Functional analysis of KSHV GPCR in human lymphocytes

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DESCRIPTION (provided by applicant): Kaposi?s sarcoma-associated herpesvirus (KSHV/HHV-8) is closely associated with Kaposi?s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman?s disease, disorders associated with HIV infection. KSHV encodes a G protein-coupled receptor (GPCR) most homologous to the human IL-8 receptors CXCR1 and CXCR2. Transcription of vGPCR has been shown in KS and PEL, and evidence in transfected animal cell lines suggests that KSHV GPCR may be involved in KSHVmediated angiogenesis and oncogenesis. vGPCR has not, however, been studied in the context of PEL cell lines or any other human cell of hematopoietic origin. Since signaling molecules behave differently in different cellular contexts, such work will be fundamental in understanding the role of vGPCR in KSHV-mediated disease. To this end, Dr. Cannon designed a novel single plasmid construct that permitted development of PEL cell lines that can be made to over-express vGPCR in a dose-dependent manner using tetracycline. Preliminary data in these cell lines already show that vGPCR has quite different downstream effects than in other cell lines. Namely, vGPCR causes activation of the mitogen-activated ERK2, downregulation of stress-related p38, and decreased cell viability. Furthermore, vGPCR upregulates NFkB and AP-1 activity, two transcription factors intimately involved in B cell physiology. Importantly, other KSHV genes have been shown to have AP-1 and NFkB-responsive promoters. These cell lines are ideal tools to study the following: 1) vGPCR signaling mechanisms and downstream effects on PEL cell proliferation, cell cycle and apoptosis, 2) vGPCR-mediated autocrine/paracrine effects including secretion of VEGF, bFGF, IL-6 and other cytokines known to play a role in angiogenesis and PEL/KS biology, 3) vGPCR-mediated effects on KSHV gene transcription and life cycle. Another major aim of the proposal is to provide didactic and research components in a phased manner to provide the applicant with effective training as a physician-scientist. Dr. Cannon will be studying at the Weill Medical College and School of Medical Sciences of Cornell University and working in the lab under the mentorship of Dr. Ethel Cesarman of the Department of Pathology.