HIF1a targeted for VHL-mediated destruction by proline hydroxylation: implications for oxygen sensing

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HIF1a targeted for VHL-mediated destruction by proline hydroxylation: implications for oxygen sensing. chemical genetic analysis describes a molecular mechanism by which translation of the HIF-2a message is maintained during conditions of cellular hypoxia through inhibition of IRP-1 dependent repression. It also provides the chemical tools for studying this phenomenon. INTRODUCTION The tumor promoting Hypoxia Inducible Factor (HIF) is a central regulator of the cellular response to hypoxia (Semenza, 2000). HIF is a heterodimeric transcription LRRC48 antibody factor consisting of hypoxia-regulated (HIF-a) and a constitutively expressed (HIF-1b) subunits (Semenza, 2000). There are two transactivating HIF-a isoforms, HIF-1a and HIF-2a, whose activity is tightly regulated by oxygen (Gordan and Simon, 2007; Raval et al., 2005). In well-oxygenated cells, the tumor suppressor protein pVHL targets HIF-a for ubiquitination and proteasomal degradation (Maxwell et al., 1999; Ohh et al., 2000). This interaction requires hydroxylation of HIF-a at conserved proline residues by iron-dependent prolylhydroxylases, termed EGLN1, 2 and 3 (Epstein et al., 2001; Ivan et al., 2001; Jaakkola et al., 2001). Hypoxia inhibits EGLN activity and disrupts the HIF-pVHL interaction. Stabilized HIF-a subunits enter the nucleus, heterodimerize with HIF-1b and bind to DNA sequences termed Hypoxia Response Elements (HREs) to transactivate genes that encode for secreted growth and pro-angiogenic factors (Maxwell et al., 2001). HIFs transcriptional activity is similarly attenuated by Factor Inhibiting HIF (FIH), a second iron-dependent enzyme that hydroxylates a conserved asparagine residue in the transactivation domain of HIF (Bruick and McKnight, 2001; Lando et al., 2002a; Lando et al., 2002b; Mahon et al., 2001). Part of the adaptive response to hypoxia in healthy cells is to conserve energy by diminishing global protein translation (Arsham et al., 2003; Bert et al., 2006; Lang et al., 2002; Liu et al., 2006; Schepens et al., 2005). This is at least in part mediated by Redd1, itself a HIF target gene, inhibiting mTOR via the tuberous sclerosis (TSC1/2) complex (DeYoung et al., 2008). However, specific messages that allow Betaxolol cells to cope with the hypoxic environment are spared this translational repression (Blais et al., 1994; Liu and Simon, 2004; Spicher et al., 1998; Thomas and Johannes, 2007; Wouters et al., 2005). The mechanisms for selectively supporting translation of certain messages in conditions of hypoxia are under investigation. Here we devised Betaxolol a cell-based assay to screen for small molecule HIF inhibitor compounds in VHL-deficient RCC cells and identified four compounds that, in multiple cancer cell lines, selectively inhibited translation of the HIF-2a message in an mTOR independent manner. We found that the HIF-2a 5-UTR is necessary and sufficient to confer compound sensitivity. Deletion analysis of the 5-UTR revealed that the minimal region necessary and sufficient for compound efficacy mapped to a newly identified Betaxolol IRE within the 5-UTR of the HIF-2a mRNA (Sanchez et al., 2007). Mutations within the conserved IRE motif abolished the effect of Betaxolol the compounds, as did knocking down the expression of the IRE binding protein, Iron Regulatory Protein 1 (IRP1). Electrophoretic mobility shift assays showed that the compounds directly promoted IRP1 binding to the HIF-2a IRE. Furthermore, we report that hypoxia de-repressed HIF-2a translation by impairing the IRP1/IRE interaction. These data explain how the HIF-2a message is translationally induced by hypoxia and provide chemical genetic tools to study this phenomenon. RESULTS High-throughput screen for small molecule HIF-2a inhibitors Functionally validated HRE and control SV40 luciferase reporter constructs were stably introduced into VHL-deficient 786-O cells to generate 7H4 and 7SV lines, respectively (Figure S1, online). These lines were used to screen five commercial small molecule libraries (NCI Diversity Set, Chembridge, Maybridge, CEREP and Peakdale) as well as the ICCB Diversity-Oriented Synthesis Diversity Set 2 (DDS2) and serine-derived peptidomimetic (SDP1) collections, totaling 58,000 compounds. Eight compounds were identified that reproducibly decreased luciferase activity by greater than 80% when applied to 7H4 cells, whilst having little if any affect on.