Oxygen tension regulates the expression of a group of procollagen hydroxylases

Abstract

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...

Hypoxia Modifies the Transcriptome of Primary Human Monocytes: Modulation of Novel Immune-Related Genes and Identification Of CC-Chemokine Ligand 20 as a New Hypoxia-Inducible Gene

“…Hypoxia increased the expression of P4HA1, P4HA2, and EGLN1, three members of the prolyl-hydroxylase family which mediate HIF-␣ hydroxylation in well-oxygenated cells targeting the protein for proteosomal degradation by the von Hippel Lindau tumor suppressor protein (pVHL) (15), and of BHLHB2, a bHLH family member implicated in the regulation of pVHL/HIF pathways (15). These data are in agreement with previous findings in other cell types (15,35,36) and suggest an O 2 -driven HIF-1-dependent autoregulatory mechanism, required to ensure fast HIF-1␣ degradation upon reoxygenation, also in Mn. Of note is the novel evidence that hypoxia promotes the expression of ATF2 and ATF5, two members of the ATF family of transcription factors (45) required for efficient activation of gene transcription by hypoxia (15).…”

“…The expression of another 46 known HMGs characterized in cells types other than mononuclear phagocytes was also triggered in hypoxic Mn (Table II), including classical genes involved in glycolytic metabolism and glucose transport (e.g., glucose transporter 1 and 3 (GLUT1, 3) (9, 13, 15, 33), or associated with nonglycolytic metabolism and ion transport (e.g., carbonic anhydrase XII (CA12) (13,33). Genes coding for two novel HMGs, hypoxia-inducible protein 2 (HIG2) (34) and HIF-1-responsive RTP801 (DDIT4) (15), and other HIF-1 target genes implicated in HIF-1␣ hydroxylation, such as EGLnine homolog-1 (EGLN1) (35) and proline 4-hydroxylases-AI,II (P4HA1, A2) (33,36), were increased in hypoxic Mn (Table II). Finally, several genes related to apoptosis, cell cycle, transcription, and immune responses, whose responsiveness to hypoxia was previously demonstrated in normal and malignant cells, were also up-regulated in hypoxic Mn.…”

Deletion ofVhlhin chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development

“…Hypoxia increases type II collagen accumulation in vitro in a HIF1α-dependent manner (Kurz et al, 2001;Pfander et al, 2003). Furthermore, recently it has been shown that hypoxia increases a group of procollagen hydroxylases that are indispensable for collagen fiber formation through stabilization of the transcription factor HIF1α (Hofbauer et al, 2003). It is thus highly possible that the increased matrix deposition observed in Vhlh null growth plates might result from accumulation of HIF1α in the mutant chondrocytes, leading to both an increased expression of type II collagen mRNA and to an enhanced synthesis of procollagen hydroxylases that are critically required for collagen triple helix formation.…”

The prolyl 3-hydroxylases P3H2 and P3H3 are novel targets for epigenetic silencing in breast cancer

“…Furthermore, a decrease in oxygen tension has been found to result in an up-regulation of P4H genes, P4HA1 and P4HA2, as they have been found to be transcriptionally activated by HIF. Although the prolyl hydroxylase reaction does require O 2 , it is thought that the over-production of P4HA1 and P4HA2 in hypoxic conditions compensates for this (Hofbauer et al, 2003;Fähling et al, 2006).…”