Hypoxic Induction of Prolyl 4-Hydroxylase α(I) in Cultured Cells

Abstract

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 ...

HIF-1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation

“…Although the HIF-1␣ prolyl hydroxylase has not yet been identified, this finding suggests that under hypoxic conditions, the regulated dissociation of HIF-1␣ from VHL might be the result of a loss of this modification. However, as has been noted recently (12), the only known prolyl hydroxylases (prolyl hydroxylases I and II, which hydroxylate collagen) can function under hypoxic conditions (14). This finding, therefore, does not readily explain the weakening of the HIF-1␣-VHL interaction, and raises the possibility that modification of other HIF-1␣ residues may contribute to the regulated interaction.…”

Brain Genomic Response following Hypoxia and Re-oxygenation in the Neonatal Rat

“…In addition to known HIF-1 target genes (VEGF, GLUT-1, prolyl 4-hydroxylase ␣, and adrenomedullin) (35)(36)(37)(38) and other known hypoxic inducible genes (MT-1 and MKP-1) (21,23,34), this study revealed new genes regulated by hypoxia/re-oxygenation in brain. For example, hypoxic regulation of CELF, 12-lipoxygenase, t-PA, CAR-1, and many other transcripts shown in the tables has not been reported.…”

“…Indeed, GLUT-1 is highly expressed in endothelial cells at the blood-brain barrier (45), and hypoxia increases this expression (46). Prolyl 4-hydroxylase ␣, an active subunit that catalyzes oxygen-dependent hydroxylation of proline residues in procollagen, is also an HIF-1 target gene (38). The loss of basal lamina, including type IV collagen, compromises microvascular integrity in cerebral ischemia that likely contributes to edema and hemorrhage (47).…”

“…In addition to known hypoxic inducible genes such as MAP kinase phosphatase-1 (MKP-1) and metallothionein-1 (MT-1) (21, 23, 34) including those under HIF-1 control (VEGF, glucose transporter-1 (GLUT-1), adrenomedullin, prolyl 4-hydroxylase ␣) (35)(36)(37)(38), this study revealed new genes induced by hypoxia and/or re-oxygenation in brain (Tables III and IV). The list of genes includes transcriptional factors, signal transduction molecules, receptors, transporters, channels, proteases, genes implicated in apoptosis, detoxification, inflammation, vasodilata- tion, collagen synthesis, and membrane trafficking.…”

Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling

“…For instance, regulation of collagen type V, alpha 1 may form part of a more general response of collagen metabolism to the hypoxia pathway. Prolyl 4-hydroxylase alpha 1, a key enzyme in collagen biosynthesis, has recently been identi®ed as a HIF-1 dependent hypoxia inducible gene (Takahashi et al, 2000). Aminopeptidase A is an ectopeptidase member of the peptidase family m1 proposed to play a role in the progression of cervical neoplasms (Fujimura et al, 2000).…”