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Down-regulation of hypoxia-inducible factor-2 in PC12 cells by nerve growth factor stimulation

Abstract:

Cellular responses to low oxygen tension are mediated, at least in part, by the activation of the hypoxia-inducible factors (HIFs). In the presence of oxygen, specific HIF residues become hydroxylated by the action of a recently described group of dioxygenases. These post-translational modifications target HIF for proteosomal degradation and prevent its transcriptional activity. Despite these detailed studies, little is known about the regulation of HIF by stimuli other than hypoxia. Here we report that, in rat pheochromocytoma PC12 cells, nerve growth factor (NGF) stimulation results in a decrease of both basal and hypoxia-induced levels of HIF-2α protein. NGF treatment did not increase HIF-hydroxylase gene expression or activity, and the reduction of the HIF-2α protein level upon stimulation was observed even in the presence of HIF-hydroxylase inhibitors such as deferoxamine or dimethyloxoglutarate. Thus, in contrast to the response to hypoxia, the effect of NGF on HIF-2α protein levels is not mediated by the HIF hydroxilases. Quantitative real time (RT)-PCR showed that NGF stimulation results in a decrease of the HIF-2α mRNA level similar to that found at the protein level. Interestingly, NGF effect was specific for HIF-2α mRNA because it did not affect HIF-1α mRNA levels. NGF treatment reduced HIF-2α mRNA levels even in the presence of actinomycin D, suggesting an effect on mRNA stability. Finally, the effect of NGF on HIF2α correlates with reduction of both basal and hypoxia-induced vascular endothelial growth factor mRNA levels. Reporter assays suggest that the reduced expression of hypoxia-inducible genes upon NGF treatment is related, at least in part, to the reduction of HIF-2α protein. Hence, in PC12 cells the level of HIF-2α protein and its effect on gene expression can be down-regulated by stimuli other than oxygen.