In this study, we have characterized the influence of hypoxia on the expression of hydroxylases crucially involved in collagen 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 lysylhydroxylases (PLOD1 and PLOD2). These effects of hypoxia were mimicked by the iron-chelator deferoxamine (100 lM) and by cobaltous chloride (100 lM). The hypoxic induction of these genes was also seen in the mouse juxtaglomerular 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 HIF1a 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 accomplished 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 procollagen 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][11][12]. For the PHD-4 (P4h) heterotetramer enzyme (a 2 b 2 ) there ex...

The formation of disulfide bonds in the endoplasmic reticulum requires protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductin 1 (ERO1) that reoxidizes PDI. We report here that the expression of the rat, mouse and human homologues of ERO1-Like protein a but not of the isoform ERO1-Lb are stimulated by hypoxia in rats vivo and in rat, mouse and human cell cultures. The temporal pattern of hypoxic ERO1-La induction is very similar to that of genes triggered by the hypoxia inducible transcription factor (HIF-1) and is characteristically mimicked by cobalt and by deferoxamine, but is absent in cells with a defective aryl hydrocarbon receptor translocator (ARNT, HIF-1b). We speculate from these findings that the expression of ERO1-La is probably regulated via the HIF-pathway and thus belongs to the family of classic oxygen regulated genes. Activation of the unfolded protein response (UPR) by tunicamycin, on the other hand, strongly induced ERO1-Lb and more moderately ERO1-La expression. The expression of the two ERO1-L isoforms therefore appears to be differently regulated, in the way that ERO1-La expression is mainly controlled by the cellular oxygen tension, whilst ERO1-Lb is triggered mainly by UPR. The physiological meaning of the oxygen regulation of ERO1-La expression likely is to maintain the transfer rate of oxidizing equivalents to PDI in situations of an altered cellular redox state induced by changes of the cellular oxygen tension.Keywords: hypoxia; HIF; protein folding; UPR; PDI.Formation of disulfide bonds is an essential event for the correct folding of proteins in the endoplasmic reticulum. It is well known that this process is catalyzed by protein disulfide-isomerase (PDI) [1]. Until a few a years ago, however, it remained unclear how PDI is reoxidized in this reaction [2]. It was the discovery of the ERO1 (endoplasmic reticulum oxidoreductin) protein in yeast [3,4] which provided evidence that this protein is essential to transfer oxidizing equivalents to PDI [5]. It turned out that ERO1 is a highly conserved endoplasmic protein and for humans and mouse two ERO1-Like proteins have meanwhile been identified, termed ERO1-La [6] and -1b [7]. The ERO1 proteins are probably flavoproteins [8] that covalently bind to PDI [9], what explains their function to transfer oxidizing equivalents to PDI. ERO1-La and -Lb display different tissue distributions [7], and moreover, appear to be differently regulated in their expression. Thus, mainly ERO1-L b transcripts are induced in the course of unfolded protein response [7]. In this pathway accumulation of misfolded proteins in the endoplasmic reticulum induces the expression of a number of proteins including those involved in the correct folding of proteins such as chaperones [10]. How the expression of the ERO1-La protein is regulated is yet unknown. Analyzing the protein expression pattern of a rat vascular smooth muscle cell line, we now found that a ERO1-Like protein highly homologous to mouse and human ERO1-La is strongly upregulated during ce...

Our study aimed to investigate the influence of tissue hypo-oxygenation on the adrenomedullin (ADM) system in vivo. For this purpose, male Sprague-Dawley rats were exposed to normobaric hypoxia (8% oxygen) or to functional anemia [0.1% carbon monoxide (CO)] or to cobalt chloride (60 mg/kg) for 6 h. Messenger RNA levels for ADM and its receptor (ADM-R) were assessed in diverse organs by RNase protection assay. Additionally, ADM protein concentrations in these organs, as in plasma, were determined by a RIA. We found that ADM mRNA abundance increased in response to hypoxia and to CO inhalation up to 15-fold in all organs examined. Similarly, ADM-R mRNA abundance increased during hypoxia and CO inhalation in all organs examined with exception of the liver. The effects of hypoxia and of CO inhalation on ADM and ADM-R mRNAs were mimicked by injection of cobaltous chloride. Hypoxia also significantly increased ADM protein content in all organs, and plasma levels of ADM rose twofold in response to hypoxia and CO inhalation. These findings indicate that tissue hypoxia leads to a widespread activation of the ADM system, which comprises a parallel stimulation of ADM and ADM receptor mRNA as enhanced ADM protein synthesis and secretion. The ADM system may, therefore, play a significant role in the physiological response to tissue hypoxia. It appears that ADM and ADM-R belong to the family of classic oxygen-regulated genes, which are activated by a decrease of the pericellular oxygen tension through the same intracellular signaling cascade.

Abstract-A body of evidence indicates that the production of adrenomedullin (ADM) in vivo is activated in states of inflammation. Our aim was to characterize the intracellular signaling pathways along which inflammation leads to a stimulation of ADM expression. For this purpose, we characterized the effects of inflammatory cytokines, tumor necrosis factor-␣ (100 g/L), interleukin-1␤ (20 g/L), and interferon-␥ (0.5 U/L) on ADM gene expression in rat aortic vascular smooth muscle cells (AVSMCs). We found that inflammatory cytokines induced a time-dependent 12-fold upregulation of ADM mRNA in AVSMCs that was paralleled by a substantial increase in inducible NO synthase mRNA expression. The stimulatory effect of cytokines on ADM gene expression was attenuated by NO deprivation induced by N-nitro-L-arginine methyl ester (1 mmol/L) and was in part mimicked by the NO donor S-nitroso-Nacetylpenicillamine (100 mol/L). The cGMP analog 8-bromo-cGMP (100 mol/L) had no effect on ADM gene expression, and inhibition of cGMP production by 1H-oxodiazolo-quinoxalin-1 (ODQ, 200 mol/L) was not able to abrogate the increase of ADM mRNA induced by NO donation using S-nitroso-N-acetylpenicillamine (100 mol/L). The significant induction of ADM gene expression by inflammatory cytokines and NO donation was also observed in mesangial cells, endothelial cells, and hepatocytes. These findings suggest that NO is a direct activator of ADM gene expression in a variety of cell types and that inflammatory cytokines stimulate ADM expression via both NO-dependent and -independent mechanisms. The stimulatory effect of NO appears to not be related to the classic guanylate cyclase-cGMP pathway.