The specific contribution of hypoxia-inducible factor-2α to hypoxic gene expression in vitro is limited and modulated by cell type-specific and exogenous factors

Cellular integrity in hypoxia is dependent on molecular adaptations dominated by the heterodimeric transcription factor hypoxia-inducible factor (HIF). The HIF complex contains one of two alternative oxygen-regulated α-subunits considered to play distinct roles in the hypoxia response. Although HIF-2α may be more important in tumour biology and erythropoiesis, the spectrum of individual target genes is still insufficiently characterized. We therefore performed an Affymetrix gene array on Hep3B cells stimulated with a hypoxia-mimetic and transfected with either HIF-1α or HIF-2α siRNA. 271 transcripts were found to be induced HIF-dependently, including most previously identified HIF targets and a number of novel genes. Most were influenced by HIF-1α knock-down, whereas a smaller number were regulated by HIF-2α. Validation of a selection of genes by RNase protection confirmed the hypoxic regulation and HIF-1α- or HIF-2α-dependency in most cases, with the latter showing a lower amplitude. Many HIF-2α targets also responded to HIF-1α knock-down. Interestingly, regulation by HIF-2α was markedly influenced not only by cell type, but also by cell culture conditions, features that were not shared with HIF-1α-regulated genes. Thus, HIF-2α effects are modulated by a number of intrinsic and extrinsic factors which may be most relevant in tumour cells.