title: Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA.
abstract: CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3'-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH-RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH-RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis.
authors.affiliation: Department of Oral and Maxillofacial Surgery, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan. seikondo12@dent.showa-u.ac.jp
authors.forename: Seiji
authors.initials: S
authors.lastname: Kondo
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kubota
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Mukudai
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishida
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Yoshihama
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Shirota
authors.forename: Satoru
authors.initials: S
authors.lastname: Shintani
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
mesh_terms: D020413:3' Untranslated Regions; D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor; D055513:Connective Tissue Growth Factor / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism*; D006801:Humans; D009243:NAD / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000235:genetics; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D050436:Regulatory Elements, Transcriptional
chemical_list: D020413:3' Untranslated Regions; C526317:CCN2 protein, human; D016877:Oxidants; D012333:RNA, Messenger; D009243:NAD; D055513:Connective Tissue Growth Factor; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)