Abstract 999: Transcriptional Regulation of HIF-1alpha by NFkappaB in Response to Hypoxia
The cellular response to hypoxia is mediated by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). HIF-1alpha has been associated with many disorders associated with chronic hypoxia including pulmonary hypertension (PH). While hypoxia has been shown to affect HIF-1alpha protein stability, a number of in vivo studies implicated that hypoxia also increases HIF-1alpha mRNA levels. Thus, we aimed to identify a mechanism by which hypoxia regulates HIF-1alpha mRNA transacription in pulmonary artery smooth muscle cells (PASMC). In PASMC hypoxia transiently elevated HIF-1alpha mRNA with maximal levels at 1 h. The mRNA increase was followed by enhanced HIF-1alpha protein levels with a maximum at 4 h. Hypoxia-dependent HIF-1alpha upregulation was completely abrogated by actinomycin D indicating a transcriptional mechanism. This was confirmed by reporter gene assays demonstrating that hypoxia stimulated HIF-1alpha promoter activity. Deletion of the HIF-1alpha promoter identified a region responsible for hypoxic induction which contained an NFkappaB consensus sequence. Indeed, hypoxia induced nuclear translocation of the NFkappaB subunits p50 and p65. Inhibition of NFkappaB using a dominant-negative IkappaB mutant diminished hypoxic induction of the HIF-1alpha promoter, mRNA and protein. Mutation of the NFkappaB binding site prevented HIF-1alpha promoter activation by hypoxia. Similarly, gel shift analyses and chromatin immunoprecipitation confirmed that NFkappaB was binding to the HIF-1alpha promoter in response to hypoxia. Finally, treatment with the PI3 kinase (PI3K) inhibitor wortmannin prevented hypoxia-dependent activation of NFkappaB and HIF-1alpha promoter activity. Together, these findings show that HIF-1alpha is a transcriptional target of NFkappaB which is activated via a PI3K-dependent pathway under hypoxic conditions. This novel pathway may explain the increased levels of HIF-1alpha mRNA in vessels from animals with hypoxia-induced PH and may play an important role in the pathogenesis of disorders associated with chronic hypoxia.