Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein

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 (HIF1 alpha or HIF2 alpha) and a beta subunit (HIF1 beta). Intriguingly, silencing of HIF1 alpha and HIF2 alpha 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 HIF1 alpha/2 alpha 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 HIF1 alpha/2 alpha reduced the methylation. ChIP assays also showed DNA methyltransferase1 (DNMT1) binding to the proximal promoter and first intron of SPRY2 and silencing HIF1 alpha/2 alpha decreased this association. Additionally, silencing of DNMT1 mimicked the HIF1 alpha/2 alpha silencing-mediated increase in SPRY2 mRNA and protein. While simultaneous silencing of HIF1 alpha/2 alpha and DNMT1 increased SPRY2 mRNA a little more, the increase was not additive suggesting a common mechanism by which DNMT1 and HIF1 alpha/2 alpha regulate SPRY2 transcription. Together these data suggest that the transcription of SPRY2 is inhibited by HIFs, in part, via DNMT1-mediated methylation.