Identification of Oxygen Sensor Expressed in Newborn Rats

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Molecular oxygen (O2) is essential for aerobic organisms. Exposure of tissues or cells to hypoxia induces a variety of adaptive or pathogenic responses. Accumulated evidence indicates that hypoxia activities collagen synthesis in tissues. To explore the molecular mechanism we screened those genes which 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) a(I), an active subunit which catalyzes the oxygen-dependent hydroxylation of proline residue in procollagen, was increased by 2 to 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, the protein level and the hydroxy proline content, an indicator of the prolyl hydroxylation, were all elevated by hypoxic culture. Analysis of the promotor region of PHa(I) gene indicated that a motif similar to hypoxia responsive element (HRE) of hypoxia-inducible genes such as erythropoietin was identified within 120-base pair sequence upstream 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 (HIF-1) was stimulated and bound to the PHa(I) HRE upon hypoxic challenge. Our results indicate that PHa(I), an essential enzyme for collagen synthesis, is a target gene for HIF-1.