Chromatin, Gene, and RNA Regulation Contribution of HIF-1a in 4E-BP1 Gene Expression

The eukaryotic translation initiation factor 4E (eIF4E) is necessary for the translation of capped mRNAs into proteins. Cap-dependent mRNA translation can be however inhibited by the eIF4E-binding protein 1 (4E-BP1). The hypophosphorylated forms of 4E-BP1 indeed sequester eIF4E and thus block translation initiation and consequent protein synthesis. Different reports indicate that, in addition to hypophosphorylation, 4E-BP1 function can be also regulated at the level of protein expression. This is the case in contact-inhibited cells or in cells exposed to hypoxia. The molecular mechanisms responsible for 4E-BP1 protein accumulation in these conditions remain however unknown. In the present study, we found that 4E-BP1 gene promoter contains a hypoxia-responsive element (HRE) that mediates 4E-BP1 gene upregulation via the hypoxia-inducible factor-1 alpha (HIF-1a) transcription factor. Gene reporter assays then revealed that the presence of such HRE in the promoter of 4E-BP1 gene is involved in 4E-BP1 accumulation in contact-inhibited cells and in cells exposed to hypoxia. We also reveal that the TGF-b–dependent transcription factor SMAD4 cooperates with HIF-1a to fully activate 4E-BP1 gene transcription under hypoxia. These data therefore suggest thatHIF-1a contributes to 4E-BP1 gene expression under different conditions. Mol Cancer Res; 11(1); 54–61. 2012 AACR. Introduction One molecular mechanism whereby cells can counteract undesired increases in proliferation rates is the inhibition of protein synthesis. In eukaryotic cells, most mRNAs are translated into proteins via the recruitment of ribosomes at the mRNA 50-cap structure. This process necessitates the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), which facilitates ribosome loading on the mRNA 50-end through its interaction with eIF4G, a large scaffolding protein. eIF4G is indeed bound to other translation initiation factors including eIF4A and eIF3, whose functions are to aid in melting mRNA 50-end secondary structures (eIF4A) or to serve as a docking site for the ribosome (eIF3; ref. 1). Assembly of this translation initiation complex is the target of tight controls that limit protein synthesis especially when there is a need to block cell proliferation. Among these mechanisms of control, sequestration of eIF4E by the eIF4E-binding protein 1 (4E-BP1) plays a prominent role (1). The binding of 4E-BP1 to eIF4E prevents eIF4G association with eIF4E and consequently precludes ribosome recruitment at the mRNA 50-cap structure. 4E-BP1 activity (i.e., sequestration of eIF4E) has been shown to be enhanced in various organisms exposed to different conditions of stress or in contact-inhibited cells (see later). The regulation of 4E-BP1 activity was initially described as being dependent mainly on phosphorylation events (2). To sequester eIF4E, 4E-BP1must be actually maintained in a hypophosphorylated state. mTOR being the major kinase of 4E-BP1, hypophosphoryation of 4E-BP1 is observed when mTOR activity is inhibited. This is the case in many conditions of stress, including nutrient deprivation (3), or exposure to oxidative (4) or genotoxic (5) compounds.More recently, however, different conditions have been shown to trigger not only 4E-BP1 hypophosphorylation, but also 4EBP1 gene expression (2). For instance inDrosophila, 4E-BP1 induction prolongs the survival of flies upon nutrient deprivation (6). Similarly, the survival of endocrine cells in the pancreas of mice exposed to a reticulum stress is dependent on the activation of 4E-BP1 expression (7). In these examples, stress-induced 4E-BP1 expression was shown to be mediated by transcription factors (FOXO inDrosophila and ATF4 in mouse endocrine pancreas) that bind to and activate the transcription of 4E-BP1 gene. On the basis of these data, stress-dependent 4E-BP1 activation, which can result from hypophosphorylation and/or enhanced expression, is believed to exert a protective effect by limiting excessive synthesis of proteins and consequently limiting cell proliferation that could otherwise have deleterious effects. Contact inhibition and hypoxia are 2 other situations in which 4E-BP1 protein expression is induced.We and others have actually observed in different cell types of various organisms that continuous cell culture and contact Authors' Affiliations: Institut national de la sant e et de la recherche medicale (INSERM) UMR-1037, Universit e Paul Sabatier; and Pôle Digestif, Centre Hospitalier Universitaire de Toulouse, Toulouse, France Current address for R. Azar: Department of Clinical Pharmacy, Faculty of Pharmacy, Lebanese University, Hadath, Lebanon. Corresponding Author: St ephane Pyronnet, Institut national de la sant e et de la recherche medicale (INSERM) U1037, BP 84225, Toulouse 31432, France. Phone: 33-561-32-24-02; Fax: 33-561-32-24-03; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-12-0095 2012 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 11(1) January 2013 54 on June 19, 2017. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst November 21, 2012; DOI: 10.1158/1541-7786.MCR-12-0095