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

RESEARCH ARTICLE Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein Xianlong Gao1☯, Kristin C. Hicks2☯, Paul Neumann2, Tarun B. Patel2* 1 Department of Surgery, Loyola University Chicago, Chicago, Illinois, United States of America, 2 Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, United States of America ☯ These authors contributed equally to this work. * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Gao X, Hicks KC, Neumann P, Patel TB (2017) Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein. PLoS ONE 12(2): e0171616. doi:10.1371/journal.pone.0171616 Editor: Jörn Karhausen, Duke University, UNITED STATES Received: November 4, 2016 Accepted: January 23, 2017 Published: February 14, 2017 Copyright: © 2017 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract 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. Funding: The authors performed the study using institutional funds. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0171616 February 14, 2017 1 / 22 Hypoxia inducible factors regulate sprouty2 expression Introduction Sprouty (Spry) proteins were first discovered in Drosophila melanogaster as inhibitors of fibroblast growth factor receptor-induced tracheal branching [1]. Subsequently, four mammalian isoforms of Sprouty (Spry1, Spry2, Spry3, and Spry4) were identified that are transcribed from four different genes. The different Spry isoforms have been shown to modulate the actions of receptor tyrosine kinases (RTKs); therefore, Spry proteins play a role in processes that require extensive RTK signaling such as organogenesis and tumorigenesis [2–5]. Specifically in development, Spry proteins have been shown to regulate the process of angiogenesis, patterning of the midbrain and anterior hindbrain, and development of the kidneys, lungs, limb buds, craniofacial features, and trunk [4,6–11]. After development Spry proteins continue to regulate angiogenesis [4,12–14], cell proliferation [15,16], migration [16–19] and survival [20,21]. Likewise, the role of Spry proteins, mainly Spry1 and Spry2, in cancer has also been investigated. Previous research has shown that the levels of Spry1 and Spry2 are decreased in cancers of the breast [22], lung [23], liver [24–28], and prostate [29–31] correlating to poor patient prognosis. Due to the important role Spry proteins play in development and tumorigenesis, it is crucial to understand how Spry levels are regulated. We focused on Spry2, which is ubiquitously expressed and most studied among the Spry isoforms. Prior studies have concentrated on the regulation of the Spry2 protein through a variety of posttranslational modifications such as ubiquitylation or phosphorylation (reviewed [32,33]). However, early on Spry expression patterns during development were assessed and showed that the transcription of SPRY is upregulated by growth factors [34–37] elevating Spry protein levels in the centers of growth factor signaling (e.g. limb buds), thereby optimizing the ability of Spry proteins to act as negative feedback inhibitors of the enhanced RTK signaling in these areas. Additionally, while many other transcription factors have been predicted to bind to the SPRY2 promoter, few have actually been shown to bind [38]. Ding et al. performed a functional analysis of the SPRY2 promoter and identified that Ap2, Ets-GATA, and SP-1 bind to the SPRY2 promoter enhancing its transcription [38]. However, the functional significance of the binding of these transcription factors to the SPRY2 promoter remains unknown. Because Spry2 levels are reduced in some forms of cancer [22–29,31], the regulation of Spry2 in cancer has been investigated. Most studies, however, have focused on the post-transcriptional regulation of Spry2 [39–43]. The few studies that have investigated transcriptional regulation of SPRY2 promoter have shown that FOXO and beta-catenin bind to the SPRY2 promoter and induce its transcription [44,45]. In terms of epigenetic modifications, the promoters of Spry4 and Spry2 have been shown to be hypermethylated in prostate cancer [46], but not breast cancer [22]. Two reports suggest that SPRY2 promoter is hypermethylated in hepatocellular carcinomas [27,47], but another report suggests otherwise [25]. In both development and tumorigenesis, cells experience a hypoxic environment to which they adapt to by upregulating the transcription factors, hypoxia inducible fac (...truncated)