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) ␣(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 PH␣(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 PH␣(I) HRE upon hypoxic challenge. Our results indicate that PH␣(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.Restricted oxygen availability is a feature of many physiologic and pathologic conditions, including high altitude residence, fetal development in the uterus, pulmonary fibrosis, wounded tissue, and neoplasm (1). Systemic and cellular responses to reduced oxygen tension (hypoxia) are initiated by activation and/or inactivation of gene expression. Hypoxia-inducible factor-1 (HIF-1), 1 which was originally found to be a critical mediator for the inducible expression of the erythropoietin (Epo) gene by hypoxia (2), is a heterodimer composed of HIF-1␣ and arylhydrocarbon receptor nuclear translocator (ARNT). HIF-1␣ and ARNT retain a basic helix-loop-helix domain and a Per-ARNT/aryl hydrocarbon receptor Sim domain in their N termini (2). Hypoxia induces stabilization of HIF-1␣ (3), heterodimerization of HIF-1␣ and ARNT (4), and the binding of the heterodimer to the hypoxia-responsive element (HRE) in the regulatory region of the target genes with the transcriptional coactivator p300/CREB-binding protein (5). Although posttranscriptional mechanisms may contribute to the induction of hypoxia-sensitive genes, activation of the HIF-1 complex is an important step leading to hypoxia-mediated induction of glycolytic enzymes (6 -9), Epo (2), vascular endothelial growth factor (10), and tyrosine hydroxylase (11).In the remodeling of the small muscular pulmonary artery observed in hypoxia-induced pulmonary hypertension, type I collagen is actively synthesized and accumulated in the media and the adventitia of the artery (12). Recent studies have revealed that in vivo exposure of rats to hypoxia increases prolyl ...

Stress proteins (heat shock proteins, HSPs) have been proposed as general biomarkers for environmental monitoring. In the present study, we evaluated the environmental stress-burden on the aquatic midge Chironomus yoshimatsui using hsp70 expression. Larvae collected from streams receiving polluted runoff (field strain) were resistant to the organophosphorus insecticide, fenitrothion (F), and the synthetic pyrethroid, ethofenprox (E), whereas a strain originally collected from an unpolluted area (susceptible strain) showed low resistance to insecticide exposure. To examine the expression of an HSP70 gene in C. yoshimatsui, an hsp70 cDNA probe was prepared using RNA obtained from the field strain larvae and used for Northern blot analyses. The expression of this HSP70 gene in larvae collected from two field sites in May about 1 week after insecticide spraying in the fields was 2.3 (p = 0.018) to 3.3 fold higher than that in the susceptible strain and was also 4.6 and 1.4 (p = 0.033) fold higher than those collected in November 3 months after the cessation of insecticide spraying. In order to identify potential inducers of the HSP70 gene of the field strain, larvae of the susceptible strain were exposed to F or E for 24 h and hsp70 mRNA levels determined. Exposures to F at 0.4 microg/L and E at 1.1 microg/L increased hsp70 mRNA levels 2.7 (p = 0.049) and 4.4 (p = 0.043) fold over controls, respectively. These results suggest that larvae collected from polluted areas are burdened by environmental stressors and the tested insecticides are potential inducers of HSP70. The results also support the suggestion that HSP70 gene expression is a sensitive indicator of low level (nonlethal) exposures to certain insecticides.

Molecular oxygen (O 2 ) is essential for aerobic organisms. Exposure of tissues or cells to hypoxia induces a variety of adaptive or pathogenic responses. To understand the mechanism and processes of cellular response to hypoxia, we exposed fetal rat lung fibroblasts to hypoxia (pO 2 Ϸ5 Pa) and screened the hypoxia-responsible gene by the differential display method. Exposure of the cells to hypoxia activated the phosphoglycerate mutase B (PGM-B) gene, resulting in the induction of PGM enzymatic activity, concomitant with elevations of PGM-B mRNA and protein levels. The mRNA level was elevated linearly with decreases in partial O 2 pressure, indicating a 2Ϫ3-fold increase in these levels after 16 h hypoxia. Up-regulation of PGM mRNA by hypoxia was obvious after 8 h exposure, reached its peak after 16 h, persisting for 40 h and returned to the basal level after reoxygenation at 20% O 2 for 16 h. Run-on and stability assays indicated that PGM-B expression is regulated mainly at the transcriptional step. These results suggest that the induction of PGM-B may contribute to the regulation of the glycolytic flux under reduced O 2 tension and play a role in the adaptation of cells to hypoxia.Keywords : hypoxia ; reoxygenation ; phosphoglycerate mutase B; differential display; fibroblast.Molecular oxygen (O 2 ) is essential for aerobic organisms. within minutes, after hypoxic exposure. It is generally accepted Principally, O 2 participates in oxidation-reduction reactions in that the Pasteur effect relates to an acute increase in the intracelthe biosystem and functions as a terminal electron acceptor of lular phosphofructokinase activity resulting from changes in the mitochondrial oxidative phosphorylation in the production of en-concentration of low molecular-mass allosteric effectors, alergy [1]. O 2 is also the means of oxygenation by which bioactive though other alterations in enzyme activities participate [6,7]. materials, such as steroid hormones and prostaglandins, are syn-Chronic hypoxic exposure of a variety of tissues and cells results thesized. Furthermore, desaturation of fatty acid and hydroxyla-in a substantial additional increase in the rate of glycolysis betion of proline also require O 2 for enzymatic reactions. The par-yond that produced by the Pasteur effect [8]. tial O 2 pressure needed for enzymatic reactions varies depending Enzymatic activities of all glycolytic components of epitheon the type of enzyme [2].lial and mesenchymal cell lines are coordinately enhanced by Ischemia and/or hypoxia cause a reduced tension of O 2 in chronic hypoxia [9,10]. Although the detailed mechanism by cells and tissues. In an acute response to reduced O 2 concentra-which the glycolytic activity is substantially increased after tions in tissues, hyperventilation is the primary systemic adapta-chronic hypoxia is unclear, some glycolytic enzymes, but not tion. It results in an increase in arterial O2 tension, enhancing all, are up-regulated at transcription levels. Steady-state mRNA delivery of O 2 to distal tissues [3]....

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