Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells.
Accumulated evidence indicates that hypoxia activates collagen synthesis in tissues. To explore the molecular mechanism of activation, we screened genes that are up-regulated or down-regulated by hypoxia. Fibroblasts isolated from fetal rat lung were cultured under hypoxia. Differential display technique showed that the mRNA level of prolyl 4-hydroxylase (PH) alpha(I), an active subunit that catalyzes the oxygen-dependent hydroxylation of proline residue in procollagen, increased 2-3-fold after an 8-h exposure to hypoxia. This elevated level was maintained over 40 h and returned to the basal level after reoxygenation. The transcription rate, protein level, and hydroxyproline content (an indicator of the prolyl hydroxylation) were all elevated by hypoxic culture. Analysis of the promotor region of PHalpha(I) gene indicated that a motif similar to hypoxia-responsive element (HRE) of hypoxia-inducible genes such as erythropoietin, was identified within a 120-base pair sequence upstream of the transcription start site. Luciferase reporter assay and mutational analysis showed that a site similar to the HRE in this motif is functionally essential to hypoxic response. Electrophoretic mobility shift assay revealed that hypoxia-inducible factor-1 was stimulated and bound to the PHalpha(I) HRE upon hypoxic challenge. Our results indicate that PHalpha(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.
Takahashi
Y
Y
Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. yuji@ls.toyaku.ac.jp
Takahashi
S
S
Shiga
Y
Y
Yoshimi
T
T
Miura
T
T
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D015687:Cell Hypoxia; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000502:physiology; D004790:Enzyme Induction; D006801:Humans; D006900:Hydroxylation; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014157:Transcription Factors; D014158:Transcription, Genetic; D015854:Up-Regulation
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D004247:DNA; D011393:Procollagen-Proline Dioxygenase