The G protein coupled receptor encoded by the Kaposi’s Sarcoma associated virus is a viral oncoprotein and an angiogenic activator

The Kaposi's Sarcoma-associated herpesvirus (KSHV-HHV-8)is a γ-2 herpesvirus that is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs), it encode putative oncogenes, and genes that may cause Kaposi's sarcoma lesions by stimulating angiogenesis. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposi's Sarcoma lesions and in PELs, and stimulates signaling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signaling by this G-Protein Coupled Receptor leads to cell transformation an tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor (VEGF), an angiogenesis and Kaposi's-spindle cell growth factor. We find that this receptor can activate ( in HEK293T) two protein kinases, JNK/SAPK and p38MAPK, by triggering signaling cascades like the ones induced by inflammatory cytokines that are angiogenic activators and mitogens for Kaposi's sarcoma-cells and B cells. Although this is a suggestive result, when we express KSHV-GPCR in different cell types it activate different signaling cascades, and induces different phenotypes. Using pathway specific dominant negatives and pharmacological inhibitors we show that, in the cell line NIH3T3, the activation of JNK and p38 play a major rol in the molecular mechanism of KSHV-GPCR induced cell transformation. Endothelial cells play a central rol in kaposi's sarcoma pathogenesis. KSHV infects endothelial cells and spindle-like cells, of suspected endothelial origin, wich are present in the kaposi's sarcoma lesions. Therefore, in the context of viral infection, KSHV-GPCR is expressed in endothelial cells. As the expression of KSHV-GPCR has different biological and biochemical effects in different cell types we decide to study the consecuences of KSHV-GPCR expression in human primary endothelial cells. KSHV-GPCR expression in endothelial cells induce strong morphologic changes, modifications in the citoskeleton structure, secretion of metaloproteases and resistence to serum starvation mediated by activation of the PI3K/AKT pathway's. All those changes are reminiscent of the transformed phenotype. Usually, the expression of transforming oncogenes in primary cells induces shortage of telomeres, premature senescence, and cell death. In sharp contrast the expression of KSHV-GPCR in primary endothelial human cells stabilize telomere length, by an alternative mechanism independent of telomerase activity (Alternative Lengthening of Telomeres or ALT ), and inmortalize endothelial cells. To our knowledge this is the first demostration that a protein encoded by KSHV is able to inmortailize primary human cells. Thus using a KSHV oncogenic protein we created a new inmortilized endothelial cell line that we call Ecs-KSGPCR. As predicted by the multistep carcinogenesis hypotesis, our preliminary experiments suggest that the mechanisms of Terminal Proliferation Arrest (TPA) are not completelly supressed by KSHV-GPCR and that additional oncogenic events are necessary in order to fully transform human primary endothelial cells. Further research will determine wether other KSHV's oncogenes can cooperate oncogenicaly with KSHV-GPCR. KDR/VEGFR-2 and Tie-2 are endothelial specific tyrosine kinase receptors that play key roles in vascular biology. These two receptors regulate cell survival and proliferation of endothelial cells during angiogenesis, as a consequence of this they appear to be minimally expressed in quiescent endothelium in vivo, and they are down regulated after a few passages, in vitro, of primary endothelial cells. Surprisingly KDR and Tie-2 are overexpressed in long term cultures of Ecs-KSGPCR. Altough in this work we show that the sustained expression of those receptors is functional, further experiments are required to determine if they play any rol in the inmortalization process. We conclude that KSHV-GPCR is a viral oncogene that can exploit cell signaling pathways to induce oncogenesis and angiogenesis in KSHV-mediated oncogenesis.Fil:Bais, Carlos E.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina