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) α(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 PHα(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 PHα(I) HRE upon hypoxic challenge. Our results indicate that PHα(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1. 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) α(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 PHα(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 PHα(I) HRE upon hypoxic challenge. Our results indicate that PHα(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1. hypoxia-inducible factor prolyl 4-hydroxylase hypoxia-responsive element arylhydrocarbon receptor nuclear translocator desferroxamine Hepa-1c1c7 cell fetal calf serum Dulbecco's modified Eagle's medium 0.1% Tween 20-Tris-buffered saline phosphate-buffered saline kilobase(s) cAMP-response element-binding protein 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.Helfman T. Falanga V. Am. J. Med. Sci. 1993; 306: 37-41Crossref PubMed Scopus (79) Google Scholar). 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.Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4995) Google Scholar), is a heterodimer composed of HIF-1α and arylhydrocarbon receptor nuclear translocator (ARNT). HIF-1α and ARNT retain a basic helix-loop-helix domain and a Per-ARNT/aryl hydrocarbon receptor Sim domain in their N termini (2.Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4995) Google Scholar). Hypoxia induces stabilization of HIF-1α (3.Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1836) Google Scholar), heterodimerization of HIF-1α and ARNT (4.Jiang B.-H. Rue E. Wang G.L. Roe R. Semenza G.L. J. Biol. Chem. 1996; 271: 17771-17778Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar), 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.Arany Z. Huang L.E. Eckner R. Bhattacharya S. Jiang C. Goldberg M.A. Bunn H.F. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12969-12973Crossref PubMed Scopus (625) Google Scholar). 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.Firth J.D. Ebert B.L. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 21021-21027Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 7.Li H. Ko H.P. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 21262-21267Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 8.Semenza G.L. Jiang B.-H. Leung S.W. Passantino R. Concordet J.-P. Maire P. Giallongo A. J. Biol. Chem. 1996; 271: 32529-32537Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar, 9.Takahashi Y. Takahashi S. Yoshimi T. Miura T. Eur. J. Biochem. 1998; 254: 497-504Crossref PubMed Scopus (34) Google Scholar), Epo (2.Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4995) Google Scholar), vascular endothelial growth factor (10.Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar), and tyrosine hydroxylase (11.Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). 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.Durmowicz A.G. Parks W.C. Hyde D.M. Mecham R.P. Stenmark K.R. Am. J. Pathol. 1994; 145: 1411-1420PubMed Google Scholar). Recent studies have revealed that in vivoexposure of rats to hypoxia increases prolyl hydroxylase activity in skeletal muscle (13.Perhonen M. Han X. Wang W. Karpakka J. Takala T.E. J. Appl. Physiol. 1996; 80: 2226-2233Crossref PubMed Scopus (10) Google Scholar) and increases the concentration of collagenous proteins in cardiac muscle (14.Ostadal B. Kolar F. Pelouch V. Widimsky J. Physiol. Res. 1995; 44: 45-51PubMed Google Scholar). Moreover, in vitro exposure of rat mesangial cells (15.Kim S.B. Kang S.A. Park J.S. Lee J.S. Hong C.D. Nephron. 1996; 72: 275-280Crossref PubMed Scopus (26) Google Scholar) and dermal fibroblasts (16.Falanga V. Martin T.A. Takagi H. Kirsner R.S. Helfman T. Pardes J. Ochoa M.S. J. Cell. Physiol. 1993; 157: 408-412Crossref PubMed Scopus (137) Google Scholar) to hypoxia results in the enhancement of type IV collagen protein level and type I procollagen mRNA level, respectively. These observations indicate that systemic and cellular hypoxia modulates collagen synthesis in several types of cells. Synthesis of collagen molecules can be regulated at several steps (17.Hay E.D. Collagen. Plenum Press, New York, NY1991Google Scholar). After translation, procollagens are hydroxylated on proline residues at the endoplasmic reticulum and form stable triple-helical trimers. The secreted procollagen trimer is proteolytically cleaved at both N-and C-propeptides in the extracellular space, where collagen trimers polymerize and are deposited nonenzymatically. These sequential steps of collagen synthesis and hypoxic effects on several stages in the expression of multiple types of collagen genes imply that hypoxic regulatory factors may be involved in common reactions during the collagen synthesis process. To explore the molecular mechanism, we screened hypoxia-responsive genes in isolated fetal rat lung fibroblasts using the differential display method. We subsequently found that prolyl 4-hydroxylase (PH) α(I), an active subunit that catalyzes oxygen-dependent hydroxylation of proline residue in procollagen, is up-regulated by hypoxia via HIF-1 transcription factor complex. Fibroblasts were isolated from the fetal rat lung at 19 days of gestation (18.Batenburg J.J. Otto-Verberne C.J. Ten-Have-Opbroek A.A. Klazinga W. Biochim. Biophys. Acta. 1988; 960: 441-453Crossref PubMed Scopus (53) Google Scholar). IMR-90 and WI-38, human fetal lung fibroblast cell lines, were obtained from the RIKEN Cell Bank (Tokyo, Japan). These cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mm sodium pyruvate, 0.1 mm minimum essential medium nonessential amino acid, 1× minimum essential medium vitamin, and 15 mmHEPES (pH 7.4). Fetal rat lung fibroblasts between the 20th and 25th passages were used for the experiments. The mouse hepatoma cell lines Hepa-1c1c7 (Hepa-1) and its ARNT-defective derivative, Hepa-c4, were generously supplied by O. Hankinson. Hepa-c4 cells lack ARNT function and also fail to express the ARNT protein (19.Hoffman E.C. Reyes H. Chu F.F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Crossref PubMed Scopus (836) Google Scholar). These cells were cultured in the minimal essential medium-α without nucleosides (Life Technologies, Inc.) supplemented with 10% FCS, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, and 15 mm HEPES (pH 7.4). At subconfluence, cells were exposed to hypoxia or treated with chemicals. Exposures of cells to hypoxia were performed as described previously (9.Takahashi Y. Takahashi S. Yoshimi T. Miura T. Eur. J. Biochem. 1998; 254: 497-504Crossref PubMed Scopus (34) Google Scholar). In some experiments, 200 μm ascorbic acid was added to the medium. We followed the fluorescent differential display method developed by T. Y. H. Y. 1994; PubMed Scopus Google Scholar) with several as described in a (9.Takahashi Y. Takahashi S. Yoshimi T. Miura T. Eur. J. Biochem. 1998; 254: 497-504Crossref PubMed Scopus (34) Google Scholar). assay was performed as described previously (9.Takahashi Y. Takahashi S. Yoshimi T. Miura T. Eur. J. Biochem. 1998; 254: 497-504Crossref PubMed Scopus (34) Google Scholar). The of PHα(I) was by with the for analysis were by with rat from the lung as previously Y. Takahashi S. Miura T. M. Appl. PubMed Scopus Google Scholar). The and were rat PHα(I), and was performed as described previously Y. Takahashi S. Yoshimi T. Miura T. T. PubMed Scopus Google Scholar). After analysis of the PHα(I) mRNA level, the was with a at The was for The of the mRNA was to the V. F. Res. PubMed Scopus Google Scholar). The fibroblast was with phosphate-buffered saline and by at 100 for at Cell were with The cell were in a of 0.1 mm (pH at 0.1% mm mm and on for followed by in a at with The was for by protein was performed as previously (9.Takahashi Y. Takahashi S. Yoshimi T. Miura T. Eur. J. Biochem. 1998; 254: 497-504Crossref PubMed Scopus (34) Google Scholar) with some of protein from were to 10% and at from the to The were with mouse at of 0.1% Tween 20-Tris-buffered saline for 1 h at and in for 15 The were with at a of for 1 After with were using an to the To proline in the fibroblasts at were cultured in 10% with or without 200 μm ascorbic acid under or the cells were maintained in 0.1% with or without 200 μm ascorbic acid under or The medium was and in for h at To the proline in the cells and fibroblasts were cultured for The medium was After with the cell was by and The proline in the was by the method PubMed Scopus Google Scholar, S. T. J. S. J. Med. PubMed Scopus Google Scholar). The content of the cell was by the method using C. Biochem. PubMed Scopus Google Scholar). The protein content was by the method using the protein assay to the was used as the The rat was screened rat PHα(I) a from of the transcription site to the was to responses of PHα(I) gene to hypoxia. Y. and T. in region from to start site was by the was to reporter and of from to and from to were and and respectively. of the HRE sequence J.D. Ebert B.L. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) at to in the of the by of the with to was by using the were by of rat lung and Hepa-c4 cells were cultured in the subconfluence, cells were by the or the with 0.1 of an of and of medium complex were added to the cultured cells. After h cells were exposed to hypoxic for and Cell were used to activity using The cell was with and After at 100 for at cell were with in for and on for with a of cell mm mm mm mm 1 mm 1 mm sodium 1× were at by multiple T. Y. H. Y. 1994; PubMed Scopus Google Scholar) passages a followed by at for 15 at The were in and at Epo J.D. Ebert B.L. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), PHα(I) to with the transcription start site as and were used as or of were with and and in mm mm and mm by at for followed by to over The were by an binding was by 15 of cell with of binding mm 1 mm mm mm of 1× for at and were added and for a at were at on in mm mm acid, mm (pH at 1 of or as were with 15 of cell at for of between were performed by the of or after analysis of were up-regulated or down-regulated by a exposure of fetal rat lung fibroblasts to were screened by the differential display technique using of and with and a at pair where the from hypoxic cells is that from cells This was by with the pair of using of this with a of sequence revealed that this was the of PHα(I) To the of PHα(I) gene expression to oxygen the level of PHα(I) mRNA was by Fibroblasts at were cultured for h at of from to The PHα(I) mRNA level increased in a hypoxic The PHα(I) mRNA to increased to the after culture. This was by hypoxic of 2 to in the PHα(I) mRNA level after hypoxia their at a that of the was in the cells cultured under 2 in the PHα(I) mRNA level after h hypoxia and its after This increased level was maintained for 40 h of hypoxic After fibroblasts were returned from h of hypoxic to the elevated level of PHα(I) mRNA after h and returned to the after h of reoxygenation. IMR-90 and WI-38, human fetal lung fibroblast cell lines, also showed increases in the PHα(I) mRNA level by the in to h of hypoxic exposure The of PHα(I) mRNA by hypoxic to a transcriptional on PHα(I) expression. To this we PHα(I) gene transcription in isolated The transcriptional of PHα(I), to the increased to and of the after and h of exposure to of to be under the a gene (6.Firth J.D. Ebert B.L. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 21021-21027Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), showed and increases in the transcription after and h of respectively. To the protein level of in we the cell by protein for the of a at in the which with PHα(I) After h of exposure to the protein level of increased to the level The protein level its after h of which was the The elevated level was maintained after h exposure to hypoxia. This protein to hypoxic was similar to that observed for mRNA of proline residue in procollagen in the endoplasmic reticulum and is important for the procollagen procollagen is for from the cell to the in which the and of procollagen are cleaved in of collagen on the cell To the in fibroblasts were cultured under or hypoxia in the or of ascorbic We the of proline residue secreted from fibroblasts the medium over the of ascorbic acid, hypoxic on the hydroxyproline content of the medium ascorbic acid the hydroxyproline content of the medium to of the ascorbic culture. without ascorbic acid, hypoxic the hydroxyproline level from to of ascorbic and We the hydroxyproline residue that accumulated in the cell or during the acid and hypoxic the hydroxyproline level in the cell and cell in the as was observed in the medium These results indicate that hypoxia-induced of PHα(I) mRNA to elevated protein which contribute to the of hydroxylation in ascorbic as as to of hydroxylation in ascorbic 1 is up-regulated by of or oxygen B.L. J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). HIF-1 is a transcription factor that hypoxic induction of a of and desferroxamine an can the HIF-1 to hypoxia M. Ratcliffe P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the HIF-1 or in is involved in the of PHα(I) by HIF-1 and and were added to the fibroblast was from cells that were treated with for h and the PHα(I) mRNA level was by and increased the PHα(I) mRNA level by in a and and and increased the PHα(I) mRNA to This was that of hypoxic Hepa-c4, the ARNT-defective and its type were exposed to and for and for PHα(I) response. The level of PHα(I) mRNA in cells was PHα(I) mRNA in Hepa-c4 cells under hypoxic This indicates that ARNT may be an essential transcription factor subunit for hypoxic induction of PHα(I) mRNA in cells. To regulatory that hypoxic of the PHα(I) we screened the rat a rat PHα(I) a from upstream of the transcription site to the was to responses of the gene to hypoxia are have deposited in the under to of rat PHα(I) promotor region was in of and the was in the fibroblasts activity cells by activity the This that the region activity to the hypoxic effects on the of the fibroblasts to hypoxia for h stimulated expression by was observed after hypoxic of cells with or and its ARNT-defective Hepa-c4, were with showed a induction of after h of hypoxic In exposure of Hepa-c4 to hypoxia expression These results that the PHα(I) may the hypoxia-responsive which the complex for binding in the region revealed binding site to the for G.L. J. Med. 1998; Full Text PDF PubMed Scopus Google Scholar) on the sequence P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which to be for hypoxic was also identified from to on the To the region that hypoxia to PHα(I), we the activity of The which the HRE showed a to hypoxic induction after h of to fetal lung fibroblasts the which the HRE activity after hypoxic culture. This HRE in the PHα(I) to hypoxia was by a of hypoxic induction in a After h of showed a induction in cells in Hepa-c4 cells of by and of reporter gene The of was to and which to and to respectively. The a sequence the This sequence was in expression Hepa-c4 and fetal rat lung fibroblasts were by the or the with an which the After h of exposure of the cells to or hypoxia for for the activity was and for to the Luciferase in hypoxic cells to in cells are of at To the HRE sequence identified in the PHα(I) was the target of cell from cells were by mobility shift The PHα(I) binding activity in cells This was in cells binding activity was in cells cultured under both and hypoxia 1 and This of the PHα(I) was also observed in cell from fibroblasts hypoxic induction of binding was identified in cell from ARNT-defective Hepa-c4 cells The of the between the and hypoxia-induced factors was by with of PHα(I) the binding of the and inducible of a PHα(I) with PHα(I) with to the HRE in the Epo a binding of the complex to PHα(I) To the hypoxia-induced cell were with a to HIF-1α the mobility shift showed that HIF-1α with the HRE sequence of PHα(I) gene used as the HIF-1 binding to the PHα(I) In the and C.J. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), and H. H. P. C. Full Text PDF PubMed Scopus (10) Google Scholar) that hypoxia increased activity of procollagen prolyl the regulatory step of the activity In this we have that exposure of fetal lung fibroblasts to hypoxia activates the PHα(I) gene the HIF-1 transcription factor the binding of which to the hypoxic element in the region of the PHα(I) gene to an in transcription rate, level of cellular protein level, and proline level in the ascorbic culture. have for gene induction by hypoxic (1.Helfman T. Falanga V. Am. J. Med. Sci. 1993; 306: 37-41Crossref PubMed Scopus (79) Google Scholar). The transcription factors HIF-1α and M. J. H. J. Y. J. PubMed Google Scholar) as or a regulatory in hypoxic gene In nuclear factor 1 can the hypoxic via a in by and 1993; PubMed Scopus Google Scholar). X. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar) that hypoxia gene expression via of a transcription factors HIF-1 have also Ratcliffe P.J. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar). The results that the and the PHα(I) mRNA level In both and PHα(I) gene expression. and hypoxic and transcription of a of genes that are by hypoxic M. Ratcliffe P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, cells of an essential of HIF-1 and transcription factor PHα(I) mRNA level and reporter gene and The of HIF-1 transcription factor complex and hypoxia-responsive element in the PHα(I) gene region were by the reporter gene assay and the mobility shift and region from to the transcription start which was to reporter the In that PHα(I) is HRE sequence and an which are similar to several of genes such as Epo and vascular endothelial growth factor (10.Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. R. J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). These results indicated that the HRE sequence in PHα(I) is and for hypoxic enhancement of the transcriptional of the PHα(I) The binding of the HIF-1 complex to the PHα(I) HRE sequence was by of the complex by the of a HIF-1α and by in binding between PHα(I) and Epo in the mobility shift These indicate that HIF-1 binding to the HRE sequence in the PHα(I) is important to hypoxic of the PHα(I) PHα(I) gene expression be also at the posttranscriptional in the of PHα(I) mRNA after h of hypoxic exposure the protein level of was elevated by h hypoxic exposure The that the protein is that of the may be to the stabilization of the protein under oxygen of cells and cells to hypoxia increases mRNA of vascular endothelial growth factor E. W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and tyrosine hydroxylase J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), respectively. in both and transcription of after hypoxic exposure contribute to their elevated the in which in the posttranscriptional steps contribute to the expression of the PHα(I) gene In collagen synthesis, is a which catalyzes the of an essential residue for the of the procollagen molecules Biol. 1998; 72: Google Scholar). The active enzyme of of and The the of the site Biochem. 1995; PubMed Scopus Google Scholar). The subunit is to the enzyme protein and is in of the Biochem. 1995; PubMed Scopus Google Scholar). the of the enzyme the of and ascorbic acid are The is during with of the the the is the on the proline residue Biol. 1998; 72: Google Scholar, Biochem. 1995; PubMed Scopus Google Scholar). Although is a for the cell by and C.J. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) showed that exposure of fibroblasts to 0.1% hydroxylation of proline in synthesized collagen in the cells. The that the proline content is by hypoxic under ascorbic acid In the ascorbic hypoxic exposure increased the proline content in with the culture. such as and can be for ascorbic acid to activity in ascorbic acid is the for both in vitro and in Biol. 1998; 72: Google Scholar, R. Biochem. Biophys. Res. PubMed Scopus Google Scholar). In in the of the to which under the in hydroxylation be to the in prolyl as as a of results that in a cell hypoxic may be the factor in procollagen hydroxylation and may the for ascorbic acid in prolyl residue is critical in triple-helical collagen In to including of pulmonary proteins and and have and are for prolyl hydroxylase Biol. 1998; 72: Google Scholar). of protein is by an of prolyl and hydroxylase P. E.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In pulmonary in the is P. A. A. J. PubMed Scopus Google Scholar), synthesis of collagen and proteins and is active in the lung E.C. Am. J. Physiol. 1995; Google Scholar, M. W. A. S. 1998; Google Scholar, J.S. Physiol. PubMed Scopus Google Scholar). this active synthesis is essential for the start of after the HIF-1 activation of PHα(I) may an important in of prolyl hydroxylation of procollagen and collagenous protein under oxygen in the fetal We O. Hankinson for in the hepatoma cell lines Hepa-1c1c7 and for the of this for and M. for