Regulation of collagen prolyl 4-hydroxylase and matrix metalloproteinases in fibrosarcoma cells by hypoxia

Comparative Biochemistry and Physiology, Part C 139 (2004) 119 – 126 www.elsevier.com/locate/cbpc

Regulation of collagen prolyl 4-hydroxylase and matrix metalloproteinases in fibrosarcoma cells by hypoxia Michael F7hlinga,b, Andrea Perlewitzb, Anke Dollerc, Bernd-Joachim Thielea,* a

Institut fu¨r Vegetative Physiologie, Charite´, Universita¨tsmedizin Berlin, Tucholskystr. 2, 10117 Berlin, Germany b Freie Universita¨t Berlin, Germany c Cardiovascular Research Center, Charite´, Universita¨tsmedizin Berlin, Germany Received 14 June 2004; received in revised form 28 September 2004; accepted 28 September 2004

Abstract The cellular response to hypoxia is characterized by an enhanced deposition of extracellular matrix (ECM) components, mainly collagens. Collagen homeostasis is determined by the rate of synthesis and degradation. In this study, we investigated the synthesis of enzymes of collagen metabolism like collagen prolyl 4-hydroxylase (P4H), matrix metalloproteinases (MMP-2 and MMP-9) and their regulatory factors MT1-MMP, TIMP-1 and TIMP-2 in HT1080 fibroblasts under the influence of hypoxia. The results indicate that hypoxia affects collagen homeostasis in a biphasic manner concerning basic mechanisms of gene expression. P4H-alpha subunits are up-regulated at the transcriptional and translational level, whereas the beta-subunit is not susceptible to hypoxia. MMP-9 is primarily regulated at the transcriptional and translational level, whereas MMP-2 is mainly controlled by proteolytic activation of the proenzyme. Our results suggest that short-term hypoxia facilitates fibrosis in HT1080 cells by activation of P4H-alpha expression and inhibition of the synthesis of MMPs. Under long-term hypoxia, however, anti-fibrotic mechanisms prevail. Although P4H-alpha expression sustains at a high level, collagenolytic activities dominate by abolishing inhibition of synthesis and activity of MMPs. D 2004 Elsevier Inc. All rights reserved. Keywords: Hypoxia; Fibrosis; Collagen; Prolyl 4-hydroxylase; Matrix metalloproteinases

1. Introduction Hypoxia is a strong physiological stimulus that affects all organisms in a unique way. Different classes of organisms cope quite differently with a changing supply in oxygen, reaching from reversible adaptation mechanisms up to irreversible detrimental processes. One of the most prominent survival strategies for many animal species is the restriction of protein synthesis leading to a depression of the rate of metabolism (Storey, 1996; Almeida-Val et al., 2000; Storey and Storey, 2004). The cellular response to hypoxia is complex and characterized by alterations in the expression of numerous genes. However, against the general tendency of down-regulation, the expression of a particular set of * Corresponding author. Tel.: +49 30 450 528184; fax: +49 30 450 528972. E-mail address: [email protected] (B.-J. Thiele). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.09.013

genes is up-regulated. Hypoxia-inducible genes can be regulated at all control levels, from transcription over translation up to posttranslational protein modification (Kumar and Klein, 2004). The molecular signal of reduced oxygen tension is mediated by a wide variety of pathways and control factors like hypoxia-inducible factor (HIF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) or transforming growth factor (TGF-beta) (Taipale and Keski-Oja, 1997; Werb and Chin, 1998; Bacakova et al., 1999; Falanga et al., 2002; Gao et al., 2003; Papakonstantinou et al., 2003). Furthermore, the role of adenosin, K+ channels, intracellular Ca2+ and phosphorylation of transcription factor cAMP response element binding protein (CREB) has been discussed (BreitnerJohnson and Millhorn, 1998; Kobayashi and Millhorn, 1999; Conrad et al., 2001). One of the most prominent cellular effects of hypoxia with pathological significance in mammals is an increase in the synthesis of extracellular

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matrix (ECM) components, mainly collagens, causing fibrosis. The steady-state concentration of the collagens depends on both the rate of their synthesis and their degradation. The impact of hypoxia on the rate of synthesis of procollagen chains is well documented in vivo (Ostadal et al., 1995; Perhonen et al., 1997) and in vitro (Falanga et al., 1993; Tamamori et al., 1997; Tajima et al., 2001). Much less, however, is known about the influence of hypoxia on the synthesis of essential enzymes involved in collagen metabolism. In this context, collagen prolyl 4-hydroxylase (P4H), matrix metalloproteinases (MMPs) and their naturally occurring inhibitors (TIMPs) are important factors. P4H is a tetrameric enzyme composed of two different subunits (alpha2beta2). Two types of alpha-subunits, coded by different genes, are, in combination with the unique beta-subunit, the structural basis for the existence of two different isoenzymes P4H(I) and P4H(II) (Myllyharju, 2003). The P4H-beta subunit has a second function: it is identical to the chaperone disulfide isomerase (PDI). The physiological significance of the existence of the two isoenzymes is not very well understood. The only currently documented differences lie in the areas of cell or tissue specificity, or enzymatic parameters (Helaakoski et al., 1995; Nokelainen et al., 2001). MMPs are a family of Zncontaining proteases specialized on the degradation of extracellular matrix proteins like collagens, elastins or laminins (Nagase and Woessner, 1999; Jones et al., 2003). MMP-1 and MMP-8 are capable of degrading native collagen I and III helices. The resulting fragments and basement membrane components, such as collagen IV, can be further degraded by MMP-2 and MMP-9, also called gelatinases A and B (Woessner, 1991; Dollery et al., 1995). The balance between collagen synthesis and degradation by MMPs, as well as the level and activity of their regulators (TIMPs), is crucial for the ECM remodeling process and the development of fibrosis (Cannon et al., 1983; Tyagi, 1997; Li et al., 2000; Ben-Yosef et al., 2002; D’Armiento, 2002; Spinale, 2002). This study was intended to elucidate differences in control mechanisms involved in the synthesis of major components of collagen metabolism that represent pro- and anti-fibrotic activities under conditions of an experimental hypoxia. As representative factors, we studied the expression of P4H subunits (P4H-alpha(I), P4H-alpha(II), P4Hbeta), MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 in HT1080 fibrosarcoma cells at the mRNA and protein level.

2. Materials and methods 2.1. Cell culture and RNA-/protein isolation Human fibrosarcoma line HT1080 (ATCC, passages 16–21) cells were maintained in DMEM (high glucose; PAA Laboratories, Cflbe, Germany) supplemented with

10% heat-inactivated fetal calf serum (FCS), 50 U/mL penicillin, 50 Ag/mL streptomycin, 15 mM HEPES and 2 mmol/L glutamine, at 37 8C, 5% CO2. Before use in experiments, cells were maintained in medium containing 0.4% FCS for at least 24 h. Measurements started after the application of fresh medium containing 0.4% FCS. For hypoxic conditions the cells were incubated in a hypoxia chamber (JOUAN IG750). Oxygen content was reduced to b1% by gas exchanges with 95% nitrogen/5% CO2. Control cells were incubated under normoxic conditions. For RNA and protein isolation, cells were quickly washed with ice-cold PBS. Cells were recovered by scraping the cell layer from culture plates with a spatula. RNA was prepared with RNAzol (Biozol Diagnostica Vertrieb, Eching, Germany) according to the manufacturer’s protocol and reverse transcribed using random hexamer primers. Protein extracts (10,000g supernatants=S10) were prepared using lysis buffer (10 mM Tris, pH 7.5, 140 mM NaCl, 1 mM EDTA, 25% glycerol, 0.1% SDS, 0.5% Nonidet NP40, 1 mM DTT, 1 mM PMSF, 1complete protease-inhibitor-mix; Roche Diagnostics). 2.2. RT-PCR Primers were designed to bridge at least one intron. PCR conditions were used as follows: 3 min 95 8C, cycles: 30 s 95 8C, 30 s annealing, 30 s 72 8C, final elongation for 2 min at 72 8C; 2.5 mM MgCl2. MMP-2 MT1-MMP TIMP-2 TIMP-1 P4H-alpha(I) P4H-alpha(II) P4H-beta

(Fw) 5V-GCCTCTCCTGACATTGACCT (Rev) 5V-AACACAGCCTTCTCCTCCTG (Fw) 5V-ATGCTGCTCTCTTCTGGATG (Rev) 5V-CGGTTCTACCTTCAGCTTCT (Fw) 5V-GAAGGAAGTGGACTCTGGAA (Rev) 5V-CTGTGGTTCAGGCTCTTCTT (Fw) 5V-ATGGAGAGTGTCTGCGGATA (Rev) 5V-CATTCCTCACAGCCAACAGT (Fw) 5V-CCACAGCAGAGGAATTACAG (Rev) 5V-ACACTAGCTCCAACTTCAGG (Fw) 5V-ACGAGATAGGAGCTGCCAA (Rev) 5V-CCATCCACAACACCGTATGA (Fw) 5V-AGACTCACATCCTGCTGTTC (Rev) 5V-TACTTGGTCATCTCCTCCTC

mRNA levels were normalized to GAPDH mRNA. 2.3. Molecular cloning and in vitro transcription Partial MMP-9 sequences (GenBank accession no. NM004994, bases 2008–2334) were amplified by PCR, cloned and transformed using the TOPO II TA Cloning Kit (Invitrogen). Positive clones were checked by sequencing. In vitro transcription was performed according to the Invitrogen protocol. In vitro transcripts were purified by BD Chroma Spink-100 (DEPC) columns (BD-Bioscience Clontech).

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2.4. Northern blot analysis

2.8. Statistical analysis

Total cellular RNA (20 Ag) was separated by electrophoresis on 1% Agarose gels containing formaldehyde. The RNA was capillary-transferred to positively charged nylon membranes (Roche Diagnostics) and the membrane was stained with ethidium bromide to document the relative level of 18S rRNA and hybridized to DIG-labeled MMP-9 anti-sense transcripts. The detection was performed using the DIG RNA Labeling Kit (Roche Diagnostics) according to the manufacturer’s protocol. mRNA levels were normalized to 18S rRNA.

Signals were scanned and quantified using the program Scion Image (Scion, Frederick, MD, USA). Results are presented as means and error bars representing the standard deviation (S.D.). Experiments were performed at least in two series containing three independent samples each. Statistical significances were analyzed using Student’s t-test.

2.5. Western blot analysis Protein extracts (30 Ag/sample) were separated by SDS– PAGE. After electrophoresis proteins were transferred to Hybondk-P membranes (Amersham Pharmacia Biotech) using a Bio-Rad Mini Trans-Blot transfer cell. The membranes were blocked for 1 h with 5% Blot-QuickBlocker (Chemicon). Following the blocking step, the membranes were incubated in 1% blocking solution (BlotQuick-Blocker diluted in TBS-T) containing primary anti MMP-2 (Sigma), P4H-alpha (Medicorp) or P4H-beta (Medicorp) antibodies for 1.5 h. The membranes were washed three times with TBS-T and incubated with a secondary anti rabbit or anti mouse antibody (Promega) for 1 h. Bands were detected using the ChemiGlowk-West Detection-Kit (Alpha Innotech). Membranes were stripped for 5 min in H2O, 5 min 0.2 M NaOH, 5 min H2O, and reprobed with anti-h-actin antibody (Chemicon) under the same conditions.

3. Results To investigate the influence of hypoxia on collagen homeostasis, we concentrated on the expression of factors representative for posttranslational modification of procollagen chains (P4H) and collagen degradation (metalloproteinases, MMPs 2 and 9, tissue inhibitors of metalloproteinases,

2.6. Enzyme-linked immunosorbent assay (ELISA) MMP-9 ELISA (Oncogenek Research Products) was carried out according to the manufacturer’s protocol. Specificity: human pro-MMP-9 protein and MMP-9/ TIMP-1 complex. Duplicate evaluations were performed for each sample. 2.7. Gelatin zymography MMP-2 and MMP-9 activities were analyzed by gel substrate zymography. Secreted proteins (30 Al supernatant/sample) were separated by electrophoresis in SDS–polyacrylamide gels containing 0.1% gelatin. After electrophoresis, gels were renatured by incubation in 2.5% Triton X-100 for 230 min at room temperature, incubated overnight in substrate buffer (50 mM Tris, pH 7.5, containing 200 mM NaCl, 5 mM CaCl2, 0.2% Brij35-solution) at 37 8C, followed by staining with 0.5% Coomassie brilliant blue. Gels were discolored until a clear zone appeared, representing the gelatinolytic activity.

Fig. 1. Expression of P4H, MMPs and TIMPs in HT1080 cells under hypoxia. (A) HT1080 cells were cultivated under hypoxia (b1% O2) up to 34 h. Total RNA was extracted and mRNAs coding for P4H subunits, MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP were analyzed by RTPCR or Northern blotting (NB) as indicated. As a loading control GAPDH was determined and 18S rRNA was stained with ethidium bromide (EtBr). (B) From cells cultivated as in (A), cytoplasmic protein extracts were prepared and P4H subunits and MMP-2 were quantified by Western blotting (WB). Beta-actin served as a control. MMP-9 was determined by ELISA. (C) The enzymatic activity of MMP-2 and MMP-9 was assessed by gelatin-zymography (Zymo). A representative set of original data is shown.

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TIMPS 1 and 2). Hypoxia experiments were performed in the fibrosarcoma cell line HT1080 and expression was studied by mRNA and protein quantification by RT-PCR and Western blotting. A representative set of original experimental results is shown in Fig. 1A–C. The data of six independent experiments were scanned, quantified and depicted as bar diagrams in Fig. 2 (P4H) and Fig. 3A–C (MMPs and TIMPs). It is obvious that P4H is not regulated as a whole by hypoxia (Fig. 2). Only the alpha subunits of P4H(I) and (II) are induced; the common beta subunit does not respond to hypoxic control. This can be seen at the mRNA as well as at the protein level. It demonstrates that not only transcription of the beta gene is insensitive to hypoxia, but also that there is no posttranscriptional activation of pre-formed mRNA. In contrast, the P4H-alpha(I) gene is induced about twofold. This is followed by a more than fivefold increase in protein concentration after 34 h. Due to the unavailability of a specific antibody, the P4H-alpha(II) subunit could not be estimated. From the mRNA data, however, it is evident that the gene is less susceptible to a hypoxic stimulus than the P4H(I) gene. The rate of induction is approximately half of that of the P4H-alpha(I) gene. Matrix metalloproteinases, especially gelatinases MMP2 and MMP-9, are important factors that determine collagen turnover. Their final action is controlled at least at four different levels: gene transcription (including extracellular matrix metalloproteinase inhibitor=EMMPRIN), mRNA

translation, activation of pro-MMPs by partial proteolysis and interaction of secreted MMPs with inhibitors (Jones et al., 2003). From Fig. 3A, it is evident that there is no dramatic increase in MMP-2 mRNA and intracellular protein by hypoxia. Long-term hypoxia (34 h), however, significantly increased the mRNA level (twofold) in contrast to the normoxic control. This increase in mRNA is not followed by an increase in the intracellular proMMP-2 protein content, which may be attributed to secretion. The major effect on MMP-2 seems to occur extracellularly. We see an extensive cleavage of pre-formed less active pro-MMP-2 by limited proteolysis generating more active MMP-2 forms (Fig. 1C). While the MMP-2 activity is inhibited at 10 h hypoxia, long-term hypoxia (34 h) leads to an about twofold elevated level of both active forms (Figs. 1C and 3A). On the contrary, MMP-9 mRNA is induced as early as at 10 h (about twofold), reaching a level of 3.5-fold at 34 h. Northern blots indicate no signs of different splice variants. The elevated mRNA level is accompanied by a delayed increase in protein concentration. Unlike MMP-2, there is only a marginal activation by pro-MMP-9 cleavage, whereas the activity of the pro-form is also inhibited at 10 h hypoxia and the effect is abolished at 34 h (Figs. 1C and 3B). The data indicate that MMP-9 is a target for translational and secretory control. The behavior of TIMP-1, TIMP-2 and MT1-MMP was finally studied at the mRNA level. As demonstrated, TIMP-1 and the membrane bound form MT1-MMP mRNA remain nearly

Fig. 2. Expression of P4H subunits; statistical analysis. HT1080 cells were cultivated under hypoxia and mRNA/protein was quantified as described in Fig. 1. Bars represent relative values obtained by comparison of P4H data with levels of GAPDH (RT-PCR), 18S rRNA (Northern blots) or beta-actin (Western blots). ELISA results of MMP-9 are given in nanograms per milliliter. All signals were scanned and quantified using the Scion Image software package. *pb0.05, **pb0.01. The numerical values are means of six independent experiments.

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Fig. 3. Expression of MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2; statistical analysis. Quantification was performed as described in the legend of Fig. 2.

unaffected by hypoxia. The level of TIMP-2 mRNA, however, was down-regulated to about a third of its original level. TIMP-2, at low levels, promotes the activation of

MMP-2 by forming a membrane complex with MT1-MMP, anchoring the pro-MMP-2 protein to the cell surface (Gomez et al., 1997; Nagase and Woessner, 1999) and

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may therefore be the main trigger in extracellular activation of MMP-2.

4. Discussion Hypoxia is a condition, which exhibits physiological (like diving mammals, or other organisms experiencing acute oxygen deficiency) as well as pathological (like ischemia) aspects. In mammals, ischemia is accompanied by the formation of connective tissue leading to fibrosis. The influence of hypoxia on the development of fibrosis in organs like lung, kidney, heart, liver or the vascular system is well documented. During hypoxia, collagens are the major extracellular matrix proteins that are up-regulated. However, the balance of collagen homeostasis is not only determined by the synthesis of the procollagen chains themselves, but also by the expression and activity of modifying and degrading enzymes like P4Hs and MMPs. In this study, we tried to gain more insight into the underlying mechanisms by detailed analysis of the synthesis at the mRNA and protein levels. The results show that P4Hs and MMPs are regulated by hypoxia at quite different control levels, in particular with respect to short-term (10 h) and long-term (34 h) hypoxia. P4Hs play a central role in the biosynthesis of collagens, as 4-hydroxyproline residues are essential for the formation of the collagen triple helix. As we can show, the alpha subunits of both isoenzymes, P4H(I) and P4H(II), are induced in HT1080 cells by hypoxia. However, the P4Halpha(I) gene is more effectively stimulated, especially by long-term hypoxia (34 h). This difference in O2 susceptibility could be of physiological relevance and might be another explanation for the existence of two P4H isoenzymes. It is important to point out that the mRNA level of P4H-alpha(II) possibly may not reflect the protein expression, since the 5V untranslated region (UTR) contains an internal ribosome entry side (IRES; UTRscan analyses, http://bighost.ba.itb.cnr.it/BIG/UTRscan/). It is known that mRNAs with IRES elements are more effectively translated under oxygen stress conditions (Stoneley and Willis, 2004). Therefore the P4H-alpha(II) subunit may be a new candidate for this kind of cap-independent translation. The P4H-beta/ PDI-gene, on the contrary, is not influenced by hypoxia (Fig. 2). This is not unexpected, as the P4H-beta subunit is expressed even under normoxic condition at more than a 10fold excess over the alpha subunits and seems not to be a limiting factor. Furthermore, this correlates with the fact that the actual identity of the beta subunit is that of a disulfide isomerase and chaperone that is also able to interact with proteins other than the P4H-alpha subunits. It is thought to maintain the catalytic alpha subunits in a soluble form rather than participating directly in catalysis (Lumb and Bulleid, 2002). From the mechanistic point of view it is noteworthy that under hypoxia the synthesis of P4H-alpha(I) protein

increases much more than its mRNA. This is most prominently seen at 34 h, when protein is elevated fivefold, whereas mRNA slightly drops under the same conditions. This clearly indicates a posttranscriptional activation of translation or increased protein stability. Our results are in contrast to the work of Takahashi et al. (2000), who studied P4H-alpha(I) expression in fetal lung fibroblasts. They also found an induction at the mRNA and protein level, while not showing activation at the translation or protein stability level. mRNA and protein increased both about twofold in a 32 h interval. This may reflect differences in the cell model, or the fact that strictly anoxic conditions (0% O2) were used. However, they also postulated a posttranscriptional control of P4H-alpha(I) under short-term anoxic conditions. For the final action of MMPs, additional principles seem to be important. As proteins localized in the extracellular space, they are subject to secretory and proteolytic activation processes. As the results show, these principles seem to differ between MMP-2 and MMP-9 under the influence of hypoxia. MMP-9 mRNA is induced by shorttime hypoxia (10 h), while MMP-2 mRNA responds only to long-term hypoxia (34 h). At the intracellular protein level, there is another significant difference: In contrast to MMP2, which steadily declines over time, MMP-9 seems to accumulate. This observation may reflect differences in secretion (Jiang and Muschel, 2002). The most prominent effect is seen at the level of pro-MMP-2 activation. Under hypoxic conditions, MMP-2 is more effectively cleaved into the active forms. However, MMP-9 expression shows an imbalance between mRNA and protein level, which may be subject to an inhibited translation of MMP-9 mRNA. Simultaneously, MMP-9 activity is decreased by hypoxia, which does not seem to be triggered by the inhibitor TIMP1. Both inhibitory processes affecting the MMP-9 expression and activation are repealed under long-term hypoxia. In combination with the enhanced MMP-2 activation, it is part of the network of anti-fibrotic control. The expression behavior of MMP-2 activator MT1-MMP and MMP-9 inhibitor TIMP-1 is unchanged at the mRNA level, suggesting that they were not involved in hypoxic activation of MMP expression. However, TIMP-2, the inhibitor of MMP-2 action, was suppressed with the result of an increase in MMP-2 activity. Hammani et al. (1996) discussed that TIMP-2 has several features of a housekeeping gene due to its constitutive promoter. The observed alteration in mRNA concentration may therefore be attributed to mRNA decay. Together, the results indicate that in hypoxic HT1080 cells, only the alpha subunits of P4H are up-regulated at the transcriptional and translational level, whereas the beta subunit remains unaffected. MMP-2 is mainly controlled by the proteolytic activation the pro-enzyme and MMP-9 by transcription, translation and secretion. Furthermore, there is evidence that at least four of the investigated genes undergo posttranscriptional control, affecting the mRNA stability and translational efficiency. Posttranscriptional activation of gene expression by hypoxia has been documented for

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typical hypoxia-inducible proteins like erythropoietin or tyrosine hydroxylase. Both are activated by mRNA binding proteins, which interact with a target sequence in the 3Vuntranslated region of the mRNAs, the so called bhypoxiainducible protein binding siteQ (HIPS) (Czyzyk-Krzeska and Bendixen, 1999; Paulding and Czyzyk-Krzeska, 1999). We are currently investigating if P4H-alpha(I), (II) and MMP-9 mRNAs are posttranscriptionally controlled by this type of mRNA/protein interaction. Our results show a biphasic effect of hypoxia on collagen homeostasis in HT1080 fibroblasts. Short-term hypoxia demonstrates that development of fibrosis is not only characterized by an increased collagen expression, but also by P4H induction and simultaneous MMP inhibition. It constitutes a rapid response to conditions where fibrosis has a positive physiological meaning, like wound healing. However, under conditions of an ischemia, this constellation is fatal due to the formation of diffusion barriers triggered by the induced fibrosis. This additionally limits the oxygen supply. In contrast, long-term hypoxia activates compensatory anti-fibrotic mechanisms, as demonstrated by abolished MMP inhibition. In long-term or chronic hypoxia, the proteolytic cleavage of pro-MMP-2, promoted by repression of TIMP-2, seems to be the main mechanism that is potentially a target for fibrosis intervention.

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