Down-regulation of Hypoxia-inducible Factor-2 in PC12 Cells by Nerve Growth Factor Stimulation

Salvador Naranjo-Suárez,María Carmen Castellanos,Miguel Álvarez-Tejado,Alicia Vara,Manuel O Landázuri,Luis Del Peso

doi:10.1074/jbc.m304079200

Salvador Naranjo-Suárez, María Carmen Castellanos + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m304079200

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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-2 alpha protein. NGF treatment did not increase HIF-hydroxylase gene expression or activity, and the reduction of the HIF-2 alpha 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-2 alpha 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-2 alpha mRNA level similar to that found at the protein level. Interestingly, NGF effect was specific for HIF-2 alpha mRNA because it did not affect HIF-1 alpha mRNA levels. NGF treatment reduced HIF-2 alpha mRNA levels even in the presence of actinomycin D, suggesting an effect on mRNA stability. Finally, the effect of NGF on HIF2 alpha 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-2 alpha protein. Hence, in PC12 cells the level of HIF-2 alpha protein and its effect on gene expression can be down-regulated by stimuli other than oxygen.

Highlights

Cellular responses to low oxygen tension are mediated, at least in part, by the activation of the hypoxiainducible factors (HIFs)
We found that nerve growth factor (NGF) treatment does not increase EGLN activity and that it reduces HIF-2␣ protein even in the presence of EGLN inhibitors
We found that NGF challenge did not affect ARD1 mRNA level, 2 supporting the lack of a role for this enzyme in the reduction of HIF-2␣ by NGF

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Reagents—The rat pheochromocytoma PC12 cells were maintained in RPMI 1640 medium with GlutaMAX-I (Invitrogen) supplemented with 10% horse serum (Invitrogen) and 5% fetal bovine serum. 8 ␮g of recombinant GST-HIF2␣ [521–542] was incubated together with 10 ␮g of lysate in a final volume of 45 ␮l of reaction buffer (20 mM Hepes, pH 7.6, 5 mM KCl, 1.5 mM MgCl2, 1 mM ␤-ME, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 260 ␮M Fe2ϩCl2, 5 mM 2-oxoglutarate, 0.6 mM ascorbic acid). Sepharose-glutathione-GST-HIF complexes were recovered by centrifugation and resuspended in 200 ␮l of binding buffer (50 mM Tris, pH 7.5, 120 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 5 mM KCl, 0.2% Nonidet P-40, 0.5 mg/ml bovine serum albumin), containing 30 ␮l of in vitro transcribed-translated (rabbit reticulocyte lysate in vitro coupled transcription/translation from Promega) [35S]VHL. Samples were incubated at 4 °C for 1 h, protein complexes were recovered by centrifugation, washed twice with binding buffer, and resuspended in 30 ␮l of 1ϫ loading buffer (2% SDS, 10% glycerol, 10 mM dithiothreitol, 62 mM Tris, pH 6.8, and 0.004% bromphenol blue).

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RESULTS

DISCUSSION