantly, it appears that the malformations of the vasculature prevent physiological gas exchange thus causing tissue necrosis throughout the embryo (34, 47). Notwithstanding, it has been shown that complete ablation of Ang-1, the Tie-2 ligand, presents a phenotype equivalent to Tie-2-/- mouse embryos (58). Intriguingly, it has been determined that in addition to Ang-1, Ang-2 also has a specific affinity for theTie-2 receptor. However, instead of activating the receptor, Ang-2 acts to block Ang-1 from binding to Tie-2 thus blocking its activity. In effect, mutant mice over-expressing Ang-2 7 exhibit a phenotype corresponding to the Tie-2 or Ang-1 deficient mice (58, 61). This receptor-ligand system perfectly demonstrates the sophisticated means in by which careful orchestration of the various molecular mechanisms and environmental cues important for angiogenesis are regulated (58, 61). Recent studies have also identified the Ephrin-B (Eph-B) receptor tyrosine kinase family and ligands, the Ephrins, as yet another receptor-ligand system important for angiogenesis (58, 60). What is interesting about this receptor-ligand system is that the ligand as well as receptor is membrane bound and binding requires cell to cell contact. This in turn suggests that bi-directional signaling between cells bearing receptor and ligand take place upon binding, a mechanism that is not fully understood. In fact, several research groups have identified specific expression patterns for the Eph- receptors and ligands (58, 60). For example, the ligand Eph-B2 is specifically expressed in arterial endothelial cells, while its corresponding receptor Eph-B4 is specifically found in venous endothelial cells (60-63). The importance of this expression specificity has been shown by Eph-B2 knockout mouse models. These studies have shown that complete ablation of Eph-B2-/- in mice leads to significant vascular defects similar to the vascular defects seen in Ang-1-/- or Tie-2-/- receptor deficient mice (58, 60-63). The interesting implication from these studies being that arteries and venous vessels might be genetically specified and that physiological stimulation received during angiogenesis only influence vessel development in a minor way. In essence, these suggest that cells might not be as plastic as we are likely to believe they are (58, 63). As previously stated, the process of angiogenesis is not limited to embryonic vascular development. It is now well documented that angiogenesis is induced by many 8 pathological conditions, such as chronic inflammation, wound healing, and tumorigenesis. In fact, a great wealth of knowledge has accumulated on the process of tumor-induced angiogenesis since the discovery that tumors are angiogenesis dependent (60-63). For instance, several groups have clearly shown the tumor capacity to switch to an angiogenic phenotype by releasing specific angiogenic growth factors and that this tumor-induced angiogenesis is no different than what is defined as a normal angiogenic process (65). In effect, a comparison analysis between healing wounds and growing tumors in terms of stroma composition revealed their multiple similarities and concluding that tumors are simply wounds that never heal. Moreover, there is also substantial evidence that a net balance of promoters and blood vessel growth inhibitors are required for normal angiogenesis to take place and only when there is an imbalance, the process is abnormally regulated inducing pathology (65, 68, 70). Thus important regulators of embryonic angiogenesis play very similar roles in angiogenesis in the adult under pathological conditions. However, the precise molecular mechanism and specific roles played in physiological and pathological angiogenesis are still not well defined. Lymphatic Development The lymphatics complement the blood vascular system making them an essential component of the vertebrate vasculature. The primary functions of Lymphatic vessels are to return fluid and macromolecules from the interstitial space to the bloodstream, absorb dietary fatty acids from the gut, and traffic immune cells from the tissues to the lymph nodes (74, 79). Although the lymphatic and blood vascular system rely on each other for the maintenance of tissue homeostasis, they develop and are structurally different. In the 9 mouse, lymphangiogenesis begins when a population of endothelial cells on the dorsolateral walls of the cardinal veins adopts a lymphatic endothelial cell (LEC) phenotype (74-76). The lymphatic phenotype specification is set off by the expression of specific transcription factors that act to reprogram the identity of endothelial cells into that of LECs at embryonic day E9.5 (18, 70, 74, 75). Once specified, LEC precursors proliferate, migrate from the veins to the mesenchyme, and go on to form the primary lymphatic sacs between embryonic days E9.5 and E11.5 (Figure 2) (63, 76, 77). As with vasculogenesis and angiogenesis, lymphangiogenic cell sprouting requires a VEGF receptor-ligand complex, namely VEGFR3 and ligand VEGF-C (33, 35). In effect, lymphangiogenesis is tightly regulated by transcription factors, co-receptors, secreted inhibitors, and alternate ligand combinations (27, 60). The transcription factors critical for LEC fate reprogramming are Sox18, Prox1, and Coup-TFII. Furthermore, recent in vitro and in vivo zebrafish studies have implicated the Notch signaling pathway in regulating Prox1 during lymphangiogenesis. Prospero-related homeobox domain 1 (Prox1) is a homeo-domain transcription factor known to play critical roles in the development of diverse organ systems including the lymphatics. Studies using various vertebrate model systems have shown that the mechanism used by Prox1 to regulate developmental cell-fate decisions is an evolutionary highly conserved mechanism. For instance, while in the Drosophila Prox1 homolog, prospero, critically regulates neural cell fate specification; in mammals Prox1 drives the differentiation of eye lens, retina, and hemopoietic compartment (18, 28, 74). Prox1 was identified as the master control transcription factor for LEC specification by studies that targeted Prox1 inactivation. These studies revealed that 10 Prox1 could serve as a marker not only for a polarized subpopulation of endothelial cells within the dorsolateral walls of the anterior cardinal vein but also for a population of sprouting endothelial cells. Prox1 expression is later restricted to the lymphatics during lymphangiogenesis. In effect, a detailed expression analysis has shown that Prox1- positive cells are true LECs. Early in lymphangiogenesis, endothelial cells in the developing cardinal vein display blood vascular endothelial cell (BEC) markers in addition to makers for LECs. LEC makers in the developing cardinal vein include lymphatic vessel endothelial receptor (LYVE)-1 and VEGFR3 (Figure2) (12, 13). LYVE-1 is a homolog of CD44+, a receptor for hyaluronan, while VEGFR3 is a lymphatic specific receptor that binds to ligands VEGF-C and D (15, 16). 11 Figure 1. Schematic representation of lymphangiogenesis. Lymphangiogenesis begins when at mouse embryonic day E9.5 to E10.5, an unknown signaling induces transcription factor Sox18 to stimulate Prox1 expression in a very specific subset of venous endothelial cells. Once Prox1-expression is fully established, these cells begin to bud-off and migrate out to form the initial lymph sacs. These budding lymphatic precursors gradually change their gene expression profile to the mature lymphatic phenotype by inducing expression of additional lymphatic-specific markers, while inhibiting expression of BEC-specific markers. The Prox1-negative venous endothelial cells continue to lose expression of LYVE-1 and VEGFR3 and further differentiate into mature blood vessels establishing the two major vascular systems, namely the blood vascular and lymphatic systems. 12 Interestingly, at mouse embryonic day E9.5 to E10.5, an unknown signaling event induces the transcription factor Sox18 to stimulate Prox1 expression in a very specific subset of venous endothelial cells (Figure 2). Once Prox1-expression is fully established, these cells begin to bud-off and migrate out to form the initial lymph sacs (17, 18). These budding lymphatic precursors gradually change their gene expression profile to the mature lymphatic phenotype by inducing expression of additional lymphatic-specific markers including the secondary lymphoid chemokines (SLC/CCL21) and neuropilin-2; while inhibiting expression of BEC-specific markers such as CD34+, laminin, and collagen type IV (10, 11, 18-20). The Prox1-negative venous endothelial cells continue to lose expression of LYVE-1 and VEGFR3 and further differentiate into mature blood vessels, in effect establishing the two major vascular systems, namely the blood vascular and lymphatic systems (10, 11, 18-20). However, the process can only take place after induction of Prox1 expression. Prox1 requirement was assessed by knockout mouse studies which showed that complete ablation of Prox1-/- critically impaired lymphangiogenesis resulting in embryonic lethality between embryonic day E (12, 13). In effect, Prox1-/- deficient mice were found to be devoid of lymphatic vessels showing massive edema throughout the embryo (76). Although endothelial cells could sprout from the dorsolateral walls of the cardinal vein, Prox1-/- deficient endothelial cells retained expression of BEC markers and failed to stimulate expression of LEC-specific markers (75). In effect these studies revealed that although Prox1 is not critical for initiating endothelial cell sprouting, it is imperative for LEC specification and organization of mature lymphatics. Furthermore, it was also discovered that VEGF-C-/- deficient mice could not undergo dorsolateral sprouting 13 despite the fact that Prox1 was properly expressed (35). In essence, these studies revealed the critical and distinct roles Prox1 and VEGFC play in cell specification and sprouting, respectively. Furthermore, several lines of evidence show that Prox1 is not only necessary but sufficient to induce LEC specification in endothelial cells. For instance, a study of forced expression of PROX1 by an adenoviral vector showed that PROX1 could effectively induce expression of lymphatic marker including Podoplanin, VEGFR3, and Nrp2, while inhibiting expression of BEC-specific markers including neuropilin-1, E-selectin and laminin (26, 58). Furthermore, an in vivo model which induced ectopic expression of Prox1 via a Tie1 promoter showed expression of LEC markers in the blood vasculature confirming the critical role PROX1 plays in LEC-fate specification (38). Despite its role in fluid homeostasis, the lymphatic system has been historically neglected by the scientific community. This indifference is not for the lack of awareness to the system since Hippocrates first described it as the "white blood vessels" around BC400 (19). However, no one took notice of it until 1627, when the Italian anatomist Gasper Asellius reintroduced it, thus calling attention to this forgotten system (78). This past decade has been the most productive period in the history of lymphatic research (10, 11). During this period, the first lymphatic specific markers such as LYVE-1, podoplanin, Prox1 and VEGFR-3 were identified and characterized (12-14, 16, 63). The first lymphatic specific growth factors, VEGF-C and -D, were described and their essential roles in lymphangiogenesis defined (79-83). The master control gene for lymphatic development, Prox1 was identified (13, 84). Moreover, defined cultures of BECs versus 14 LECs were established and successfully propagated without compromising their lineage- specific phenotypes (85, 86). Combined with advances in microarray technology, this facilitated genome-wide comparisons of transcriptional profiles of human dermal BECs versus LECs from the same donors (22, 87, 88). In addition, recent studies of the transcriptome of uncultured, freshly isolated BECs and LECs along with peptide mapping of endothelial surface molecules provided improved snapshots about the expression of endothelial lineage- specific markers (89-91). Furthermore, the genome-wide analyses revealed that embryonic programs for endothelial cell differentiation could be recapitulated in post- developmental settings. In effect, Prox1 was shown to reprogram post-developmental BECs into adopting lymphatic phenotypes by modulating a significant fraction of genes reported to be differentially expressed between the two cell types (21-23, 87, 88, 92). Similar to the vasculature, endothelial cells are the essential constituent of the lymphatic system (10, 11). Despite the fact that the blood vasculature and the lymphatics have a common progenitor and make use of very complex networks for a common interest, namely tissue perfusion and fluid circulation, they have very unique ways of accomplishing this task due to their specialized anatomical structures. The blood vascular system is a closed circular network, where blood leaves the heart, flows through arteries into tissue capillaries and veins to finally return back to the heart (Figure 3) (13). In contrast, the lymphatic system is a blunt-ended linear system, which begins at the interstitial spaces of peripheral tissues and organs and end at the thoracic duct, which is the thickest lymphatic vessel that connects to the left subclavian vein (56). This specialized network is designed to allow extravasated cells, fluids and large molecules 15 (collectively called lymph) that end up in the periphery to drain back into lymphatic capillaries for recirculation through the vascular system. In effect, the primary function of the lymphatic system is to control tissue-fluid homeostasis, absorption of lipids and large molecules in the digestive system, and trafficking of immune cells to regional lymph nodes (10, 11). Unfortunately, due to its accessibility to the various organs, the lymphatic system also presents an avenue for malignant tumors dissemination. 16 Figure 2. Schematic representation of the blood vascular system and lymphatic system. While the blood vascular system is a close circular network, the lymphatics are comprised of open-ended vessels. 17 Molecular Interplay in Cell Reprogramming Embryonic development occurs when pluripotent stem cells undergo cellular differentiation induced by activation of specific molecular mechanisms that stimulate gradual acquisition of complex epigenetic modifications, including changes in DNA methylation and histone-tail modifications. These modifications establish heritable gene expression programs compliant with a specific cell identity (87, 72). Recent studies have established that somatic adult tissue is composed of unipotent as well as multipotent cell types, whose main function is to support growth and repair throughout the lifetime of the organism (67, 82). In effect, the successful generation of animals by somatic cell nuclear transfer experiments revealed that cellular differentiation depends on epigenetic differences between the genome of differentiated and embryonic cells that can be reset by exposure to the oocyte cytoplasm. Furthermore, it was discovered that in essence, cellular reprogramming refers to the rewiring of epigenetic and transcriptional networks of one cell type to that of a different cell type (88). These studies have shown that the reprogramming of cells is effectively achieved through the manipulation of specific molecular mechanisms. For instance, several groups have shown that reprogramming of fibroblasts into myoblast-like cells only requires ectopic expression of the MyoD transcription factor. Furthermore, recent studies show that T- and B-cells can be made to adopt a macrophage-like phenotype by the simple over-expression of myeloid transcription factor CCAAT/enhancer-binding protein (C/EBPα) (76, 91). At the molecular level, the cause of cell reprogramming is presumably a change in the expression of master regulatory genes whose normal function is to distinguish between distinct tissues during embryogenesis (1, 3). In effect, the process of 18 embryogenesis involves cell differentiation from pluripotent stem cells to tissue-specific cells via induction of specific combinations of master switch genes in response to inductive signals. Since transdifferentiation appears to be the result of a single gene modification, it is not surprising to conceive that cell transdifferentiation can only occur between cells that originated from neighboring tissues thus generated by a common progenitor. There are now a growing number of models, both in vitro and in vivo that demonstrates the plasticity of differentiated cells (89, 92). However, there is yet a lot to be determined regarding successful cell reprogramming but is unquestionable the therapeutic potential tissue reprogramming and cell replacement can have for disease therapies. As our understanding of the molecular regulation of tissue development and maintenance increases, the promise of controlling gene expression to influence cell phenotype and tissue reprogramming presents a worthy challenge. 19 CHAPTER II: HISTORY OF KAPOSI'S SARCOMA Kaposi's Sarcoma Kaposi Sarcoma (KS) is a soft-tissue neoplasm distinguished by the prominent nodular lesions in the skin and oral cavity, often disfiguring and radically impacting the quality of life of infected individuals (Figure 4) (71). What is more, late-stage forms are extremely aggressive and extend lesion-dissemination to internal organs such as the lymph nodes, lungs, liver, and GI tract (71,72). KS dissemination to these vital organs critically impairs organ function resulting in high mortality rates among afflicted individuals (67). To date, four different variants of KS have been identified, namely classic, epidemic, endemic and iatrogenic KS (75). Classic KS was the first to be described by the Hungarian dermatologist Moritz Kaposi in 1872. Dr. Moritz's descriptions and observations were fundamental in classifying classic KS as indolent and rare disease that primarily inflicted older men of Mediterranean and Middle Eastern descent (81, 99). Due to its rarity and slow progression, the neoplasm received little attention from the scientific community until the 1980s when an epidemic form of KS rapidly surged devastating a very specific group of individuals (66, 72). In contrast to classic KS, this more aggressive form of KS was physically impairing and often fatal with predilection for young HIV-infected homosexual men. Naturally, with the advent of the HIV epidemic KS became of great concern to the medical professionals and scientists alike. Nevertheless, despite heroic attempts at deciphering the cause of epidemic KS, it was not until 1994 that Chang et al. made news when they reported a novel human herpesvirus-8 (HHV-8) or Kaposi Sarcoma-Associated Herpesvirus (KSHV) to be the offending agent (63, 78). 20 A B C D Figure 3. Kaposi's Sarcoma nodular lesions of the skin and oral cavity. Panels A and B depict classical KS nodular lesions of the skin. Panel A depicts typical skin lesions seen on HIV-infected individuals, while panel B shows the type of lesions that are seen in children of KS endemic countries. Panels C and D show classical KS lesions of the oral cavity. Panel C is a representation of KS tumors normally seen in the mouth, while panel D shows KS lesions on the esophagus. 21 Regardless of the KS variant, the tumors are highly vascularized with extensive neo-angiogenesis, massive number of infiltrated inflammatory cells and prominent vascular slits that promote leakage and inflammation, a hallmark of the disease (Figure 5). However, the most prominent characteristic of this unique tumor is the spindle cell. These cells are the driving force for KS and therefore believed to be the tumor source (Figure 5). Given the role of the spindle cell, naturally scientist focused most of their efforts at elucidating its origin and important molecular mechanisms critical for its oncogenic function (63-68). However, this task proved to be more difficult than anticipated. Since its discovery, controversy has surrounded the origin of these cells. This controversy is not without reason since these cells display a very unique genetic profile. Intriguingly, these cells appear to recapitulate an immature cell-state that hasn't quite made its mind as to a lineage commitment and displays a chimerical identity (71, 74). Besides classical endothelial cell markers, these cells display specific markers for blood endothelial cells (BEC), (including CD31+ and CD34+) as well as lymphatic endothelial cells (LEC), such as LYVE-1 and PROX1 (74-76). As can be surmised, the nature of its genetic profile has made it difficult to ascertain with conviction the endothelial cell origin of these cells. Notwithstanding, the research field began to disentangle the mystery surrounding KS spindle cell identity by gathering evidence to support one cell-specificity versus another thus creating a divide. Since KS spindle cells have been shown to express elevated levels of vascular endothelial growth factor receptor 3 (VEGFR3), LYVE-1 and Prox1, specific lymphatic markers, a group of researchers began to support the hypothesis of a lymphatic lineage origin (72, 75, 78). Not 22 surprisingly, similar evidence was put forward by supporters of a blood endothelial origin hypothesis. Figure 4. Kaposi's Sarcoma tumor histology. Characteristic features of KS are massive infiltration of immune cells, multiple vascular slits, and the proliferative spindle cells thought to be the KS tumor cells. 23 Kaposi's Sarcoma-Associated Herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as the human herpesvirus-8 (HHV8), is a novel human herpesvirus responsible for KS pathogenesis (44, 45). KSHV is the first identified human γ2-herpesvirus with sequence and biological features similar to the human tumor virus Epstein-Barr virus (EBV) and Herpesvirus Saimiri (HVS) (44-46). KSHV has a large linear double stranded DNA in the viral episome; however, upon infection the genome circularizes and forms a double-stranded episome in the nucleus of the host cell without ever integrating with the host genome (44). The estimated genome size is approximately 165 kb, containing well over 90 open reading frames (ORFs) coding for viral proteins (44-46). Not unlike other herpesviruses, KSHV has two distinct phases in its life cycle, namely latency and lytic replication. Latency is its default program and is characterized by the persistent expression of six latent viral genes. Three of these [Latency-Associated Nuclear Antigen (LANA), v-cyclin, and v-Flip] are encoded from one transcriptional unit and the others (Kaposin A, B, and C) are encoded from a second, independent promoter (46-48). As mentioned, KSHV adopts a latent program by default once it enters the host mainly to avoid detection by the host's immune surveillance. Nevertheless, upon viral reactivation, the virus abandons its latency to enter its lytic phase activating expression of most viral genes in a regulated orderly fashion (immediate-early, early, and late), leading to the production of infectious virions and cell death (44, 46, 51). It has been challenging to determine the mode of KSHV transmission since viral DNA has been difficult to detect in human fluids with current detection methods. Sexual contact has had more traction as the potential means of transmission of epidemic KS (48, 24 61). The factors that appear to be important for KSHV transmission to date include promiscuity, history of other sexually transmitted diseases, and HIV-1 in addition to other chronic viral infections. Although sexual contact appears to be an important mode of transmission, semen has not been found to contain KSHV DNA in asymptomatic individuals, as analyzed by current PCR methods (61, 73). As a result, it has been difficult ascertain KSHV transmission between asymptomatic individuals. In addition, there is evidence that KSHV-infected individuals carry low levels of KSHV DNA in their saliva and not surprisingly seen as another mode of viral transmission. In effect, the presence of KSHV in saliva could partially explain the strong dose-dependent association of oral-genital contact with KSHV seroconversion among HIV-negative individuals. In fact, at present there's only limited evidence for sexual transmission of KSHV among heterosexuals or asymptomatic individuals (68,74). Interestingly, KSHV seroprevalence in endemic countries, unlike developed countries, appears to be the same for men as for women, eliminating the infection preference KSHV shows for men in epidemic countries (78). In addition, transmission in childhood appears to account for much of the spread of KSHV in endemic countries. Given that KSHV infection is low in children less than two years of age and that it increases with age argues against transmission through breast feeding and in favor of spread through close contact and shared household utensils (74, 78). In addition, KSHV seroprevalence in Ugandan children have also been correlated with hepatitis B infection. These studies suggest that the living conditions that predispose children to hepatitis B infection can also promote KSHV dissemination. Yet another predisposing factor for KSHV infection is organ transplantation (69, 73, 78, 79). 25 Initial efforts to identify the KSHV gene(s) responsible for the initiation of KS focused primarily on viral latent genes (LANA, v-cyclin, vFLIP, and kaposin) (82). Surprisingly, Montaner et al. found that although several of the latent genes were expected to play an important role in driving KS tumorigenesis, none appeared to be sufficient alone or in combination to initiate endothelial cell transformation (82). Moreover, they found that the viral G-protein coupled receptor (vGPCR), a lytic gene, was not only sufficient but necessary to induce endothelial cell transformation. The KSHV vGPCR is a member of the family of seven transmembrane receptors, CXC chemokine G-protein-linked receptors, with significant homology to human CXCR1 and CXCR2 (2). Unlike cellular GPCRs, vGPCR has a mutation in its second transmembrane helix that renders it constitutively active. Interestingly, vGPCR signaling has been reported to be susceptible to modulation by various host ligands (such as IL-8, Groα, IP-10 and SDF1), and the ability to be modulated may prove to be essential to its pathogenic role in KSHV-mediated disease (88, 91). Different intracellular signaling molecules have been shown to be activated by vGPCR (via the heterotrimeric Gαi, Gαq, and Gα13 subunits), including MAPK, p38, and JNK, which in turn may control the expression of growth-promoting genes (91, 94). In addition, activation of Akt by vGPCR may represent a critical intracellular pathway in the blockade of cell death as this kinase acts on a large number of target molecules involved in the control of apoptotic signals and in the promotion of cell survival (100). Furthermore, it has also been shown that vGPCR induces the secretion of angiogenic growth factors from vGPCR-expressing cells, including VEGF, IL-8, and Groα, suggesting that vGPCR may serve direct and indirect roles in cell transformation (100). 26 Origin of KS Tumor Cells The origin of the KS spindle-shaped cell is controversial due to the fact that these cells histologically resemble a heterogeneous population with distinct phenotypes. While some KS spindle-shaped cells in the tumor express characteristic features resembling activated endothelial cells, other KS spindle-shaped cells bear macrophage and dendritic cell antigens, and yet others express a hybrid of macrophage and endothelial cell markers (100). Despite the inconsistency in the spindle-shaped cell identity, there is some consensus that the origin of these cells is from endothelium based on immunohistochemical and gene expression microarray studies. What is not clear, however, is whether these cells are of vascular or lymphatic endothelial origin. Nevertheless, regardless of its origin, there is no doubt that the spindle-shaped cells represent the KS tumor cells. There are several lines of evidence to support this view: KS spindle cells can generate tumors in immunodeficient mice (95, 100). KS spindle cells can invade reconstituted basement membrane by constitutively producing type IV collagenase. And finally, (3) KS spindle cells can escape apoptosis by over-expressing the anti-apoptotic protein Bcl-2 or Bcl XL (100). Early studies focused on identifying KS spindle cell origin classified them as endothelial cells based on specific expression of Factor VIII-related antigen (FVIIIRA) and Weibel-Palade bodies, as determined by enzymatic and immunohistochemical methods (75-79). As better endothelial cell markers appeared in the 1990s, these only confirmed KS spindle cell endothelial origin. However, as lymphatic specific markers became available, several groups reported important evidence showing these cells resembling an expression pattern that more closely resembled lymphatic tissue than blood 27 vessels (59). This new hypothesis gained further support when other groups performed immunohistochemical analyses on KS biopsies using antibodies against the pan- endothelial EN4 antigen and the BEC-specific PAL-E that is not expressed in LECs, along with other markers (60, 61). KS tumor cells were demonstrated to be positive for EN-4 and UAE-1, but negative for PAL-E and FVIIIRA (60, 61). Furthermore, additional support was provided by anatomical observations in which the unique distribution of KS lesions in the skin and certain viscera was believed to be due to pre- existing lymphatic channels (62). The proposed lymphatic origin of KS gained enormous support in the late 1990s with the arrival of novel lymphatic-specific markers such as LYVE-1, VEGFR-3, podoplanin (antigen for D2-40 antibody) and Prox1 (52-54, 56, 63, 64). These newly identified LEC markers were reported to be prominently expressed in the majority of KS tumor specimens tested (63, 65-68). Moreover, the lymphatic specific VEGF-C was shown to stimulate the migration and proliferation of cultured tumor cells freshly isolated from KS lesions (69). Interestingly, an alternative thought for the origin of KS cells was put forward when aberrant lymphatic-venous connections were reported in the early stage of KS lesions (70, 71). This alternative idea was backed by observations showing that KS tumor cells displayed dual immunohistochemical properties of BECs and LECs. KS tumor cells were alleged to originate from ECs present in the abnormal (lymphatico- venous) junctions between lymphatic vessels and venous capillaries (61, 72-74). Although this interesting idea did not receive additional scientific support due to lack of further evidence, its underlying premise is very similar to that of pathological endothelial plasticity and very closely matches our current view of the origin of KS tumor cells. 28 The dispute over the origin of KS cells was difficult to resolve due to the variability, inconsistency and even irreproducibility of several immunohistochemical studies of KS lesions. These were attributed to variations in embedding and fixation protocols, staining methods as well as the sensitivity of adopted experimental modalities. Moreover, the poor credibility of some the histochemical markers used in earlier studies also made it hard to reach a general consensus on the origin of KS tumor cells. For example, FVIIIRA, thrombomodulin and CD31+ are now known to also be expressed in LECs (75-77). As a result, some twenty years since KS tumor cells was determined to be of EC origin, the question surfaced again as to whether these ECs were BECs, LECs or both. The mode of infection employed by KSHV is extraordinary in that it induces the reprogramming of the transcriptional profile of infected cells to resemble a lymphatic- like phenotype. This atypical mode of infection was initially inferred by our lab and others when it was discovered that blood endothelial cells infected with KSHV were systematically down regulating BEC specific markers while up-regulating those of LEC. KS development is a multi-step process involving viral and cellular factors. There is evidence that infection of spindle cells by the KSHV/HHV-8 is necessary but not sufficient for KS development which points to the necessity of other factors including angiogenic factors, inflammatory cytokines, chemokines and immunodeficiency. In advanced KS lesions, spindle-shaped cells become the predominant cell type and there is evidence to suggest that these are the KS tumor cells. One of the hallmarks of KS tumor cells is the expression of lymphatic endothelial cell (LEC)-markers and because of this, KS tumor cells were thought to be derived from KSHV-infected LECs. However, we and 29 others have shown that KSHV infection reprograms the transcriptional profile of blood vascular endothelial cells (BECs) to adopt a lymphatic endothelial cell (LEC)-like phenotype by inducing the over-expression of Prox1, which is essential in the genetic reprogramming process (Fig.1). KSHV-induced cell fate reprogramming is a novel concept in KS tumorigenesis and the mechanism or pathological benefits of this KSHV- mediated endothelial cell reprogramming event remains to be defined. However, we hypothesize that KSHV-induced Prox1 activates the expression of LEC-specific genes to promote lymphatic differentiation of the KSHV-infected cells, which might create an ideal microenvironment for KSHV infection and disease progression. 30 Figure 5. Current view on KSHV infection and reprogramming of infected BECs. Once KSHV enters the blood stream through various means, (i.e. blood transfusion, syringe exchange, sexual transmission, etc.) the virus has the opportunity to infect the blood endothelial cells lining the blood vessels. Shortly after the virus enters the cell, it induces a multitude of host cellular signaling pathways resulting in the activation and expression of transcription factor PROX1. In turn, PROX1 expression induces expression of additional lymphatic specific markers (including LYVE-1) reprogramming infected BECs to resemble a lymphatic-like phenotype. The infected cells then go on to proliferate and form KS tumor lesions. 31 Unlike other herpesviruses, KHSV employs extensive molecular piracy of critical host cell regulatory genes (101). These pirated viral genes however, are not susceptible to host cellular regulation and are either constitutively active or modulated by viral proteins. Not surprisingly, a group of genes highly altered are those involved in angiogenesis, including VEGFR-1 (Flt1) (95, 100). A comprehensive understanding of how KSHV induces infection and disease progression involves a critical analysis of the genotypic and phenotypic changes altered by viral infection. To this end, several research groups have intensified their efforts in elucidating the global changes in gene expression corrupted by the virus. In conjunction with genomics technology, several groups have reported the modulation of global gene profiles in ECs upon KSHV infection (93-98). Genome-wide analyses of gene expression profiles changed by KSHV infection provide a comprehensive and in depth understanding of KS pathogenesis. Genome-wide comparison of the transcriptome between normal BECs and those that were KSHV - infected revealed that KSHV -infection modulates several groups of genes including those responsible for interferon (IFN)-response, cell signaling, angiogenesis, lymphangiogenesis, cell cycle progression and apoptosis (93-99). These studies showed that KSHV-infection results in host cell reprogramming, in which the expression profile of BECs resembles that of LECs, a process that is highly similar to that of embryonic LEC-differentiation (101). KSHV-infection of BECs significantly up-regulates the expression of Prox1, a gene not usually expressed in normal BECs. KSHV-induced Prox1 has been shown to up-regulate approximately 70% of lymphatic-associated genes including LYVE-1, podoplanin, and VEGFR-3 (101, 102). It appears that Prox1 plays an 32 essential role in KSHV- mediated cell reprogramming (101, 102). KSHV-mediated up- regulation of Prox1 can be observed as early as 2 hours post infection (99). Although the underlying mechanism is not fully understood, the JAK2/STAT3 and PI3K/Akt cell signaling pathways have been implicated in the reprogramming process (103). Although Wang et al found that KSHV can infect LECs as efficiently as BECs (100), a higher copy number of the viral genome is maintained in LECs than in BECs per a given amount of virus particle. This is possibly due to improved KSHV replication, more efficient maintenance of episomal DNA, or increased viral entry into LECs (100). It is known that both cultured tumor cells isolated from KS lesions and mature ECs in vitro infected with KSHV fail to sustain a persistent viral infection (107). These findings raised the possibility that less differentiated ECs, namely circulating EPCs in peripheral blood, may serve as a third type of HHV8-host in addition to residential BECs and LECs. In fact, the proliferative potential, EC-like characteristics and participation of EPCs in post-developmental vascular development have made these cells an attractive host cell candidate for KSHV. KSHV has consistently been reported to be detected in peripheral blood mononuclear cells (PBMC) (108-111). Pellet et al investigated KSHV viral load in PBMCs from KS patients after magnetic cell sorting and found that CD146- positive circulating EPCs harbored the KSHV genome (98). Moreover, a recent study showed that KSHV-infected EPCs could be cultured from peripheral blood of classical KS patients, and then differentiated into mature ECs with serial expansion without losing HHV8 genome (98, 99). These studies strongly indicate that HHV8 may infect circulating EPCs causing them to differentiate into endothelial cancer cells, thereby forming KS lesions. 33 CHAPTER III: PROX1'S ROLE IN KSHV-MEDIATED ONCOGENESIS The discovery that KSHV pathologically hijacks a PROX1-mediated physiological process to lymphatically reprogram BECs has provided a novel insight into the genetic interaction between virus and host cells. We have recently defined the molecular mechanism underlying PROX1-upregulation by KSHV (106). However, the pathological benefit resulted from the PROX1-upregulation and concomitant lymphatic reprogramming remains to be elucidated. More specifically, it would significantly advance our understanding of KS tumorigenesis to discover how PROX1-mediated lymphatic differentiation contributes to endothelial cell transformation. In the current study, we present data showing that KSHV-induced PROX1 prominently promotes the transformation activity of vGPCR by repressing the expression of RGS4, a cellular antagonist for KSHV vGPCR. Furthermore, this finding suggests that the lymphatic microenvironment may be more favorable for KSHV pathogenesis and thus KSHV pathologically induces lymphatic reprogramming of host cells to promote its oncogenesis. In effect, our study provides an important insight towards defining the mechanism and pathological benefits of KSHV-mediated endothelial cell reprogramming in KS tumor development. KSHV Selectively Represses the Expression of RGS4 We and others previously reported that KSHV infection of BECs results in lymphatic reprogramming of the host cells, characterized by PROX1-mediated upregulation of a number of LEC-specific genes (23-26). However, it remains unclear as 34 to why the oncogenic virus goes through the trouble of inducing lymphatic reprogramming of BECs. In order to better understand the pathological benefits of this lymphatic reprogramming of host cells by KSHV, we performed a genome-wide comparative survey to search for endothelial-lineage genes that are commonly regulated by KSHV and PROX1. To this end, we used two independent sets of reported microarray data defining target genes of KSHV (3) or PROX1 (88). This survey identified a group of endothelial lineage genes that may be important for KS tumor development. Among the identified genes, RGS4 was found to be of particular interest for its profound role in regulating the activity of cellular GPCRs. The Regulator of G-protein signaling (RGS) proteins have been found to attenuate the signaling activities of many GPCRs via their action as GTPase-activating proteins (GAP) for Gα subunits, resulting in faster deactivation of Gα (123, 124). In effect, more than twenty RGS family members have been described so far with nine structural classes according to the presence and nature of protein motifs other than the RGS domain. RGS proteins have been shown to regulate a wide range of cellular functions, including cell migration, proliferation and survival. Furthermore, specific studies on RGS4 showed that it could inhibit epithelial and endothelial cell tubulogenesis by effectively inhibiting G- protein and VEGF-mediated activation of MAPK including ERK1/2 and p38. This inhibition in effect resulted in a diminished cell proliferation, migration, and invasion (95). Based on these findings we hypothesize that RGS4 antagonizes various cellular and perhaps viral GPCRs signaling pathways, which may be essential for KSHV pathogenesis. In effect, we found that KSHV significantly down-regulates the expression of RGS4 and that among the over twenty members of the RGS superfamily that is repressed 35 by the virus and known to antagonize the activity of GPCRs (Supplemental Table 1). We further confirmed this KSHV-mediated down-regulation of RGS4 mRNA and protein by quantitative real-time RT-PCR (qRT-PCR) and western blot analyses, respectively (Fig.6A-C). We then asked whether RGS4 down-regulation was limited to KSHV- infected cells, or if it extended to uninfected neighboring cells via a paracrine effect. Immunofluorescent analyses performed on KSHV-infected cultured BECs showed that RGS4 down-regulation was observed only in latency-associated nuclear antigen (LANA)-positive cells and not in neighboring cells (Fig.6D). Together, these data demonstrate that KSHV down-regulates RGS4 at the transcriptional level and that this repression occurs only in KSHV-infected cells, suggesting that viral infection may be required for RGS4 down-regulation. 36 Figure 6. RGS4 is down-regulated in BECs by KSHV infection. Primary human dermal BECs were infected with KSHV for two days and the expression of LANA (A) and RGS4 (B) was determined by quantitative real time RT-PCR (qRT-PCR). More than three independent experiments were performed and results are shown as average ± standard deviation (SD) of one representative experiment done in triplicates. Triple asterisks indicate p < 0.001 against the mock infection control. (C) Western blot analyses confirmed the down-regulation of RGS4 protein in KSHV-infected BECs. (D) Immunofluorescent staining against LANA and RGS4 revealed that RGS4 is down- regulated in only LANA-positive, KSHV-infected BECs (marked with an arrow), but not uninfected neighboring cells. 37 RGS4 is Predominantly Expressed in BECs We next studied the expression pattern of RGS4 in cultured primary BECs and LECs, isolated from neonatal human foreskins. A series of qRT-PCR and western blot analyses revealed that while PROX1 was selectively expressed in LECs, but not in BECs (Fig.7A), RGS4 was predominantly expressed in BECs, but not in LECs (Fig.7B) showing a mutually exclusive expression pattern. To validate these in vitro findings, a human foreskin skin section was stained for RGS4 along with CD31, an endothelial cell marker that is highly expressed in BECs but only weakly in LECs (22, 23). Consistent with our in vitro data, RGS4 was predominantly expressed in the CD31-high blood vessels, and not in the CD31-low lymphatic vessels (Fig.7C-F). Furthermore, we employed the RGS4-promoter-GFP bacterial artificial chromosome (BAC) transgenic mouse (24) to investigate the tissue-specific GFP expression pattern directed by the RGS4 promoter. Indeed, GFP-positive blood vessels in the embryonic back skin of the RGS4-GFP mouse (Fig.7G) were found to be morphologically distinct from the GFP- positive lymphatic vessels of a lymphatic-specific PROX1-GFP BAC transgenic mouse we recently reported (25) (Fig.7H). Moreover, we also confirmed GFP-positive blood vessels in the retina and the trachea of the adult RGS4-GFP BAC transgenic mouse (Fig.7I,J). Taken together, our in vitro and transgenic animal data demonstrate that RGS4 is predominantly expressed in blood vessels and not in lymphatic vessels. 38 Figure 7. RGS4 is predominantly expressed in BECs over LECs. Expression of PROX1 (A) and RGS4 (B) in human primary dermal BECs and LECs was determined by qRT-PCR and western blot analyses (insets). Data are shown in average relative mRNA expression ± standard deviation (SD). More than three experiments were performed. (C- F) Immunofluorescent analyses with DAPI (C), anti-CD31 (D) and anti-RGS4 (E) antibodies on a neonatal human foreskin section show that RGS4 is mainly expressed in CD31-high blood vessels (arrows), but not in CD31-low lymphatic vessels (arrowheads). A merged image (F) is also shown. Bar, 20 µm. (G) Embryonic back skin from the RGS4-GFP transgenic embryo (E15.5) shows blood-vessel specific expression of the GFP reporter. Bars, 50 µm. (H) In comparison, a lymphatic-specific GFP transgenic embryo (E15.5) shows the GFP-positive lymphatic vessels in the back skin. Bars, 20 µm. (I,J) GFP-positive blood vessels in the retina (I) or trachea (J) of RGS4-GFP transgenic adult mouse. Bars, 20 µm. 39 PROX1 Down-Regulates RGS4 Promoter Activity PROX1 has been shown to regulate a number of BEC/LEC-specific genes (26-28). The finding of the BEC-specific expression of RGS4 prompted us to question whether down-regulation of RGS4 by KSHV could be mediated through PROX1 (3-6). We became particularly interested in studying the significant down-modulation of RGS4 due to the fact that RGS4 is a GTPase that inactivates cellular G-protein coupled receptors (GPCR). We wondered if RGS4 was a negative regulator of the viral GPCR, which is an important viral protein capable of cell transformation, and whether KSHV was down- regulating RGS4 to facilitate unperturbed vGPCR activity and therefore KS pathogenesis. Although vGPCR has been reported to be constitutively active, its activity can be circumvented by ligand binding in a positive (IL-8, Gro-alpha) or in a negative manner (IL-10, SDF-1). Given that Prox1 is a major player in KSHV-mediated cell-fate reprogramming, we questioned whether Prox1 had a role to play in modulating RGS4 expression. To answer this question, we adenovirally induced the expression of Prox1 in BECs and found that when we ectopically expressed Prox1 in BECs, RGS4 expression was significantly reduced (Figure 8A). Conversely, when we knocked-down Prox1 expression via siRNA, we observed that RGS4 expression was up-regulated in LECs, which normally do not express RGS4 (Figure 8B). These findings revealed that Prox1 was indeed a negative regulator of RGS4 in endothelial cells. In order to determine the pathological significance of this finding we decided to investigate Prox1-mediated repression of RGS4 in the context of KSHV infection. For this experiment, we pretreated BECs with siRNA against Prox1 or scramble and either infected or not these cells with KSHV. To our delight, we found that the KSHV-mediated down-regulation of RGS4 40 was abrogated by Prox1-siRNA pretreatment (Figure 14A). Thus suggesting that in deed Prox1 could modulate RGS4 expression in a negative manner. Figure 8. Regulation of RGS4 by PROX1. (A) qRT-PCR analysis of primary BECs adenovirally-induced to expressed PROX1 show down-regulation of RGS4 mRNA levels in BECs. (B) Expression of RGS4 mRNA in primary LECs after 48-hr treatment with SiRNA duplex against fire-fly luciferase (siCTR) or PROX1 (SiProx1) show an up- regulation of RGS4 mRNA levels in these cells. 41 PROX1 Represses RGS4 at the Promoter Level In determining the mechanism by which Prox1 represses RGS4 expression in BECs, we decided to investigate the RGS4 promoter for Prox1 putative binding sites. Given that Prox1 consensus sequence is loosely defined, we found many Prox1 biding sites throughout the RGS4 promoter. To investigate whether or not Prox1 could modulate RGS4 expression at the promoter level, we decided to perform Luciferase reporter assays using RGS4 promoter constructs of various sizes (Figure 9). We transfected NIH 3T3 with pcDNA (control) or wild type Prox1 in addition to the various RGS4 promoter constructs and determine luciferase activity. As expected, we found that the expression of Prox1 markedly reduced luciferase activity in all RGS4 promoter constructs including our shortest construct (0.3kb) when compared to our control (Figure 10B). Since Prox1 has been reported to bind various nuclear receptors via its NR1/NR2 binding motif, we wanted to know if Prox1 was inducing RGS4 promoter repression alone or in collaboration with other nuclear receptors. To determine if Prox1 was repressing RGS4 promoter activity in concert with other nuclear receptors, we obtained a Prox1 NR1/NR2-binding mutant and performed luciferase reporter assays (Figure 11A). We found that although Prox1 could not interact with any of its nuclear receptor binding partners via its NR1/NR2 binding motif, it could still repress RGS4 promoter activity (Figure 11B). This finding was highly suggestive that Prox1 alone could modulate RGS4 promoter activity. We next wanted to investigate whether or not physical interaction between Prox1 and RGS4 promoter was necessary for RGS4 promoter repression. We obtained a Prox1 DNA-binding mutant and found that Prox1 physical interaction with 42 RGS4 promoter was necessary for Prox1-mediated repression of RGS4 promoter activity (Figure 12). In addition, we further investigated the molecular interaction between recombinant PROX1 protein and the RGS4 minimal promoter by performing an electrophoretic mobility shift assay (EMSA), and found that PROX1 indeed binds to the RGS4 promoter (Fig.13). To corroborate this molecular interaction, we carried out PROX1-chromatin immunoprecipitation (ChIP) against the RGS4 promoter region using cultured human primary LECs (in vitro) and also mouse whole organ lysates prepared from the brain and intestine (Fig.13B). Both in vitro and in vivo ChIP assays confirmed that the PROX1 protein was physically associated with the RGS4 promoter regions in cultured human LECs and in mouse brain and intestine cells. Taken together, these data demonstrate that PROX1 binds to the promoter of RGS4 and directly represses its transcription. 43 Figure 9. RGS4 promoter analysis. Several RGS4 promoter constructs ranging from full length (3,400 base pairs) to minimum promoter length of 300 base pairs were used to determine functional PROX1 binding sites. 44 Figure 10. RGS4 promoter analysis by luciferase assay. (A) Putative PROX1 binding sites in RGS4 promoter. (B) RGS4 promoter constructs of various lengths were co- transfected with a control (pcDNA) or Prox1 expressing vector into NIH3T3 cells and luciferase activity was measured after 48-hrs post-transfection. Analysis shows that PROX1 expression down-regulates RGS4 promoter activity. 45 Figure 11. RGS4 promoter analysis by luciferase assay. (A) RGS4 promoter activity was analyzed 48-hrs post-transfection with a control (pcDNA) or Prox1 NR1/NR2 binding mutant. (B) Prox1 NR1/NR2 binding mutant was able to inhibit RGS4 promoter activity. 46 Figure 12. RGS4 promoter analysis by luciferase assay. (A) RGS4 promoter activity was analyzed 48-hrs post-transfection with control (pcDNA) or Prox1 DNA-biding mutant. (B) Prox1 DNA biding mutant failed to down-regulate RGS4 promoter activity. 47 Figure 13. PROX1 protein down-regulates the RGS4 expression by binding to it promoter. (A) EMSA showing direct binding of recombinant PROX1 protein to the RGS4 proximal promoter. DNA sequences of each probe (P1~P4) are shown in the Materials and Methods section. (E) PROX1 ChIP performed against the RGS4 promoter in cultured LECs (LEC ChIP). Two sets of primers (RGS4 #1 and RGS4 #2) were used to detect the RGS4 promoter. As negative controls, a normal IgG antibody and primers for the FER and HS3ST2 genes were used. (F) PROX1 ChIP performed against the RGS4 promoter in the brain and intestine cells (In vivo organ ChIP). A normal IgG and primers for the ROSA26 locus were used as negative controls. The outcome of qRT-PCR and luciferase assays was displayed as average ± standard deviation (SD). *, p < 0.05. 48 KSHV Down-Regulates RGS4 Through the PROX1-LRH1 Complex We then wondered if we could impede KSHV-mediated repression of RGS4 by treating cells with SiRNA against PROX1 prior to KSHV infection. Not surprisingly, we determined that prevention of PROX1 upregulation in BECs by PROX1 knockdown clearly abrogates KSHV-mediated RGS4 repression (Fig.14A). We thus concluded that RGS4-downregulation seen in KSHV-infected BECs is primarily caused by the PROX1 upregulation induced by KSHV. PROX1 has been previously reported to interact with various nuclear receptors such as COUP-TFII/NR2F2, LRH1/NR5A2 and HNF4A/NR2A1 and to function as a co-regulator for their target gene expression (18, 30- 33). Therefore, it was important to ascertain if any of these nuclear receptors could be involved with PROX1 to effectively mediate repression of RGS4. To this end we set out to investigate if KSHV could induce expression of any of the PROX1-interacting nuclear receptors in addition to PROX1 to facilitate collaboration between them to repress RGS4 expression. Interestingly, we found from our previous KSHV-microarray studies (3) that, among the 24 different nuclear receptor family members tested, LRH1/NR5A2 is the only nuclear receptor that is significantly upregulated by KSHV infection in BECs (Supplemental Table 2). We subsequently confirmed this LRH1 upregulation by KSHV using qRT-PCR and western blot analyses (Fig.14B). Moreover, LRH1 was also found to be able to repress all RGS4 promoter-constructs previously tested by PROX1 (Fig.14C). Finally, we found that when PROX1 and LRH1 were co-transfected at a low concentration into BECs, RGS4 was synergistically down-regulated (Fig.14D). Taken together, our findings demonstrate that the nuclear receptor LRH1 and its co-regulator 49 PROX1 are upregulated by KSHV and that they collaboratively repress the expression of RGS4, likely through formation of a protein complex. 50 Figure 14. Cooperative regulation of RGS4 by KSHV-upregulated PROX1 and LRH1. (A) PROX1 is required for KSHV-induced RGS4 repression. The PROX1 expression was prevented by transfecting siRNA into BECs 18-hour prior to KSHV infection. After 48-hours of KSHV infection, the expression of RGS4 was determined by qRT-PCR or western blot analyses. (B) Upregulation of LRH1/NR5A2 by KSHV in BECs. KSHV induced the expression of LRH1 mRNA and protein based on qRT-PCR and western blot analyses. Expression of the viral LANA protein was detected to confirm KSHV infection. (C) Repression of the RGS4 promoter constructs by LRH1. A set of the RGS4 promoter-reporter constructs was transfected along with a LRH1-expressing vector into HEK293 cells and the luciferase activity was determined after 48 hours. (D) Concerted repression of RGS4 by PROX1 and LRH1 in BECs. To detect a cooperative repression of RGS4 by PROX1 and LRH1, a low amount of plasmid vector expressing either PROX1 or LRH1 was transfected alone or in combination into BECs for 48 hours, followed by western blotting analyses. The outcome of qRT-PCR and luciferase assays was displayed as average ± standard deviation (SD). *, p < 0.05. 51 RGS4 Attenuates vGPCR-Mediated Activation of Akt Studies have shown that the viral G-protein coupled receptor (vGPCR) of KSHV is not only necessary, but sufficient for endothelial cell immortalization and tumor formation (9, 19, 34-38). In effect, vGPCR has been shown to activate the PI3K/Akt pathway and its downstream effects including upregulation of VEGF and activation of cell proliferation and migration (10, 12, 37, 39). Since RGS4 inhibits activation signaling mediated by both the Gαi and Gαq classes of Gα subunits, subunits found to be utilized by both cellular and viral GPCRs to mediate cell proliferation and invasion (11, 40); we wondered if RGS4 could act as a negative regulator of vGPCR. To address this question, we transfected either an RGS4-expressing vector or a control vector into vGPCR- expressing mouse endothelial cells (SVEC/vGPCR) (12, 19), to establish SVEC/vGPCR/RGS4 or SVEC/vGPCR/CTR stable cell lines, respectively. For this study, we first evaluated the effect of RGS4 expression on the behavior of vGPCR- expressing cells. To our delight, we found that indeed RGS4 expression significantly reduced cell proliferation of vGPCR-expressing cells (Fig.15A). Moreover, we found that RGS4 was also able to inhibit cell migration of vGPCR-expressing cells regardless of the presence or absence of Gro-α, a potent ligand for vGPCR. Notably, the expression and secretion of VEGF was shown to be increased by the stable expression of vGPCR (37). However, we found that vGPCR-induced secretion of VEGF was notably diminished by the stable expression of RGS4 (Fig.15C) in these cells. Finally, we observed that RGS4 expression strongly reduced the Gro-α-induced phosphorylation of Akt (Fig.15D). Together, these data enable us to conclude that RGS4 can serve as a 52 potent inhibitor against the vGPCR-induced activation of Akt signaling and the accompanying downstream cellular effects. 53 Figure 15. RGS4 inhibits not only proliferation and migration of the vGPCR- expressing cells, but also their VEGF secretion and Akt activation. (A) Stable expression of RGS4 resulted in a strong inhibition of cell proliferation of the vGPCR- expressing SVECs. vGPCR/CTR and vGPCR/RGS4 represent SVEC/vGPCR cells that were transfected with a control or an RGS4-expressing vector, respectively. (B) Expression of RGS4 reduced cell migration of vGPCR-expressing cells in the presence or absence of Gro-α. (C) RGS4 inhibits the secretion of vascular endothelial growth factor (VEGF) by the vGPCR-expressing cells into the culture media based on ELISA. (D) Stable expression of RGS4 prevented the Gro-α-induced phosphorylation of Akt (S473) in the vGPCR-expressing cells. The outcome of proliferation, migration and ELISA assays was displayed as (percent) average ± standard deviation (SD). *, p <0.05; **, p <0.01. 54 RGS4 Antagonizes vGPCR Tumor Formation To further corroborate our in vitro data showing the RGS4-mediated inhibition of vGPCR activity, we next investigated whether RGS4 could inhibit vGPCR-induced tumor formation using two different immunodeficient mouse models, athymic nude mice (Nu/Nu) and NOD-SCID IL2Rγ null mice (NSG). In agreement with our previous reports (19), control parental SVECs did not form any detectable tumors in athymic nude mice (data not shown). We thus grafted vGPCR/CTR and vGPCR/RGS4 cells on the right and left flank areas, respectively, of the same mouse (Fig.16A). In athymic nude mice, while the control vGPCR-expressing SVEC cells (vGPCR/CTR) formed palpable tumors within two weeks, the RGS4 and vGPCR-expressing SVEC cells (vGPCR/RGS4) gave rise to considerably smaller tumors only after the 4 th week (Fig.16B). Similarly, in NOD- SCID IL2Rγ null mice, the control vGPCR-expressing SVEC cells (vGPCR/CTR) began to form tumors after day 7, whereas the RGS4 and vGPCR-expressing SVEC cells (vGPCR/RGS4) started to form visible tumors only after day 14 these tumors were significantly smaller than control tumors in the same mouse (Fig.16C). Subsequent immunohistochemical analyses of these tumors revealed that RGS4 strongly repressed the tumor-associated angiogenesis induced by vGPCR (Fig.16D). Notably, both vessel number and size were significantly reduced in RGS4-expressing tumors as compared to control tumors. Taken together, these in vivo studies demonstrate that RGS4 can antagonize vGPCR-mediated tumor formation by suppressing tumor-associated angiogenesis. 55 56 Figure 16. Inhibition of vGPCR-mediated tumor formation by RGS4 (A) Tumor formation by SVECs expressing vGPCR alone (vGPCR/CTR) or together with RGS4 (vGPCR/RGS4) was evaluated in athymic nude mice. This representative photograph was taken at 6 th week post subcutaneous inoculation of the cells. Arrow indicates a vGPCR/RGS4 tumor and arrowhead points a vGPCR/CTR tumor in the same mouse. (B,C) Tumor growth curves of vGPCR/CTR and vGPCR/RGS4 cells in athymic nude (Nu/Nu) (B) or in NOD-SCID IL2Rγ null mice (NSG) (C). Equal numbers of vGPCR/CTR and vGPCR/RGS4 cells were subcutaneous injected at the right and left flank area, respectively, of five female mice per each mouse background. Experiments were performed twice with similar results and tumor volume was shown as average ± standard deviation (SD) of one experiment. (D) Reduced tumor-associated angiogenesis by RGS4. Sections prepared from paraffin-embedded tumors harvested from athymic nude mice at Day 42 were subjected to immunohistochemical analysis for CD31. Number and size of the CD31-positive vessels were analyzed using the NIH ImageJ program and expressed as relative percent values ± standard deviation (SD). Bars, 100 µm; *, p <0.05; ***, p <0.001. 57 CHAPTER IV: DISCUSSION In general, host cells are usually equipped with various defense mechanisms against viral infection and dissemination. As a result, many pathogenic viruses have acquired an equal number of counteracting tactics to nullify or constrain host defense responses. In effect, there is a wealth of knowledge extensively describing the immunological viral-host interactions critical for pathogen clearance or persistence of the various pathogen categories. To date, numerous studies support a convectional oncogenic viral-host response for KSHV infection. Furthermore, it is believed that KSHV vGPCR, a viral homologue of the host CXCR2 protein, plays a “necessary and sufficient” role in KSHV-mediated endothelial cell transformation by activating important signal cascades including AKT, ERK1/2 and p38, and by stimulating production of pro- angiogenic factors such as VEGF, angiopoietin-2, and angiopoietin-like (Angptl)-4, (9-14, 38, 41, 42). Although the vGPCR is constitutively active, its activity can be modulated by the agonists or inverse agonists of CXCR2. While the CXC chemokines CXCL1/Gro-α and CXCL8/IL-8 function to further enhance activation of vGPCR-mediated downstream signaling, other CXC chemokines such as CXCL10/IP-10 and CXCL12/SDF-1α act as inverse agonists to inhibit activated signaling (43). Given the implication of cellular GPCRs in a vast number of cellular functions, cells exercise various mechanisms to ensure receptor regulation and inactivation. To this end, an important mechanism is to desensitize the receptor by internalizing it. In general, GPCR desensitization is carried out by two molecular players: the GPCR kinases (GRKs), which phosphorylate intracellular serine and threonine 58 residues of activated GPCRs, and the arrestins, which uncouple phosphorylated GPCRs from heterotrimetic G protein complexes (44). The sequential action of GRKs and the arrestins result in a rapid attenuation of GPCR-mediated signaling, followed by receptor internalization. Notably, the carboxyl terminal tail of vGPCR was found to be the target site for this GRK/arrestin-mediated receptor desensitization mechanism (45, 46). In keeping with this observation, PMA-induced activation of protein kinase C (PKC) and the expression of GRK4 were shown to inhibit KSHV vGPCR-mediated signaling (10, 47). In this study, we explore the GAP-dependent cellular receptor inactivation mechanism specifically mediated by RGS4. RGS4 is a member of the RGS superfamily, which utilizes a desensitization mechanism different from that of the GRK/arrestin proteins do. RGS family members act as GTPase-activating proteins (GAP) for Gα subunits and accelerate deactivation of Gα by promoting the hydrolysis of Gα-GTP to Gα-GDP (13, 14). Among the RGS proteins, we found RGS4 to be expressed in vascular endothelial cells and played an inhibitory role in cell proliferation, migration, and invasion (15). Importantly, we discovered that RGS4 is predominantly expressed in BECs and not in LECs, and that KSHV infection of BECs significantly inhibits RGS4 expression. Moreover, this repression of RGS4 by KSHV was found to be mediated by the lymphatic-specific regulator PROX1 and its interacting nuclear receptor LRH1. Our current study shows that PROX1 directly binds to the RGS4 promoter repressing transcription of RGS4 through collaboration with LRH1. It is worth noting that our microarray study found that KSHV does not alter the expression of GRK5 or arrestins (3), 59 indicating that KSHV may employ a specific mechanism for RGS4 regulation. Together, our previous and current studies put forward an interesting hypothesis, namely that KSHV-mediated upregulation of PROX1 results in suppression of RGS4 expression, which may otherwise antagonize the activity of vGPCR and thus inhibit KSHV-induced endothelial transformation (Fig.17). 60 Figure 17. Working hypothesis of PROX1-mediated inhibition of RGS4 expression to protect the vGPCR activity in KS tumorigenesis. Acting a GTPase-activating protein (GAP) of various cellular GPCRs, RGS4 can also antagonize the KSHV viral GPCR that is necessary and sufficient for endothelial transformation. In order to maximize the transforming activity of vGPCR in the KSHV-infected BECs, KSHV up- regulates a nuclear receptor LRH1 and its interacting co-regulator PROX1, which cooperatively suppress the gene expression of RGS4. 61 The data presented in this study provide the starting point for further detailed analysis of this novel view on viral infection and pathogenesis. In essence, KSHV exploits vulnerable cellular mechanisms set in place to allow cells the flexibility and plasticity needed to effectively perform essential tasks (24, 68). KSHV has found the way to silently infect cells and remain dormant for the life of the host if unperturbed. It does so by masterfully manipulating infected cells to reprogram their phenotype and attain the necessary elements for infection susceptibility and essential microenvironment for effective dissemination and persistence (66, 72, 78). Since KSHV is an opportunistic virus, once the host becomes immune-compromised, it takes advantage of the host's vulnerable immune-responses to effectively invade the host by aggressively disseminating to vital organs resulting in fatal outcomes for the afflicted. To date, there are no effective therapeutic interventions for all forms of KS. Several promising approaches are currently under intense investigation. These approaches cover a wide range of mechanisms including specifically targeting KSHV with antiviral agents, reconstituting host's immune system, and molecularly targeting vGPCR-mediated signal transduction pathways (68, 70-73). However, until now none of these approaches have translated into successful therapeutic treatments. Our study focuses on exploring the therapeutic potential of a GAP-dependent system directed by RGS4 (123). Given the enormous body of evidence pointing to vGPCR as the viral oncogene driving KS pathogenesis, we believe that our study provides a novel view on KSHV infection tactics and disease dissemination and it highlights a potential alternative therapeutic mechanism for treatment of KS and other angioproliferative diseases. 62 CHAPTER V: MATERIALS AND METHODS Cells and Animals. Normal C57BL/6 mice, RGS4-GFP BAC mice (B6.Cg-Tg(Rgs4- EGFP)4Lvt/J) and immunodeficient NOD-SCID IL2Rγ null mice (NSG; NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) were purchased from the Jackson Laboratory. Athymic nude mice (Crl:NU-Foxn1<nu>/Foxn1<+>) were purchased from Charles River Laboratories. All mouse works have been approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC). Primary blood and lymphatic endothelial cells were isolated from de-identified human foreskins and cultured in endothelial basal medium (EBM, Lonza) supplemented with 20% fetal bovine serum (FBS) and other supplements as previously described (17, 18). Isolation and culture of human endothelial cells were approved by the University of Southern California Institutional Review Board (IRB). Primary human BECs and LECs were transfected by electroporation (Nucleofector II, Amaxa Biosystems) and other cell lines were by Lipofectamine 2000 (Invitrogen). SV40 large T-antigen immortalized murine endothelial cells (SVEC) and its vGPCR-expressing derivative cell line (SVEC-vGPCR) were generously provided by Dr. Silvia Montaner (University of Maryland) and cultured as previously described (19). SVEC-vGPCR cells were transfected by either a human RGS4-expressing vector (Cat. No. RGS040TN00, Missouri S&T cDNA Resource Center) or pcDNA3.1 (Invitrogen) together with a hygromycin-resisant vector (pIRESHyg2, Clontech) at a molar ratio of 10:1, and then selected for hygromycin to generate SVEC-vGPCR cells expressing RGS4 or not, termed SVEC-vGPCR/RGS4 and SVEC-vGPCR/CTR, respectively. PROX1- expressing adenovirus was previously described (17). 63 Cell proliferation assay, scratch assay and chromatin immunoprecipitation (ChIP). Proliferation and scratch assays were performed essentially as previously described (20). Cells were seeded and various time points investigated (24, 48 and 72 hours) using WST- 1 assay (TaKaRa MK400). For scratch assay, cells were grown in a 6-cm dish to a 90- 95% confluence and then the cell monolayer was scratched by using a 1ml pipette tip. The scratched monolayer was pre-treated with mitomycin C (10 µg/mL) prior to activation with Gro-α (50mg/ml) or not in a serum-free media for 24 hours. The area of wound was photographed at 0, 2, 4, 8, 12, and 24 hours and measured by using NIH ImageJ software. ChIP assay was prepared as previously described (17) by using rabbit anti-PROX1 antibody (generated by the authors) or a normal rabbit IgG (Sigma) against LEC cell lysates (in vitro ChIP) or mouse organ lysates (in vivo ChIP). Primers used for in vitro ChIP were as follows: human RGS4 (#1, TGACATTGGTGGAGACATTGA/GTGAACGAGCAGAGAAAATCC; #2, TGACATTGGTGGAGACATTGA/TGACGCATCAGCAATGTTAAGTG), human FER (CACCCTCGAATAATGACGCATA/AACCCAAACGGGTCTGCTCT) and human HS3ST2 (CCCTGGTAGGTGGTCTTTGA/GCACTTCAGAAAAGCCTTGG). Primers used for in vivo ChIP were against mouse RGS4 (AACGCCAAAGCTGGACTAGA/ACACGGAGGGATGTGGATAG) and mouse ROSA26 (AAGGGAGCTGCAGTGGAGTA/CCGAAAATCTGTGGGAAGTC). To prepare the organ lysates, the brain and intestine isolated from a normal C57BL/6 mouse were ground by homogenizer, strained through 70-µm strainers and centrifuged. The 64 pallets were resuspended with DMEM with 10% calf-serum, incubated with 1% Formaldehyde at 37 ⁰C for 10 minutes and subjected to the standard ChIP protocol. Western blot, luciferase and Immunofluorescence (IF) assays. Western blot assays were performed as previous described (17), except that the cell lysis buffer (RIPA; 20mM Tris-Cl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 150mM sodium chloride, and 1mM ethylene glycol tetraacetic acid) was supplemented with phenylmethylsulfonyl fluoride, phosphatase inhibitors and proteasome inhibitor MG-132 (20 µM, Sigma). Sources of the antibodies used are PROX1 (Reliatech), RGS4 (Santa Cruz), whole AKT and phospho-AKT (S473) (Cell signaling) and whole and phospho-ERK1/2 (Cell signaling). The human RGS4 promoter luciferase constructs (21) were kindly provided by Dr. Vishwajit L. Nimgaonkar (University of Pittsburgh). Luciferase reporter assays were carried out after 48 hours post transfection and luciferase activity was measured in triplicates using Bright-Glo reagent (Promega), followed by normalization by total protein amount used. Immunofluorescent staining was performed as previously described (17) and antibody sources are CD31 (Upstate Biotech), RGS4 (Santa Cruz) and LANA (Advanced Biotechnologies Inc., Columbia, MD). Isolation of infectious KSHV. BCBL-1 cells were cultured to the density of 10 million cells/ml and then activated with TPA (20 ng/ml) and sodium butyrate (NaB, 3 mM). At 48-hours post TPA/NaB virion induction, culture media was replaced with normal media and cells were incubated for an additional 3 days. Culture media was then collected and filtered through 0.45-mm filter and centrifuged for 30 minutes at 4 ⁰C at 4,000 rpm to 65 remove cell debris. Supernatant was then further centrifuged for 5 hours at 4⁰C at 10,000 rpm to pellet the virus. Virus-containing pellet was resuspended in endothelial cell media. Infectivity was measured by immunohistochemistry for LANA after a 5 day infection. Electro-mobility shift assay (EMSA). EMSA was performed as previously described (17). Double-stranded oligonucleotides with the following DNA sequences spanning the 0.3-kb RGS4 promoter were annealed and extended with the klenow fragment (New England Biolab) in the presence of 32 P dCTP: (P1, TTTTCAGAAGGATTTTCTCTGCTCGTTCACTTAACATTGC, TGACGCATCAGCAATGTTAAGTGAACGAGCAGAGAAAATC; P2, ATTTTTTCCCATATCCCTACTTTTCAGAAGGATTTTCTCT, GTGAACGAGCAGAGAAAATCCTTCTGAAAAGTAGGGATAT; P3, TGATGCGTCAGTCTTTTCTTCCTCATCTCTTTCAGGGGCT, CTGCCTCTCCAGCCCCTGAAAGAGATGAGGAAGAAAAGAC; P4, GGAGAGGCAGAGGGAGACAGAGGAGCTGGTACTGCAGAGC, TCAGACGACCGCTCTGCAGTACCAGCTCCTCTGTCTCCCT). Fully extended labeled probes were purified by polyacrylamide gel electrophoresis, followed by dialysis. 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Note that RGS4 is the only member of the RGS family genes that is down-regulated by KSHV with a Present (P) call. These array data have been deposited in NCBI Gene Expression Omnibus (GEO) (DataSet Record GDS940). Array-1 Array-2 Probe ID Gene Detection Call* Log FC^ Detection Call* Log FC^ 202988_s_at RGS1 A 0.3 A -0.1 202989_at RGS1 A 0 A -0.1 216834_at RGS1 A -1.8 A -0.4 202388_at RGS2 P -0.6 P -0.6 203823_at RGS3 P 0.2 P 0 220300_at RGS3 P 1.1 P 0.9 204337_at RGS4 P -1.8 P -1.8 204338_s_at RGS4 P -2.6 P -2.6 204339_s_at RGS4 P -2 P -2.2 209070_s_at RGS5 A -4.4 A -3.8 209071_s_at RGS5 A -2.2 A -2.8 218353_at RGS5 A -1.6 A -1.3 210270_at RGS6 A 0 A 0 211448_s_at RGS6 A 0.4 A -1.8 214538_x_at RGS6 A 0.4 A -0.2 206290_s_at RGS7 P -0.4 P -0.4 216970_at RGS7 A -1.6 A 0 204316_at RGS10 M -0.2 A -1.1 204319_s_at RGS10 P -0.5 P -0.8 214000_s_at RGS10 A -0.6 A -2.9 206107_at RGS11 A 0.5 A 0.2 211872_s_at RGS11 A 1.6 A 0 205823_at RGS12 A -0.4 A 0 97 209637_s_at RGS12 A 0.5 P 0.1 209638_x_at RGS12 A 0.9 A 0.5 209639_s_at RGS12 A 0.3 A 0.7 214361_s_at RGS12 A -0.3 A -0.8 214362_at RGS12 A -1.3 A 0.5 210258_at RGS13 A 0.1 A -0.7 204280_at RGS14 A -0.3 A 0.5 211021_s_at RGS14 A 0.1 A -0.6 38290_at RGS14 A -1.1 A 0.5 209324_s_at RGS16 A -0.3 A -1.2 209325_s_at RGS16 A 0.5 A -0.2 204336_s_at RGS19 P 0.1 P -0.1 210138_at RGS20 P -0.5 A -0.6 * Detection Call: A = Absent, P = Present, M = Marginal ^Log FC, Log Fold Change in KSHV-infected BECs over uninfected BECs 98 APPENDIX B: SUPPLEMENTARY TABLE 2 Regulation of expression of various nuclear receptors by KSHV in BECs. Note that NR5A2 is the only nuclear receptor that is significantly upregulated by KSHV with a Present (P) call. These array data have been deposited in NCBI Gene Expression Omnibus (GEO) (DataSet Record GDS940). Array-1 Array-2 Probe ID Gene Detection Call* Log FC^ Detection Call* Log FC^ 206644_at NR0B1 P 0.5 A 0.3 206645_s_at NR0B1 A 1.0 A 1.6 206410_at NR0B2 A 1.2 A -0.2 204760_s_at NR1D1 A 0.2 A 0.3 217476_at NR1D1 A 1.6 A 0.0 31637_s_at NR1D1 A 0.2 A 0.1 209750_at NR1D2 P 0.2 P -0.1 218215_s_at NR1H2 P 0.1 P 0.2 203920_at NR1H3 P 1.0 P 0.9 206340_at NR1H4 A 0.1 A -0.3 207202_s_at NR1I2 A 0.3 A -0.1 207203_s_at NR1I2 A 1.2 A 2.1 207007_at NR1I3 A 0.4 A 0.1 204791_at NR2C1 P 0.2 P 0.4 210530_s_at NR2C1 A 1.8 A -1.9 210531_at NR2C1 A 0.3 A -0.3 206038_s_at NR2C2 P 0.1 P 0.0 207443_at NR2E1 A 0.6 A 0.2 208385_at NR2E3 A 0.4 A 0.1 208388_at NR2E3 A 0.6 A -0.7 209505_at NR2F1 P 0.8 P 0.8 209506_s_at NR2F1 P 0.8 P 0.4 209119_x_at NR2F2 P 0.0 P 0.1 99 209120_at NR2F2 P -0.1 P 0.1 209121_x_at NR2F2 P 0.3 P 0.0 215073_s_at NR2F2 P -0.2 P 0.1 209261_s_at NR2F6 A 0.2 A 0.2 209262_s_at NR2F6 A -0.5 A 0.2 213354_s_at NR2F6 A -0.1 A -0.5 201865_x_at NR3C1 P -0.1 P -0.1 201866_s_at NR3C1 P 0.1 P 0.1 211671_s_at NR3C1 P -0.2 P 0.1 216321_s_at NR3C1 P -0.6 P -0.2 205259_at NR3C2 P -1.0 P -0.7 202340_x_at NR4A1 A 1.1 A -0.1 210226_at NR4A1 A -1.1 A 0.0 211143_x_at NR4A1 A 1.5 A -0.1 204621_s_at NR4A2 A 0.6 A -0.1 204622_x_at NR4A2 A -0.4 A 1.3 216248_s_at NR4A2 A 1.2 A 1.0 207978_s_at NR4A3 A 0.1 A 0.1 209959_at NR4A3 A 0.6 A -0.2 216979_at NR4A3 A -0.6 A -1.2 210333_at NR5A1 A 1.6 A 0.5 208337_s_at NR5A2 P 2.5 P 2.0 208343_s_at NR5A2 P 6.3 P 5.3 210174_at NR5A2 P 2.0 P 2.2 207742_s_at NR6A1 A 0.6 A 0.9 210391_at NR6A1 A 0.2 A -0.3 210392_x_at NR6A1 A -0.6 A 0.2 211402_x_at NR6A1 A 0.1 A 0.6 * Detection Call: A = Absent, P = Present ^Log FC, Log Fold Change in KSHV-infected BECs over uninfected BECs
Abstract (if available)
Abstract
Kaposi Sarcoma (KS) is the most prevalent neoplasm within HIV-infected patients and transplant recipients. Kaposi's Sarcoma-Associated Herpesvirus (KSHV) causes the disease by using a novel mechanism that reprograms endothelial cells making them susceptible targets for viral infection and dissemination. We and others reported that KSHV induces lymphatic differentiation of blood vascular endothelial cells (BECs), by inducing PROX1 up-regulation. Importantly, KSHV G-protein coupled receptor (vGPCR) has been identified as the major viral gene responsible for cellular transformation and disease maintenance. Given that PROX1 is an important mediator of KSHV-induced cell reprogramming, we set out to determine if it had other functional implications in KS pathogenesis. In this study, we report that the regulator of G-protein signaling (RGS)-4 is selectively expressed in BECs and not in LECs, and acts as a cellular agonist against the transformation function of vGPCR. In effect, we found that RGS4 is able to suppress cell proliferation, migration, VEGF-expression and activation of vGPCR-expressing cells in vitro. In accordance, RGS4 significantly antagonized vGPCR-induced tumor growth in two models of immune-deficient mice with reduced tumor-associated angiogenesis. Finally, we demonstrate that KSHV-up-regulated PROX1 and LRH1 cooperate to repress RGS4 expression in KSHV-infected BECs. Together, our study identifies a novel viral strategy that hinders a major host-GPCR regulatory mechanism that may function as an inhibitor for vGPCR activity. Based on our data, we propose a novel hypothesis that KSHV obstructs RGS4-mediated host inhibition of vGPCR transforming activity by up-regulating its transcriptional repressors, PROX1 and LRH1, in order to facilitate KS tumorigenesis.
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Creator
Aguilar, Berenice
(author)
Core Title
Kaposi's sarcoma associated herpes-virus induces cellular proxi expression to modulate host gene expression that benefits viral infection and oncogenesis
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
05/09/2012
Defense Date
12/22/2011
Publisher
University of Southern California
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University of Southern California. Libraries
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Tag
blood endothelial cells,endothelial cells,herpesvirus,Kaposi's sarcoma,KSHV,LRH1,lymphatics,OAI-PMH Harvest,oncogenesis,PROX1,RGS4,vGPCR
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English
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Hong, Young-Kwon (
committee chair
), Stallcup, Michael R. (
committee chair
), DeClerck, Yves A. (
committee member
), Ou, J.-H. James (
committee member
)
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baguilar@usc.edu,bernice.ac@gmail.com
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https://doi.org/10.25549/usctheses-c3-37966
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UC11288343
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Aguilar, Berenice
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Tags
blood endothelial cells
endothelial cells
herpesvirus
Kaposi's sarcoma
KSHV
LRH1
lymphatics
oncogenesis
PROX1
RGS4
vGPCR