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 gen es that are up-regulated or down-regulated by hypoxia, Fibroblasts isolated from fetal rat lung were cultured under hypoxia, Differential display tech nique 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 hypoxi a, This elevated level was maintained over 40 h and returned to the basal l evel after reoxygenation, The transcription rate, protein level, and hydrox yproline content tan indicator of the prolyl hydroxylation) were all elevat ed by hypoxic culture. Analysis of the promotor region of PH alpha(I) gene indicated that a motif similar to hypoxia-responsive element (HRE) of hypox ia-inducible genes such as erythropoietin, was identified within a 120-base pair sequence upstream of the transcription start site. Luciferase reporte r assay and mutational analysis showed that a site similar to the HRE in th is motif is functionally essential to hypoxic response. Electrophoretic mob ility shift assay revealed that hypoxia-inducible factor-1 was stimulated a nd bound to the PH alpha(I) HRE upon hypoxic challenge. Our results indicat e that PHa(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.