Abstract
Inactivating mutations within the von Hippel-Lindau (VHL) tumor suppressor gene predispose patients to develop a variety of highly vascularized tumors. pVHL targets alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), a critical regulator of energy metabolism, angiogenesis, hematopoiesis, and oxygen (O(2)) delivery, for ubiquitin-mediated degradation in an O(2)-dependent manner. To investigate the role of Vhl in cellular proliferation and tumorigenesis, we utilized mouse embryonic fibroblasts (MEFs), a common tool for analyzing cell cycle regulation, and generated Vhl(-)(/)(-) MEF-derived fibrosarcomas. Surprisingly, growth of both Vhl(-)(/)(-) MEFs and fibrosarcomas was impaired, although tumor vascularity was increased. Decreased proliferation of Vhl(-)(/)(-) MEFs was correlated with an overexpression of cyclin kinase inhibitors (CKIs) p21 and p27. The transcription of p21 and p27 is inhibited by c-Myc; therefore, the induction of CKIs was attributed to the ability of HIF to antagonize c-Myc activity. Indeed, p21 mRNA levels were elevated under normoxia in Vhl(-)(/)(-) MEFs, while c-Myc transcriptional activity was markedly reduced. Gene silencing of HIF-1alpha by small interfering RNA reduced p21 and p27 protein and mRNA levels in Vhl(-)(/)(-) MEFs. The induction of p21 and p27, mediated by constitutive activation of the HIF pathway, provides a mechanism for the decreased proliferation rates of Vhl(-)(/)(-) MEFs and fibrosarcomas. These results demonstrate that a loss of pVHL can induce growth arrest in certain cells types, which suggests that additional genetic mutations are necessary for VHL-associated tumorigenesis.