Hypoxia inducible factor 1alpha : molecular mechanisms of regulation

Abstract

Hypoxia-Inducible Factor 1alpha (HIF-1alpha) is an important protein in the homeostatic control of genes in response to low oxygen, or hypoxia. It is thus also a key factor in the regulation of angiogenesis in response to hypoxia, including induction of vascular endothelial growth factor (VEGF) gene expression. HIF-1alpha is a member of the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) family of transcription factors, which are involved in many diverse biological processes.

A common thread is that these proteins are inducible intracellular transcription factors. Moreover, they bind DNA as heterodimers, dimerising first with a partner protein belonging to the ARNT subfamily of bHLHPAS proteins. HIF-1alpha is an intriguing protein, as it is regulated intensively by proteolysis. During normoxia, it is barely detectable within the cell, but under conditions of hypoxia, it is stabilised and rapidly accumulates in the nucleus. By proteosome inhibition, we could isolate poly-ubiquitinated HIF1alpha protein from cells, which seemed to he unavailable for nuclear translocation and any subsequent activation of transcription. The 'pool' of HIF-1alpha created by hypoxia, however, manages to escape ubiquitination and can participate in nuclear translocation and transcription.

To further investigate the actual 'trigger' for this process, we examined the effect of the serine/threonine kinase inhibitor quercetin on HIF-1alpha activity. Quercetin treatment induced substantial transcriptional activity of HIF1alpha, even greater than hypoxic induction of the same system. It was further observed that quercetin could activate HIF-1alpha along its pathway of activation from the cytoplasm to the nucleus; enhancing protein stability, nuclear translocation and upregulation of target genes. Quercetin is a dietary flavonoid, and thus, the present results expose a novel pathway of activation of regulation of HIF-1alpha function linked to dietary compounds. Yet another mechanism by which HIF-1alpha is regulated involves dimerisation with the dominant negative factor, inhibitory PAS protein (IPAS). Our research shows that this protein is a splice variant of the HIF-3alpha gene, and its production is favoured during hypoxia in the specific tissues in which it is expressed.

During a genome wide bioinformatic investigation of human bHLH-PAS factors, regions of conserved synteny between the three HIF-alpha and neuronal PAS protein (NPAS) loci were discovered. These two bHLH-PAS factors have paralogues on three different human chromosomes (2p21,14q22,19q13.3) and together share loci with paralogues of both the sine oculis (SIX) and protein kinase C (PKC) gene families. Remarkably, this duster of loci is semi-conserved within the mouse genome, indicating a close evolutionary history for these genes. As the genes within this conserved region have orthologues identified in Caenorhabditis elegans and Drosophila melanogaster, it allows us to reconstruct the evolutionary history of these particularly interesting bHLH-PAS family members.