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Sprouty2 Protein Regulates Hypoxia-inducible Factor-? (HIF?) Protein Levels and Transcription of HIF?-responsive Genes.

ABSTRACT: The ?-subunits of hypoxia-inducible factors (HIF1? and HIF2?) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation by a number of different mechanisms. Because of the seemingly opposite actions of HIF? subunits and Spry2 on cellular processes, we investigated whether Spry2 regulates the levels of HIF1? and HIF2? proteins. In cell lines from different types of tumors in which the decreased protein levels of Spry2 have been associated with poor prognosis, silencing of Spry2 elevated HIF1? protein levels. Increases in HIF1? and HIF2? protein levels due to silencing of Spry2 also up-regulated HIF? target genes. Using HIF1? as a prototype, we show that Spry2 decreases HIF1? stability and enhances the ubiquitylation of HIF1? by a von Hippel-Lindau protein (pVHL)-dependent mechanism. Spry2 also exists in a complex with HIF1?. Because Spry2 can also associate with pVHL, using a mutant form of Spry2 (3P/3A-Spry2) that binds HIF1?, but not pVHL, we show that WT-Spry2, but not the 3P/3A-Spry2 decreases HIF1? protein levels. In accordance, expression of WT-Spry2, but not 3P/3A-Spry2 results in a decrease in HIF1?-sensitive glucose uptake. Together our data suggest that Spry2 acts as a scaffold to bring more pVHL/associated E3 ligase in proximity of HIF1? and increase its ubiquitylation and degradation. This represents a novel action for Spry2 in modulating biological processes regulated by HIF? subunits.

SUBMITTER: Hicks KC

PROVIDER: S-EPMC4974391 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Publications

Sprouty2 Protein Regulates Hypoxia-inducible Factor-α (HIFα) Protein Levels and Transcription of HIFα-responsive Genes.

Hicks Kristin C KC Patel Tarun B TB

The Journal of biological chemistry 20160608 32

The α-subunits of hypoxia-inducible factors (HIF1α and HIF2α) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation by a number of different mechanisms. Because of the seemingly opposite actions of HIFα subunits and Spry2 on cellular processes, we investigated whether Spry2 regulates the levels of HIF1α and HIF2α proteins. In cell lines from different types of tum ...[more]

PMID: 27281823

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Project description:Receptor Tyrosine Kinase (RTK) signaling plays a major role in tumorigenesis and normal development. Sprouty2 (Spry2) attenuates RTK signaling and inhibits processes such as angiogenesis, cell proliferation, migration and survival, which are all upregulated in tumors. Indeed in cancers of the liver, lung, prostate and breast, Spry2 protein levels are markedly decreased correlating with poor patient prognosis and shorter survival. Thus, it is important to understand how expression of Spry2 is regulated. While prior studies have focused on the post-translation regulation of Spry2, very few studies have focused on the transcriptional regulation of SPRY2 gene. Here, we demonstrate that in the human hepatoma cell line, Hep3B, the transcription of SPRY2 is inhibited by the transcription regulating hypoxia inducible factors (HIFs). HIFs are composed of an oxygen regulated alpha subunit (HIF1α or HIF2α) and a beta subunit (HIF1β). Intriguingly, silencing of HIF1α and HIF2α elevates SPRY2 mRNA and protein levels suggesting HIFs reduce the transcription of the SPRY2 promoter. In silico analysis identified ten hypoxia response elements (HREs) in the proximal promoter and first intron of SPRY2. Using chromatin immunoprecipitation (ChIP), we show that HIF1α/2α bind near the putative HREs in the proximal promoter and intron of SPRY2. Our studies demonstrated that not only is the SPRY2 promoter methylated, but silencing HIF1α/2α reduced the methylation. ChIP assays also showed DNA methyltransferase1 (DNMT1) binding to the proximal promoter and first intron of SPRY2 and silencing HIF1α/2α decreased this association. Additionally, silencing of DNMT1 mimicked the HIF1α/2α silencing-mediated increase in SPRY2 mRNA and protein. While simultaneous silencing of HIF1α/2α and DNMT1 increased SPRY2 mRNA a little more, the increase was not additive suggesting a common mechanism by which DNMT1 and HIF1α/2α regulate SPRY2 transcription. Together these data suggest that the transcription of SPRY2 is inhibited by HIFs, in part, via DNMT1- mediated methylation.

Dataset Information

Sprouty2 Protein Regulates Hypoxia-inducible Factor-? (HIF?) Protein Levels and Transcription of HIF?-responsive Genes.

Publications

Sprouty2 Protein Regulates Hypoxia-inducible Factor-α (HIFα) Protein Levels and Transcription of HIFα-responsive Genes.

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