James Gnarra, Ph.D.
Associate Professor
Ph.D., University of Virginia Medical School
Email: gnarrajr@upmc.edu
Research Interest:
The von Hippel-Lindau (VHL) tumor suppressor is inactivated in patients with von Hippel-Lindau disease, an inherited cancer syndrome, and in most sporadic clear cell renal carcinomas (RCC). VHL-inactivated tumors over-express many hypoxia-responsive gene products, including the pro-angiogenic vascular endothelial growth factor (VEGF). Re-expression of wild type VHL in RCC cell lines results in down-regulation of expression of these gene products. A long-term goal of our work is to understand the mechanisms through which VHL controls expression of hypoxia-responsive angiogenic growth factors and how VHL inactivation leads to tumorigenesis.
Hypoxic induction of VEGF in normal cells occurs transcriptionally through the binding of hypoxia-inducible factor (HIF) to promoter enhancer elements and post-transcriptionally through cis-acting A+U-rich elements (AREs) in the 3' untranslated region (UTR) of the VEGF mRNA. Under normal oxygen conditions (normoxia), VEGF exhibits low levels of transcription and the mRNA that is expressed undergoes rapid decay. Under hypoxic conditions, however, VEGF transcription increases and the mRNA becomes stable. The 3' UTR AREs and the association of ARE-binding proteins mediate the post-transcriptional stabilization of VEGF mRNA. While several VEGF ARE binding proteins have been identified, the role that these proteins play in controlling VEGF mRNA stability and the hypoxic response remains unclear.
The VHL protein associates with a complex that exhibits E3 ubiquitin ligase activity. In normal kidney cells, VHL controls VEGF expression at the transcriptional level through regulation of HIF- subunit levels. Under normoxia, VHL binds directly to HIF- , targeting the protein for ubiquitylation and subsequent proteasomal degradation. Thus, the lack of functional VHL in RCC results in constitutively elevated HIF- subunit levels and the subsequent induction of HIF-mediated transcription. VHL also controls VEGF expression at the post-transcriptional level: VEGF mRNA half-life in VHL-negative RCC cells is ~4 hours, while VEGF mRNA half-life in RCC cells expressing wild type VHL is ~30 minutes.
Our ongoing studies focus on the effects of VHL inactivation and hypoxia on post-transcriptional regulation of VEGF mRNA. We recently found that VEGF AREs conferred VHL- and oxygen-dependent regulation in kidney cells. RNA probes containing VEGF AREs exhibited VHL-dependent degradation in vitro, as well as VHL- and oxygen-dependent turnover of heterologous mRNAs in vivo. The ARE binding proteins, AUF1 and HuR, associated with VEGF ARE RNA in vitro and in vivo under both normoxic and hypoxic cell culture conditions. In addition, VHL formed a stable complex with AUF1 and HuR. We found that the VHL-AUF1 complexes associated with VEGF mRNA in cells cultured under normoxic conditions, but in hypoxia, complexes lacked VHL. AUF1 was partially down-regulated in RCC cells in a VHL-dependent manner, and we could demonstrate VHL-dependent polyubiquitylation of AUF1 in vitro. Our data suggest that VHL, in a ternary complex with ARE binding proteins AUF1 and/or HuR and VEGF ARE RNA, drives the decay of VEGF mRNA under normal oxygen conditions. Under hypoxic conditions, AUF1 and HuR bind VEGF AREs and stabilize VEGF mRNA in the absence of VHL. Our future studies will extend these observations and determine the role of VHL ubiquitin ligase activity on ARE binding protein activity as well as how ubiquitylation may control the fate of ARE-containing mRNAs.
Recent Publication
Zanesi, N., Mancini, R., Sevignani, C., Valtieri, M., Vecchione, A., Kaou, M., Calin, G.A., La Rocca, G., Fidanza, V., Gnarra, J.R., Croce, C.M., and Huebner, K. Lung cancer in Fhit deficient mice: synergism with Vhl. Cancer Research, 65, 6576-6582, 2005.
Kleymenova, E., Everitt, J.I., Pluta, L., Portis, L.M., Gnarra, J.R., and Walker, C.L. Susceptibility to vascular neoplasms but no increased susceptibility to renal carcinogenesis in Vhl knockout mice. Carcinogenesis, 25, 309-315, 2004.
Mack, F.A., Arsham, A., Keith, B., Rathmell, W.K., Gnarra, J., Keith, B., and Simon, M.C. Loss of pVHL is sufficient to cause HIF dysregulation in primary cells but does not promote tumor growth. Cancer Cell, 3, 75-88, 2003.
Katner, A.L., Gootam, P., Quoang, P., Jaruga, E., Ma, Q., Gnarra, J.R., and Rayford, W. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27Kip1. The Prostate, 15, 77-87, 2002.
Katner, A., Gootam, P., Quoang, Q.B.L., Gnarra, J.R., and Rayford, W. A recombinant adenovirus expressing p27Kip1 induces cell cycle arrest and apoptosis in human 786-O renal carcinoma cells. J. Urol., 168, 766-773, 2002.
