Oxygen tension regulates the expression of a group of procollagen hydroxylases Karl-Heinz Hofbauer 1 , Bernhard Gess 1 , Christiane Lohaus 2 , Helmut E. Meyer 2 ,Do¨ rte Katschinski 3 and Armin Kurtz 1 1 Institut fu ¨ r Physiologie der Universita ¨ t Regensburg, Germany; 2 Medizinisches Proteom, Center der Ruhr, Universita ¨ t Bochum, Germany; 3 Abteilung Zellphysiologie der Martin-Luther Universita ¨ t Halle, Germany In this study, we have characterized the influence of hypoxia on the expression of hydroxylases crucially involved in col- lagen fiber formation, such as prolyl-4-hydroxylases (Ph4) and procollagen lysyl-hydroxylases (PLOD). Using the rat vascular smooth muscle cell line A7r5, we found that an hypoxic atmosphere caused a characteristic time-dependent five- to 12-fold up-regulation of the mRNAs of the two P4h a-subunits [aI (P4ha1) and aII (P4ha2)] and of two lysyl- hydroxylases (PLOD1 and PLOD2). These effects of hyp- oxia were mimicked by the iron-chelator deferoxamine (100 l M ) and by cobaltous chloride (100 l M ). The hypoxic induction of these genes was also seen in the mouse juxta- glomerular As4.1 cell line and mouse hepatoma cell line Hepa1 but was almost absent in the mutant cell line Hepa1C4, which is defective for the hypoxia-inducible transcription factor 1 (HIF-1). In addition, the enzyme expression was induced by hypoxia in mouse embryonic fibroblasts but not in embryonic fibroblasts lacking the HIF- 1a subunit. These findings indicate that hypoxia stimulates the gene expression of a cluster of hydroxylases that are indispensible for collagen fiber formation. Strong indirect evidence, moreover, suggests that the expression of these enzymes during hypoxia is coordinated by HIF-1. Keywords: prolyl hydroxylase; lysyl hydroxylase; protein disulfide isomerase; hypoxia inducible transcription factor. In a variety of tissues, an hypoxic environment favors the formation of collagen deposits. Such an hypoxia-related collagen formation has a clear (patho)physiological impact for wound healing in the skin, for the remodeling of small muscular pulmonary arteries in hypoxia-induced pulmonary hypertension and possibly also for cardiac hypoxia. The formation of collagen fibers and deposits is a multi-step event that includes procollagen protein synthesis, prolyl hydroxylation as requirement for triple helix formation, lysyl hydroxylation, protein folding, maturation and secretion, and finally covalent cross-bridging between collagen fibers through the activity of the lysyloxidase. Which of these steps are directly triggered by hypoxia and how this is accom- plished is not well understood. It has been reported that hypoxia increases mRNA levels for different procollagens in the lung [1,2] and heart in vivo [3]. In vitro studies suggest that this effect of hypoxia on procollagen gene expression might be isoform and cell-type specific. Thus, hypoxia stimulates procollagen I formation in renal [4], dermal [5], and cardiac fibroblasts [6], but neither in fetal lung fibroblasts [7] nor in 3T3 fibroblasts [8]. 3T3 fibroblasts [8], like renal mesangial cells [9], however, increase the gene expression of procol- lagen IV in response to hypoxia. The effect of hypoxia on the activity of the prolyl-4-hydroxylase (PHD-4 or P4h) is clearer; it is crucially required to enable triple helix formation and has been found to be increased in its activity in response to hypoxia [7,10–12]. For the PHD-4 (P4h) heterotetramer enzyme (a 2 b 2 ) there exist two isoforms with a variable a-subunit (aIoraII) and a constant b-subunit, which is identical to protein disulfide-isomerase (PDI) [13]. In vitro studies have shown recently a moderate increase of aI protein and gene expression in fetal lung fibroblasts during hypoxia [14], which is likely mediated by the hypoxia inducible transcription factor HIF-1 [14]. Whether hypoxia also triggers the gene expression of aII is not yet known. Although PDI as the b-subunit is considered to be expressed in excess, there is a report that hypoxia also causes a delayed increase of PDI expression in cultured astrocytes [15]. Whether such an hypoxic stimulation of PDI expression is a more general phenomenon and what the possible underlying mechanism could be, is also unknown. In addition to prolyl hydroxylation, maturation of procollagen also requires the hydroxylation of lysin residues mediated by procollagen lysyl-hydroxylases (PLOD), for which three isoforms exist [16], two of which, namely PLOD1 and PLOD2, are more closely related and colocalize with P4h in the endoplasmic reticulum [17]. Whether the homodimeric PLODs are triggered by hypoxia is also unknown. Screening a rat vascular smooth muscle cell line for hypoxia-induced proteins revealed a clear stimulation of P4ha1 and P4ha2 protein expression that was absent in a cell line defective for HIF-1. As these findings suggested a more Correspondence to A. Kurtz, Institut fu ¨ r Physiologie, Universita ¨ t Regensburg, D-93040 Regensburg, Germany. Fax: + 49 941 9434315, Tel.: + 49 941 9432980, E-mail: armin.kurtz@vkl.uni-regensburg.de Abbreviations: HIF-1, hypoxia inducible transcription factor 1; PDI, protein disulfide isomerase; Ph4, prolyl-4-hydroxylases; PLOD, pro- collagen lysyl-hydroxylases; SDS/PAGE, sodium dodecyl sulfate/ polyacrylamide gel electrophoresis; UPR, unfolded protein response. (Received 31 July 2003, revised 8 September 2003, accepted 19 September 2003) Eur. J. Biochem. 270, 4515–4522 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03846.x general HIF-1-related effect of hypoxia on the expression of the critical hydroxylases for collagen fiber formation, this study aimed to characterize the influence of hypoxia on the gene expression of these hydroxylases in more detail. Materials and methods Cell cultures Rat aortic vascular smooth muscle cells (A7r5) from BDXI rats (ATCC CRL 1444) were cultured in 75 cm 2 flasks (Sarstedt) with 15 mL Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and penicillin/strepto- mycin (P/S; 10 U/10 lgÆmL )1 )(Biochrom), kept in an atmosphere of 10% CO 2 (v/v), 21% O 2 and 69% N 2 at 37 °C. Medium was changed every second day and cells were confluent on days 7–10 after splitting, which was achieved with trypsin–EDTA for 5 min at 37 °C. For the experi- ments, cell cultures (triplicates) were incubated in 21% O 2 (i.e. normoxia) or 1% O 2 , 10% CO 2 , 89% N 2 (i.e. hypoxia) for up to 24 h. Additional culture dishes were incubated at 21% O 2 with either cobalt(II) chloride (100 lmolÆL )1 )or with desferoxamine (200 lmolÆL )1 ) for 12 h. Mouse As4.1 cells [18] were incubated under the afore- mentioned conditions for 4.5 h. Mouse hepatoma Hepa1 cells, and their subclone Hepa1C4, which produces defective ARNT (HIF-1b)[19] due to a point mutation [20] rendering the cells unable to form active HIF [21], were grown under the above mentioned conditions. For the experiments the cells were incubated either at 0.5% O 2 ,10%CO 2 balance N 2 (i.e. hypoxia) or at 21% O 2 , 10% CO 2 balance N 2 with deferoxamine (200 lmolÆL )1 ) for 24 h. Mouse embryonic fibroblasts with normal (+/+) and with a disrupted (–/–) gene for HIF-1a [22] were grown under the above-mentioned conditions. The cells were incubated either at 0.5% O 2 (i.e. hypoxia) or at 21% O 2 with deferoxamine (100 lmolÆL )1 ) for 24 h. Preparation of protein samples After removal of cell culture medium, cell were washed three times with ice-cold NaCl/P i andthenscrapedoffinlysis buffer (300 lL per 75 cm 2 flask) consisting of 7 molÆL )1 urea, 2 molÆL )1 thiourea, 2% CHAPS, 1% dithiothreitol, Pharmalyte TM pH 3–10 (Pharmacia, Uppsala, Sweden), supplemented with protease inhibitors (CompleteÒ, Boeh- ringer Mannheim, Germany). The material was then homo- genized with an Ultraturrax (3 · 10 s) and further sonicated for 3 · 10 s. The homogenate was then allowed to stand at room temperature for 60 min prior to ultracentrifugation at 80 000 g at 15 °C for 1 h. Aliquots of the clear supernatant were frozen in liquid nitrogen and stored at )80 °C. For determination of the protein concentration, protein was precipitated with 10% trichloroacetic acid in acetone and resuspended in 0.1 M NaOH. Protein concentration was then determined with the Bio-Rad protein assay (Bio-Rad, Int.). Two-dimensional PAGE Protein (150 lg) for silverstained gels and for Coomassie- blue staining (600 lg) were loaded for each sample onto the first dimension strips. A linear immobilized pH gradient (pH 5.0–6.0 IPG buffer 18; Pharmacia) was used as the first dimension. Hydration of gel strips and sample application was performed at 50 V for 15 h. For protein separation, a step-voltage protocol was applied (1 h 150 V, 3 h 500 V, 1 h 1000 V, gradient to 8000 V within 0.5 h). A total volt– hour product of 60 kVh was used for 150 lgprotein and 110 kVh for 600 lg protein. Afterwards, the strips were incubated in 50 mmolÆL )1 Tris/HCl (pH 6.8), urea 6molÆL )1 , glycerol 30%, dithiothreitol 65 mmolÆL )1 ,2% sodium dodecyl sulfate (SDS) for 20 min at room tempera- ture followed by incubation in 50 mmolÆL )1 Tris/HCl (pH 8.8), urea 6 molÆL )1 , glycerol 30%, iodoacteamide 140 mmolÆL )1 , and 2% SDS for another 20 min. For the second dimension, a vertical gradient slab gel of 8–18% acrylamide was used and SDS/PAGE was performed at 8mApergelat13°C for 4 h followed by 30 mA for 12 h. At the end of the second dimension, the gels were removed from the glass plates. Staining of two-dimensional PAGE The gels were fixed and stained with silver according to standard protocols [23]. The gels were then scanned (Image Scanner Sharp JX-330, Amersham Biosciences) and ana- lyzed with the IMAGE 3.1 analysis software package (Amer- sham Bioscience). Each spot was matched from one gel to another and the relative volume of matched spots was compared. For preparative protein analysis higher amounts of protein were loaded for two-dimensional PAGE and the protein spots were then stained with colloidal Coomassie- blue. Protein sequence analysis Coomassie-blue stained spots were excised from the gels and were subjected to ESI-MS analysis [24]. Sequences obtained with ESI-MS analysis were then compared with the mouse and rat subset of the NCBInr.fasta protein database. RNA isolation Total RNA was extracted from freshly harvested cells and from frozen tissues according to the protocol of Chomczynski and Sacchi [25]. Real time PCR analysis Real time PCR was performed in a Light Cycler (Roche, Germany). All PCR experiments were performed using the Light Cycler DNA Master SYBR Green I kit provided by Roche Molecular Biochemicals (Mannheim, Germany). Each reaction (20 lL) contained 2 lLcDNA,3.0m M MgCl 2 , 1 pmol of each primer and 2 lLofFastStarter Mix (containing buffer, dNTPs, SYBR Green and hotstart Taq polymerase). The primers used are summarized in Table 1. The amplification program consisted of one cycle at 95 °C for 10 min, followed by 40 cycles with a denaturing phase at 95 °C for 15 s, an annealing phase of 5 s at 60 °C and an elongation phase at 72 °C for 15 s. A melting curve analysis was carried out after amplification to verify the 4516 K H. Hofbauer et al. (Eur. J. Biochem. 270) Ó FEBS 2003 accuracy of the amplicon. For verification of the correct amplification, PCR products were analyzed on an ethidium bromide stained 2% agarose gel. In each real-time PCR run for each gene product under investigation and for b-actin a calibration curve was included, that was generated from serial dilutions (1 : 1, 1 : 10, 1 : 100, 1 : 1000) of a cDNA generated from the pooled RNA of the normoxic (control) cultures (at the different time points) of the respective experimental series (standard cDNA). Analysis of the individual unknowns therefore yielded values relative to this pool. Data are presented as the relative mRNA/b-actin mRNA ratio. The mRNA/b-actin mRNA ratio of the time standards (pools) cDNA was set to 1.0 (i.e. normoxia, 21% oxygen). Data are therefore expressed as relative values related to normoxia. Statistics Levels of significance between groups were calculated using ANOVA test followed by Bonferoni’s reduction for multiple comparisons. P < 0.05 was considered significant. Results Screening the rat vascular smooth muscle cell line A7r5 for hypoxia (1% oxygen for 24 h) induced proteins by two- dimensional electrophoresis revealed a highly reproducible two- to fourfold up-regulated abundance of two proteins (Fig. 1). One of them appeared with two slightly different masses around 60 kDa on SDS/PAGE with a pI around pH 5.7. Analysis of tryptic peptides by ESI-MS identified these proteins as rat prolyl-4-hydroxylase aI subunit (P4ha1). The slightly different molecular masses of the aI subunit probably result from different glycosylation [26]. The second protein appeared as a single spot with a somewhatsmallermolecularmassonSDS/PAGEthanthe prolyl-4-hydroxylase aI subunit, but with a rather similar pI of pH 5.7. This spot was identified by ESI-MS as rat prolyl- 4-hydroxylase aII subunit (P4ha2). Repeated analysis of different gel runs using different protein extracts from A7r5 cells indicated that the prolyl-4-hydroxylase aI spot was increased two to 2.5-fold and the prolyl-4-hydroxylase aII spot was increased three- to fourfold after exposure of the cells to 1% oxygen for 24 h. Table 1. Primers used for real-time PCR of procollagen, prolylhydroxylases, PDI and lysylhydroxylases mRNAs. Mouse (gi:836897) prolylhydroxylase alpha I Sense: 5¢-CGGGATCCTAGACCGGCTAACAAGTA-3¢ Antisense: 5¢-GGAATTCCAAGCAGTCCTCAGCTGT-3¢ Rat (gi:474939) prolylhydroxylase alpha I Sense: 5¢-CGGGATCCTCGGACACCCTGTAAATG-3¢ Antisense: 5¢-GGAATTCCAAGCAGTCCTCAGCTGT-3¢ Mouse (gi:6754969) prolylhydroxylase alpha II Sense: 5¢-CGGGATCCTGCAGGCAGAATTCTTCA-3¢ Antisense: 5¢-GGAATTCCCAGTCTGTGTTCAACCG-3¢ Rat (gi: 6754969) prolylhydroxylase alpha II Sense: 5¢-CGGGATCCTGCAGGCAGAATTCTTCA-3¢ Antisense: 5¢-GGAATTCGCTGAACTGAGAGGTTAG-3¢ Mouse (gi: 20913928) and rat (gi: 6981323) protein disulfide isomerase Sense: 5¢-CGGGATCCAGCAGTATGGTGTCCGTG-3¢ Antisense: 5¢-GGAATTCACCGTCACTTCGCTTGAG-3¢ Mouse (gi: 6755105) lysylhydroxylase I Sense: 5¢-AACTGGTGGCCGAGTGGG-3¢ Antisense: 5¢-GCAGGGTGTCATAGGCCA-3¢ Rat (gi: 409058) lysylhydroxylase I Sense: 5¢-AACTGGTGGCCGAGTGGG-3¢ Antisense: 5¢-GCCCATTTCAAACTTGAG-3¢ Mouse (gi: 6755107) lysylhydroxylase II Sense: 5¢-GCACATTGGGAAACGCTA-3¢ Antisense: 5¢-AATTTTGCATTTGTGATC-3¢ Rat (gi: 6755107) lysylhydroxylase II Sense: 5¢-CCTGTTGTGCACATTGGG-3¢ Antisense: 5¢-AATTTTGCATTTGTGATC-3¢ Mouse (gi: 424103) procollagen Col1a1 Sense: 5¢-ACCTCAAGATGTGCCACT-3¢ Antisense: 5¢-TCCATCGGTCATGCTCTG-3¢ Rat (gi: 807263) procollagen Col1a1 Sense: 5¢-ACCTCAAGATGTGCCACT-3¢ Antisense: 5¢-GATGTACCAGTTCTTCTG-3¢ Mouse and rat (gi: 6671508) b-actin Sense: 5¢-CGGGATCCCCGCCCTAGGCACCAGGGTG-3¢ Antisense: 5¢-GGAATTCGGCTGGGGTGTTGAAGGTCTCAAA-3 ¢ Ó FEBS 2003 Hypoxia and procollagen hydroxylases (Eur. J. Biochem. 270) 4517 To investigate the underlying mechanism for the increased expression of the P4h subunits in response to hypoxia, we next analyzed mRNA expression for P4ha1 and P4ha2 mRNA. Real-time PCR analysis revealed a characteristic time-dependent increase of the mRNA abun- dance in A7r5 cells incubated at 1% oxygen for P4ha1 and P4ha2, starting around 4 h of hypoxia, the induction of P4ha2 mRNA being stronger than that of P4ha1 (Fig. 2A). In view of this concordant regulation, we further considered the possibility that also the expression other hydroxylases involved in collagen fiber formation might be regulated by the cellular oxygen tension. In fact, it turned out that also the mRNAs for lysyl hydroxylases I and II (PLOD1 and -2) increased clearly during hypoxia, in a very similar fashion to the mRNAs for the prolyl hydroxylases. In addition, also the mRNA for protein disulfide isomerase (PDI), as the b-subunit of prolyl hydroxylases, increased in A7r5 cells, although significantly delayed and to a lesser extent. After 24 h of hypoxia the mRNA abundance was five-, 12-, six, seven- and fivefold increased for P4ha1, P4ha2, PDI, PLOD1 and PLOD2, respectively. Notably the abundance of procollagen Ia was not changed by hypoxia (Fig. 2B). Very similar results to those with hypoxia were obtained, when A7r5 cells were incubated with the iron-chelator deferoxamine (100 lmolÆL )1 ) at ambient oxygen tension (21% O 2 ). After 24 h mRNA abundance was increased five-, 16-, two-, five- and 10-fold, for P4ha1, P4ha2, PDI, PLOD1 and PLOD2, respectively (Fig. 3A), whilst the mRNA abundance for procollagen Ia was unchanged. Also, cobalt(II) chloride (100 lmolÆL )1 ) moderately increased the mRNAs of the hydroxylases, but not of procollagen Ia mRNA (Fig. 3B). To test for the cell and species specificity of the changes of the enzyme expression in response to hypoxia, we also analyzed the mouse juxtaglomerular cell line As4.1. As we have recently found that these cells respond to hypoxia rather rapidly [27], we exposed the cells to either hypoxia (0.5% oxygen) or deferoxamine (100 lmolÆL )1 ) for only 4.5 h and assayed the mRNA levels of the procollagen hydroxylases. By these maneuvers, P4ha1 mRNA increased five- to eightfold, P4ha2 mRNA 25- to 33-fold, PDI mRNA twofold, PLOD1 mRNA fivefold, and PLOD2 mRNA twofold (Fig. 4). As the combination of the stimulatory effects of hypoxia, deferoxamine and cobalt suggested a possible involvement of the hypoxia-inducible transcription factor (HIF) in the activation of P4h, and PLOD gene expression by hypoxia, we further examined the expression of these genes in a cell line with a defective HIF, namely the murine hepatoma cell line Hepa1C4. In the control cell line, Hepa 1 both hypoxia (0.5% oxygen) and deferoxamine (100 lmolÆL )1 ) clearly induced P4ha1 (four- and sevenfold) and P4ha2 mRNA (seven- and fourfold) and to a lesser extent also PDI mRNA (two- and 1.5-fold), whilst procollagen Ia mRNA remained unchanged after 12 h of stimulation (Fig. 5A). The stimu- latory effect of hypoxia and of deferoxamine on P4ha1 and P4ha2 mRNA and PDI mRNA was almost abrogated in Hepa1C4 cells (Fig. 5B), supporting the assumption that the expression of these genes was driven by HIF. Unfor- tunately, mRNA levels for the PLOD mRNAs were too low to allow reasonable semiquantification in both Hepa1 and Hepa1C4 cells. This first indication about an essential role of HIF in the triggering of prolylhydroxylase gene expression was further Fig. 1. Two-dimensional electrophoresis of proteins isolated from the rat vascular smooth muscle cell line A7r5. The indicated protein spots were up-regulated by exposure of the cells to hypoxia (1% O 2 ) for 12 h. 4518 K H. Hofbauer et al. (Eur. J. Biochem. 270) Ó FEBS 2003 corroborated in mouse embryonic fibroblasts lacking the HIF-1a subunit. As shown in Fig. 6 hypoxia, deforoxamine and cobalt stimulated the expression of P4ha1, P4ha2, PDI, PLOD1 and PLOD2 mRNAs in wild-type embryonic fibroblasts, but not in embryonic fibroblasts lacking the HIF-1a subunit. As the enzymes involved in collagen formation are important for the correct folding of the protein, it is in principle conceivable, that their expression is also triggered Fig. 3. P4ha1, P4ha2, PDI and PLOD1, PLOD2, and procollagen Ia mRNA in A7r5 cells after exposure to cobalt(II) chloride (100 lmolÆL -1 ) (A) or to deferoxamine (100 lmolÆL -1 ) (B) for 12 h at 21% O 2 . Data are means ± SEM of five experiments each. *P <0.05 vs. control (21% O 2 ). Controls are the means of five experiments and the average mRNA/b-actin mRNA ratio is set to 1 (dotted line). Fig. 4. P4ha1, P4ha2, PDI, PLOD1 and PLOD2 mRNA in mouse As4.1 cells after exposure to hypoxia (0.5% O 2 ), to deferoxamine (100 lmolÆL -1 ) and to cobalt(II) chloride (100 lmolÆL -1 )(at 21%O 2 )for 4.5 h of incubation. Data are means ± SEM of five experiments each. *P < 0.05 vs. control (21% O 2 ). Controls are the means of five experiments and the average mRNA/b-actin mRNA ratio is set to 1 (dotted line). Fig. 5. P4ha1, P4ha2, PDI mRNA and procollagen Ia mRNA in Hepa1 (A) and in Hepa1C4 cells (B) after exposure to hypoxia (0.5% O 2 )orto deferoxamine (100 lmolÆL -1 )at21%O 2 . Data are means ± SEM of five experiments each. *P <0.05 vs. control (21% O 2 ). Controls are the means of five experiments and the average mRNA/b-actin mRNA ratio is set to 1 (dotted line). Fig. 2. Time course of P4ha1, P4ha2 mRNA (A) and PLOD1, PLOD2 mRNA (B) and PDI, procollagen Ia mRNA (C) in A7r5 cells after exposure of the cells to 1% O 2 . Data are means ± SEM of five experiments. *P < 0.05 hypoxia (1% O 2 ) vs. normoxia (21% O 2 ). Controls are the means of five experiments and the average mRNA/b- actin mRNA ratio is set to 1 (dotted line). Ó FEBS 2003 Hypoxia and procollagen hydroxylases (Eur. J. Biochem. 270) 4519 by a disturbance of protein folding due to energy depletion in the course of cellular hypoxia. This so-called unfolded protein response (UPR) can also be elicited by tunicamycin at normal oxygen tensions [28], as shown in Fig. 7. Tunicamycin gave a clear twofold increase of PDI mRNA, a more moderate increase of the mRNAs for prolyl hydroxylases, and did not increase the mRNA abundance of the other enzymes. Discussion Our data show that the expression of a functional cluster of hydroxylases enzymes crucially required for procollagen maturation and collagen fiber formation are regulated in concert by the tissue oxygen tension, in the way that they are up-regulated at low oxygen tensions, i.e. hypoxia. This observation thus confirms the previous notion about an increased prolyl hydroxylase activity in hypoxic tissues in situ [10], as well as the up-regulation of P4ha1 mRNA and protein in response to hypoxia in cell culture [14]. As to the induction of P4ha1 mRNA and protein our data obtained in A7r5 cells are almost identical with regard to time course and to ability to stimulate to those reported by Takahashi and coworkers for fetal lung fibroblasts [14]. As to the induction of PDI mRNA, our data are also in good accordance regarding the delayed time course and the differential ability to stimulate by hypoxia, iron chelation and cobalt with those reported by Tanaka and coworkers for cultured astrocytes [15]. Our findings thus on the one hand fully support these previous findings and on the other hand extend them by far and set them in a more general context, as both a-subunits of P4h, PDI as the b-subunit of P4h [13] and the collagen procollagen lysyl hydroxylases PLOD1 and PLOD2 are oxygen regulated. As these effects were seen in different mouse and rat cell lines, we infer that the up-regulation of the procollagen hydroxylases during hypoxia is a more general effect with physiological relevance. Such a concerted regulation of these key enzymes of collagen formation appears reasonable from a physiological point of view, as all of these enzymes use oxygen directly as a common substrate and because the formation of collagen requires the coordinated activities of all the enzymes. Such a common regulation of a functional cluster of genes by the oxygen tension has already been found for the enzymes involved in the glycolytic cascade [29] and also for the key players of angiogenesis [30]. Considering the parallel up-regulation of enzymes involved in collagen fiber formation raises the question of whether this up-regulation is physiologically primarily meant to increase collagen formation in hypoxic tissues, or if it reflects more a compensatory change of the concentration of enzyme molecules to maintain a given normal hydroxylation rate at altered substrate (oxygen) concentrations (Fig. 8). The explanation as a compensatory increase of gene expression was previously also presented for the increased expression of the endoplasmic oxido- reductase Ero1-L during hypoxia which transfers oxidizing equivalents onto PDI [27]. Such a view would be supported by the observation that the expression of the procollagen (Ia) gene itself was not regulated by the oxygen tension, which is also in accordance with data obtained by others [7]. As the genes for P4ha1, P4ha2, PDI, and PLOD 1,2 are localized on different chromosomes the question arises concerning the mechanisms underlying the orchestrated expression of these enzymes by the oxygen tension. The findings that the effect of hypoxia on the gene expression of the hydroxylases was mimicked by the iron Fig. 6. P4ha1, P4ha2, PDI, PLOD1 and PLOD2 mRNA in mouse embryonic fibroblasts with intact and with disrupted HIF-1a gene after exposure to hypoxia (0.5% O 2 ) or to deferoxamine (100 lmolÆL -1 )at 21%O 2 for 24 h. Data are means ± SEM of five experiments each. *P < 0.05 vs. control (21% O 2 ). Controls are the means of five experiments and the average mRNA/b-actin mRNA ratio is set to 1 (dotted line). Fig. 7. P4ha1, P4ha2, PDI, PLOD1, PLOD2, and procollagen I(a) mRNA in mouse As4.1 cells after incubation with tunicamycin (10 lgÆmL -1 ) for 24 h at 21% O 2 . Data are means ± SEM of five experiments each. *P < 0.05 vs. control (21% O 2 ). Controls are the means of five experiments and the average mRNA/b-actin mRNA ratio is set to 1 (dotted line). 4520 K H. Hofbauer et al. (Eur. J. Biochem. 270) Ó FEBS 2003 chelator deferoxamine and the divalent cation cobalt, suggest that these genes are under the control of the hypoxia inducible transcription factor HIF. HIF is a heterotetramer consisting of an a-andab-subunit [31]. The protein abundance of this protein dimer is inversely related to the cellular tension, because the a- but not b-subunit protein stability is dependent on the oxygen tension, in the way that the a-subunit is more stable at low oxygen tensions. The reason for this behavior is an oxygen dependent prolyl-hydroxylation of the a-subunit, which finally directs the protein to proteasomal degradation [32]. The assumption that HIF could in fact be a main trigger of the procollagen hydroxylases is further corroborated by the findings that the stimulatory effect of hypoxia on gene expression was absent in cells with a functional inactive HIF or lacking the HIF-1a protein in general. In fact, for the P4ha1I the involvement of HIF in the activation of gene expression during hypoxia has recently been directly dem- onstrated [14]. A search for the HIF-binding consensus sequence CGTG revealed six, six, two, nine and 10 theoretical HIF binding sites within the first 1 kb of the 5¢-promoter region of mouse P4ha1, P4ha2, PLOD1, PLOD, and PDI, respectively. Although prolyl hydroxylation is a critical event for both procollagen triple helix stabilization on the one hand and for the stability of HIF-1a protein, different prolyl hydroxylases appear to be required for these processes. HIF-a prolyl hydroxylation is managed by PHD-1, -2 and -3 [33,34], whilst procollagen prolylhydroxylation is performed by P4h [13], which does not accept HIF-a as a substrate [31]. Interestingly, the expressions of PHD-3 [35,36] and eventually of PHD-2 itself are also oxygen sensitive [35,36]. They are up-regulated by hypoxia by a process critically involving HIF, which in turn is also the substrate of PHD-2 and PHD-3. Thus, PHD-2, PHD-3 and P4h expression appear to be subject to a common control by oxygen, whilst the expression of PHD-1 is not. The physiological meaning of this differential regulation of PHD-gene expression remains to be clarified. In spite of the similar regulation of PHD-2, PHD-3 and P4h expression by oxygen, not only the protein target but also the intracellular localization is different between the two groups of enzymes. Whilst PHD-1, -2 and -3 are mainly cytosolic and nuclear proteins [37], P4h is like the lysyl hydroxylases PLOD1 and -2, being localized within the endoplasmic reticulum [13,38]. This localization could be of some interest, as HIF-regulated genes, as identified so far, encode mainly for cytosolic or for secreted proteins [28,39]. Constituents of the endoplasmic reticulum as being HIF-regulated have not yet been frequently reported [39]. We have recently obtained evidence that the expres- sion of endoplasmic oxidoreductase Ero1-L, which oxid- izes PDI, is also controlled by HIF [27]. Given the conjunction of PDI with PDH-4 and the conjunction of PDI with Ero1-L as an essential oxidizing enzyme of PDI, there arises the concept a network of endoplasmic enzymes that mediate oxygen dependent reactions and that are in turn regulated by the oxygen tension in an inverse fashion most likely regulated by the transcription factor HIF-1. It must be considered in this context that the expression of endoplasmic proteins with folding or chaperone function might also be indirectly triggered by cellular hypoxia through the UPR [40] induced by cellular energy depletion [41,42]. We have addressed this issue therefore by investi- gating the influence of the UPR for the expression of the procollagen hydroxylases. 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