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Negative regulation of HIF-1? by an FBW7-mediated degradation pathway during hypoxia.

ABSTRACT: Hypoxia inducible factor-1? (HIF-1?) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1? is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1? in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3? (GSK3?) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1? phosphorylation by GSK3? in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3? reduced HIF-1? protein and transcriptional activity and increased ubiquitination of HIF-1? in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3? promoted HIF-1? stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1? stabilization when FBW7 was overexpressed and both the elevation of HIF-1? levels and decrease in ubiquitinated HIF-1? when FBW7 was suppressed. Furthermore, HIF-1? associated with FBW7? by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3? phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3? or FBW7. These data introduce a new mechanism for regulation of HIF-1? during hypoxia that utilizes phosphorylation to target HIF-1? for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.

SUBMITTER: Cassavaugh JM

PROVIDER: S-EPMC3202039 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Negative regulation of HIF-1α by an FBW7-mediated degradation pathway during hypoxia.

Cassavaugh Jessica M JM Hale Sarah A SA Wellman Theresa L TL Howe Alan K AK Wong Cheung C Lounsbury Karen M KM

Journal of cellular biochemistry 20111201 12

Hypoxia inducible factor-1α (HIF-1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3β (GSK3β) and targets them for destruction. This study ...[more]

PMID: 21964756

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Project description:BackgroundUnder conditions of hypoxia, cancer cells with hypoxia inducible factor-1α (HIF-1α) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1α under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited.MethodsIn this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown.ResultsHere we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1α. We also demonstrated that NADH functions as a signal for conveyance of HIF-1α degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1α via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1α is recogonized by VHL, which leads to degradation of HIF-1α via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1α was independent of proline hydroxylation.ConclusionsOur findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1α stability. These results also demonstrate the involvement of VHL in degradation of HIF-1α through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1α acetylation in hypoxia.

Dataset Information

Negative regulation of HIF-1? by an FBW7-mediated degradation pathway during hypoxia.

Publications

Negative regulation of HIF-1α by an FBW7-mediated degradation pathway during hypoxia.

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