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

Abstract

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 alpha-subunits considered to play distinct roles in the hypoxia response. Although HIF-2alpha 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-1alpha or HIF-2alpha 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-1alpha knock-down, whereas a smaller number were regulated by HIF-2alpha. Validation of a selection of genes by RNase protection confirmed the hypoxic regulation and HIF-1alpha- or HIF-2alpha-dependency in most cases, with the latter showing a lower amplitude. Many HIF-2alpha targets also responded to HIF-1alpha knock-down. Interestingly, regulation by HIF-2alpha was markedly influenced not only by cell type, but also by cell culture conditions, features that were not shared with HIF-1alpha-regulated genes. Thus, HIF-2alpha effects are modulated by a number of intrinsic and extrinsic factors which may be most relevant in tumour cells.