Differential Roles of Hypoxia-Inducible Factor 1α (HIF-1α) and HIF-2α in Hypoxic Gene Regulation

Abstract

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF), a heterodimer of HIF-␣ and the aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1␣ and HIF-2␣ subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes. Previous studies using Hif-1␣ ؊/؊ embryonic stem and mouse embryonic fibroblast cells show that loss of HIF-1␣ eliminates all oxygen-regulated transcriptional responses analyzed, suggesting that HIF-2␣ is dispensable for hypoxic gene regulation. In contrast, HIF-2␣ has been shown to regulate some hypoxia-inducible genes in transient transfection assays and during embryonic development in the lung and other tissues. To address this discrepancy, and to identify specific HIF-2␣ target genes, we used DNA microarray analysis to evaluate hypoxic gene induction in cells expressing HIF-2␣ but not HIF-1␣. In addition, we engineered HEK293 cells to express stabilized forms of HIF-1␣ or HIF-2␣ via a tetracycline-regulated promoter. In this first comparative study of HIF-1␣ and HIF-2␣ target genes, we demonstrate that HIF-2␣ does regulate a variety of broadly expressed hypoxia-inducible genes, suggesting that its function is not restricted, as initially thought, to endothelial cell-specific gene expression. Importantly, HIF-1␣ (and not HIF-2␣) stimulates glycolytic gene expression in both types of cells, clearly showing for the first time that HIF-1␣ and HIF-2␣ have unique targets.Oxygen (O 2 ), the final electron acceptor during oxidative phosphorylation, is absolutely required for invertebrate and vertebrate life. The immediate response to O 2 deprivation (hypoxia) is a defense phase, which suppresses ATP consumption by arresting protein translation and ion channel activity, two major ATP sinks during normoxia. During a rescue phase, in spite of a general reduction in RNA synthesis, transcription of some genes increases dramatically under low O 2 (21, 34). These hypoxia-responsive genes are involved in glucose transport, glycolysis, erythropoiesis, angiogenesis, vasodilation, and respiratory rate, and together they function to minimize the effects caused by low O 2 at cellular, tissue and systemic levels (93, 106).The activation of many O 2 -regulated genes is mediated by hypoxia-inducible factor (HIF), a heterodimer consisting of HIF-1␣ and HIF-1␤ (also called the aryl hydrocarbon receptor nuclear translocator [ARNT]) in most cells (52,104,105). Both HIF-1␣ and ARNT belong to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of transcription factors, which share several conserved structural domains, including a bHLH region for DNA binding and two PAS domains for target gene specificity and dimerization (102). Although ARNT is absolutely required for HIF activity (63, 110), HIF function is primarily regulated by HIF-1␣ protein stability (37,46,84). Under normoxia, HIF-1␣ is ubiquitinated through interaction with the von Hippel-Lindau tumor suppr...

Target gene selectivity of hypoxia-inducible factor-α in renal cancer cells is conveyed by post-DNA-binding mechanisms

“…Taken together, these findings strongly suggest that HIF-2a has pro-tumorigenic actions in RCC that are isoform specific, and not shared by HIF-1a. In keeping with this, HIF-1a and HIF-2a show clear transcriptional selectivity, with studies to date defining at least two types of isoform-specific responses; certain genes appear exclusively responsive to HIF-1a in both RCC and non-RCC cells, whereas others respond to both HIF-1a and HIF-2a in non-RCC cells, and are dominantly regulated by HIF-2a in RCC cells (Hu et al, 2003;Sowter et al, 2003;Raval et al, 2005). Hence, RCC cells display marked HIF-a chain selectivity in target gene responses that likely underlie differences in the role of these molecules in promoting tumour growth.…”

“…Previous work with non-RCC cells has indicated that although the large majority of hypoxia inducible genes are responsive to HIF-1a (Hu et al, 2003;Park et al, 2003;Sowter et al, 2003;Manalo et al, 2005;Wang et al, 2005), two distinct patterns of response are observed with some genes being exclusively responsive to HIF-1a and some responding to both HIF-1a and HIF-2a. In RCC cells, this transcriptional selectivity is more marked with the former group of genes remaining exclusively responsive to HIF-1a and the latter becoming dominantly, or even exclusively, responsive to HIF-2a (Sowter et al, 2003;Raval et al, 2005).…”

Hypoxia-associated markers in gastric carcinogenesis and HIF-2α in gastric and gastro-oesophageal cancer prognosis

“…Hypoxia-inducible factor-2a has been shown to regulate a number of the same hypoxia-inducible genes as HIF-1a (Hu et al, 2003). However, it is now known that the various hypoxia-inducible genes vary in their sensitivity to HIF-1a and HIF-2a (Wang et al, 2005) and therefore different downstream pathways can be preferentially activated (Hu et al, 2003;Sowter et al, 2003). The classical 'hypoxic' expression of HIF-2a in tumour sections was not found in our study as there was no association with necrosis or distance from blood vessels.…”

The role of oxygen availability in embryonic development and stem cell function

“…We have shown that HIF-2α, but not HIF-1α, binds to the Oct4 promoter and induces Oct4 expression in hypoxic cells if the Oct-4 locus is in an 'open' configuration and not embedded in heterochromatin 75,84 . There are several putative HREs in the promoter region of Oct4 that are conserved between mice and humans 85 .…”