Hepatitis B virus X protein enhances transcriptional activity of hypoxia-inducible factor-1α through activation of mitogen-activated protein kinase pathway

Hepatitis B virus X protein (HBx) of the hepatitis B virus was strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Here, we explored the possibility of cross-talk between HBx and hypoxia-inducible factor-1α (HIF-1α), a potent transcriptional inducer of angiogenic factors. First, we showed that stability of HIF-1α protein was increased by HBx in HBx-inducible Chang liver cells as well as in transient HBx expression system of non-hepatic cells. Immunofluorescence studies revealed that the HBx-induced HIF-1α was partially translocated into the nucleus in majority of cells while additional CoCl₂-induced hypoxic condition caused complete nuclear translocation. Second, HBx induced both phosphorylation of HIF-1α and activation of p42/p44 mitogen-activated protein kinases (MAPKs), which were synergistically enhanced in the presence of CoCl₂. Furthermore, HBx enhanced transcriptional activity of HIF-1αa in the reporter genes encoding hypoxia response element or VEGF promoter. Either treatment of MEK inhibitor PD98059 or coexpression of dominant-negative MAPK mutants abolished the HBx-induced transcriptional activity and protein stability as well as nuclear translocation of HIF-1α, suggesting that HBx activates HIF-1α through MAPK pathway. Third, the association of HIF-1α with von Hippel-Lindau was decreased but the association with CREB-binding protein was enhanced in the presence of HBx, suggesting the molecular mechanism by which HBx enhances the protein stability and transactivation function of HIF-1α. Finally, we demonstrated that expression of HIF-1α and vascular endothelial growth factor was increased in the liver of HBx-transgenic mice, suggesting that the cross-talk between HIF-1α and HBx may lead to transcriptional activation of HIF-1α target genes, which play a critical role in hepatocarcinogenesis.