title: Expression of Iron Regulatory Protein 1 Is Regulated not only by HIF-1 but also pCREB under Hypoxia.
abstract: The inconsistent of responses of IRP1 and HIF-1 alpha to hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). We demonstrated that 1) Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3 DNA-protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under hypoxia could be prevented by LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2 siRNA had no effect on the expression of pCREB and IRP1 proteins in cells treated with hypoxia for 8 hours. Our findings evidenced for the involvement of pCREB in IRP1 expression and revealed a dominant role of PI3K/Akt pathway in CREB activation under hypoxia and also suggested that dual-regulation of IRP1 expression by HIF-1 and pCERB or other transcription factor(s) under hypoxia might be a common mechanism in most if not all of hypoxia-inducible genes.
authors.affiliation: Department of Biochemistry, Institute for Nautical Medicine, Nantong University, Nantong, 226001, China; Laboratory of Neuropharmacology, FudanUniversity School of Pharmacy,826 Zhang Heng Road, Pu Dong, Shanghai201203, China.
authors.forename: Qian-Qian
authors.initials: QQ
authors.lastname: Luo
authors.affiliation: Department of Biochemistry, Institute for Nautical Medicine, Nantong University, Nantong, 226001, China; Laboratory of Neuropharmacology, FudanUniversity School of Pharmacy,826 Zhang Heng Road, Pu Dong, Shanghai201203, China.
authors.forename: Zhong-Ming
authors.initials: ZM
authors.lastname: Qian
authors.affiliation: Laboratory of Neuropharmacology, FudanUniversity School of Pharmacy,826 Zhang Heng Road, Pu Dong, Shanghai201203, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, NT, Hong Kong.
authors.forename: Yu-Fu
authors.initials: YF
authors.lastname: Zhou
authors.affiliation: Laboratory of Neuropharmacology, FudanUniversity School of Pharmacy,826 Zhang Heng Road, Pu Dong, Shanghai201203, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, NT, Hong Kong.
authors.forename: Meng-Wan
authors.initials: MW
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, Institute for Nautical Medicine, Nantong University, Nantong, 226001, China.
authors.forename: Dang
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Institute for Nautical Medicine, Nantong University, Nantong, 226001, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Laboratory of Neuropharmacology, FudanUniversity School of Pharmacy,826 Zhang Heng Road, Pu Dong, Shanghai201203, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, NT, Hong Kong.
authors.forename: Ya
authors.initials: Y
authors.lastname: Ke
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005455:Fluorescent Antibody Technique; D056945:Hep G2 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D035941:Iron Regulatory Protein 1 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D035941:Iron Regulatory Protein 1; D034741:RNA, Small Interfering; D000099162:Endothelial PAS Domain-Containing Protein 1
keywords: HepG2 cells.; Hypoxia; cyclic AMP-responsive element-binding protein (CREB); hypoxia-inducible factor-1 (HIF-1); iron regulatory proteins 1