Proteasomal Inhibition Attenuates Transcriptional Activity of Hypoxia-Inducible Factor 1 (HIF-1) via Specific Effect on the HIF-1α C-Terminal Activation Domain

Abstract

The ubiquitin-proteasome pathway (UPP) is involved in regulation of multiple cellular processes. Hypoxia-inducible factor 1α (HIF-1α) is a prototypic target of the UPP and, as such, is stabilized under conditions of proteasomal inhibition. Using carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) expression as paradigmatic markers of HIF-1 activity, we found that proteasomal inhibitors (PI) abrogated hypoxia-induced CAIX expression in all cell lines tested and VEGF expression in two out of three. Mapping of the inhibitory effect identified the C-terminal activation domain (CAD) of HIF-1α as the primary target of PI. PI specifically inhibited the HIF-1α CAD despite activating the HIF-1α coactivator p300 and another p300 cysteine/histidine-rich domain 1-dependent transcription factor, STAT-2. Coimmunoprecipitation and glutathione S-transferase pull downs indicated that PI does not disrupt interactions between HIF-1α and p300. Mutational analysis failed to confirm involvement of sites of known or putative posttranslational modifications in regulation of HIF-1α CAD function by PI. Our data provide evidence for the counterintuitive hypothesis that inhibition of HIF-1 function could be responsible for at least some of the antitumor effects of proteasomal inhibition. Further studies of the mechanism of the PI-induced attenuation of HIF-1α will provide important, potentially novel insight into regulation of HIF-1 activity and possibly identify new targets for HIF-directed therapy.

This study was supported by grants from the California Cancer Research Program (00-00789V-20240) and The Avon Foundation.

We thank J. Pastorek and S. Pastoreková for the CAIX Ab. We also thank N. Sang for the Gal4-HIF-1α and pFR-Luc constructs and A. Giordano for the Gal4-p300 construct. P. Kaiser provided the ubiquitin Ab.