title: Glutathione peroxidase 8 is transcriptionally regulated by HIFα and modulates growth factor signaling in HeLa cells.
abstract: GPx8 is a mammalian Cys-glutathione peroxidase of the endoplasmic reticulum membrane, involved in protein folding. Its regulation is mostly unknown. We addressed both the functionality of two hypoxia-response elements (HREs) within the promoter, GPx8 HRE1 and GPx8 HRE2, and the GPx8 physiological role. In HeLa cells, treatment with HIFα stabilizers, such as diethyl succinate (DES) or 2-2'-bipyridyl (BP), induces GPx8 expression at both mRNA and protein level. Luciferase activity of pGL3(GPx8wt), containing a fragment of the GPx8 promoter including the two HREs, is also induced by DES/BP or by overexpressing either individual HIFα subunit. Mutating GPx8 HRE1 within pGL3(GPx8wt) resulted in a significantly higher inhibition of luciferase activity than mutating GPx8 HRE2. Electrophoretic mobility-shift assay showed that both HREs exhibit enhanced binding to a nuclear extract from DES/BP-treated cells, with stronger binding by GPx8 HRE1. In DES-treated cells transfected with pGL3(GPx8wt) or mutants thereof, silencing of HIF2α, but not HIF1α, abolishes luciferase activity. Thus GPx8 is a novel HIF target preferentially responding to HIF2α binding at its two novel functional GPx8 HREs, with GPx8 HRE1 playing the major role. Fibroblast growth factor (FGF) treatment increases GPx8 mRNA expression, and reporter gene experiments indicate that induction occurs via HIF. Comparing the effects of depleting GPx8 on the downstream effectors of FGF or insulin signaling revealed that absence of GPx8 results in a 16- or 12-fold increase in phosphorylated ERK1/2 by FGF or insulin treatment, respectively. Furthermore, in GPx8-depleted cells, phosphorylation of AKT by insulin treatment increases 2.5-fold. We suggest that induction of GPx8 expression by HIF slows down proliferative signaling during hypoxia and/or growth stimulation through receptor tyrosine kinases.
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Bosello-Travain
authors.affiliation: Life and Environmental Sciences, University of California at Merced, Merced, CA 95344, USA.
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Forman
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Roveri
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Toppo
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Ursini
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy.
authors.forename: Rina
authors.initials: R
authors.lastname: Venerando
authors.affiliation: Department of Nephrology and Hypertension, Translational Research Center, University Hospital Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Warnecke
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Zaccarin
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy. Electronic address: matilde.maiorino@unipd.it.
authors.forename: Matilde
authors.initials: M
authors.lastname: Maiorino
mesh_terms: D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000595:Amino Acid Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D015687:Cell Hypoxia; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D016415:Sequence Alignment; D015398:Signal Transduction; D013386:Succinates / Q000494:pharmacology; D014158:Transcription, Genetic; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D015082:2,2'-Dipyridyl; D051792:Basic Helix-Loop-Helix Proteins; D005346:Fibroblast Growth Factors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007328:Insulin; D008156:Luciferases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010544:Peroxidases; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013386:Succinates; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C558642:GPX8 protein, human; C535150:MAPK1 protein, human; C052819:diethyl succinate
keywords: Endoplasmic reticulum; Free radicals; GPx8; Hydroperoxide; Hypoxia; Receptor tyrosine kinase; Redox; Signaling