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Cardiovascular Research 2004 61(4):736-744; doi:10.1016/j.cardiores.2003.12.018
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Copyright © 2004, European Society of Cardiology

Differential roles of extracellular signal-regulated kinase 1/2 and p38MAPK in mechanical load-induced procollagen {alpha}1(I) gene expression in cardiac fibroblasts

Jenny Papakrivopoulou*, Gisela E Lindahl, Jill E Bishop and Geoffrey J Laurent

Centre for Cardiopulmonary Biochemistry and Respiratory Research, University College London Medical School, The Rayne Institute, 5, University Street, London WC1E 6JJ, UK

* Corresponding author. Tel.: +44-207-679-6976; fax: +44-207-679-6973. jpapakri{at}yahoo.co.uk

Received 10 April 2003; revised 25 November 2003; accepted 5 December 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective and methods: We have previously demonstrated that mechanical loading of cardiac fibroblasts leads to increased synthesis and gene expression of the extracellular matrix protein collagen. We hypothesised that the upregulation of procollagen gene expression in cardiac fibroblasts, in response to cyclic mechanical load, is mediated by one or more members of the MAP kinase family. To test this hypothesis, the effect of mechanical load on the activation of extracellular signal-regulated kinase (ERK) 1/2, p46/54JNK, and p38MAPK was examined in rat cardiac fibroblasts. Results: Peak phosphorylation of ERK 1/2, p38MAPK kinases, and p46/54JNK was observed following 10–20 min of continuous cyclic mechanical load. Mechanical load significantly increased procollagen {alpha}1(I) mRNA levels up to twofold above static controls after 24 h. This increase was completely abolished by the MEK 1/2 inhibitor U0126, with no effect on basal levels. In contrast, SB203580, a specific inhibitor of p38MAPK, enhanced both basal and stretch-stimulated levels of procollagen mRNA. Consistent with this finding, selective activation of the p38MAPK signalling pathway by expression of MKK6(Glu), a constitutive activator of p38MAPK, significantly reduced procollagen {alpha}1(I) promoter activity. SB203580-dependent increase in procollagen {alpha}1(I) was accompanied by ERK 1/2 activation, and inhibition of this pathway completely prevented SB203580-induced procollagen {alpha}1(I) expression. Conclusions: These results suggest that mechanical load-induced procollagen {alpha}1(I) gene expression requires ERK 1/2 activation and that the p38MAPK pathway negatively regulates gene expression in cardiac fibroblasts. These pathways are likely to be key in events leading to matrix deposition during heart growth and remodelling induced by mechanical load.

KEYWORDS Extracellular matrix; Fibrosis; Mechanotransduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The heart is continually subjected to mechanical forces due to changes in blood volume and pressure. As the physical properties of the heart are so important to its function, so is the extracellular matrix fundamental in maintaining the structure and function of the myocardium. Fibrillar collagens are the most abundant components of the extracellular matrix in the heart [1], and contribute significantly to the physical properties of tissues [2]. During pathophysiological conditions such as hypertension, the mechanical environment of the heart changes, resulting in tissue remodelling. In vivo, this remodelling includes enlargement of myocytes, proliferation of fibroblasts [3], and increased procollagen synthesis [4–6]. In vitro, mechanical loading induces cell hypertrophy and activation of a genetic program (e.g., activation of c-jun and c-fos) similar to that seen in the hypertrophic myocardium in vivo [7]. In fibroblasts, we have previously shown that mechanical load stimulates cardiac fibroblast procollagen gene expression and synthesis [8,9]. Although the effects of mechanical load on fibroblast function are becoming increasingly documented, the signal transduction pathways that mediate these responses are still poorly understood.

Mechanical loading of myocytes stimulates a variety of signal transduction pathways including the mitogen-activated protein kinase (MAPK) family member extracellular signal-regulated kinase (ERK) 1/2, and its upstream activators MEK 1 and Raf-1 [10]. c-Jun NH2–terminal kinase (JNK) can also be activated by mechanical forces in cardiac myocytes [11] and endothelial cells [12]. Activation of these signalling pathways by mechanical load, as well as activation of protein kinase C and Rsk [13], have each been implicated in signalling one or more components of the hypertrophic phenotype. In cardiac fibroblasts, MacKenna et al. [14] demonstrated that static mechanical load activates ERK 1/2 and JNK1, although the downstream biological events were not assessed. Components of the MAPK/ERK 1/2 cascade have been shown to contribute to the regulation of procollagen gene transcription in transforming growth factor-β1 (TGF-β1) stimulated 3T3 cells and activated rat hepatic stellate cells [15,16]. However, the signalling pathways mediating the effects of mechanical load on collagen gene expression remain as yet largely unknown. p38MAPK is activated by a variety of stimuli such as phenylephrine [17], endothelin-1, oxidative stress [18], and hypoxia [19]. Activation of specific isoforms of p38MAPK has been implicated in the development of cardiomyocyte hypertrophy and apoptosis [20], but has been little studied in the context of collagen gene regulation.

To test whether strain-induced upregulation in procollagen {alpha}1(I) gene expression is mediated via the MAP kinases, we studied firstly the activation of these kinases by mechanical load and then used pharmacological inhibitors of the ERK 1/2 and p38MAPK to determine their importance in mediating this response. We report here that three members of the MAP kinase family, ERK 1/2, p46/54JNK, and p38MAPK, are activated by cyclic mechanical load in cardiac fibroblasts. Inhibition of ERK 1/2 activity completely abrogated the mechanical load-induced procollagen {alpha}1(I) gene expression, whereas inhibition of p38 MAPK activity potentiated this process. These observations demonstrate a key role for the ERK 1/2 and p38MAPK pathways in the regulation of extracellular matrix synthesis by mechanical load.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Materials
All tissue culture reagents were purchased from GIBCO-BRL (Paisley, UK). Anti-phospho-ERK 1/2, p38 antibodies, and a nonradioactive kit to measure JNK activity were from New England Biolabs (Hertfordshire, UK). Secondary IgG conjugated to horseradish peroxidase was from DAKO (Bucks, UK). The BCA protein assay reagent was from Pierce and Warriner (Chester, UK). SB203580 was from Calbiochem-Novabiochem (Nottingham, UK). The U0126 MEK inhibitor and the calcium phosphate ProFection Mammalian Transfection System were from Promega (Madison, WI, USA). Chloramphenicol acetyl transferase (CAT) protein levels were measured by enzyme-linked immunosorbent assay (ELISA; Roche Diagnostics, Mannheim, Germany). [{alpha}32P]dCTP, the DNA Megaprime labelling kit, and the enhanced chemiluminescence (ECL) system were purchased from Amersham (Little Chalfont, Bucks, UK). All other reagents were from Sigma (Dorset, UK).

2.2 Cell culture and mechanical loading
The investigation conforms with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Primary cultures of foetal rat cardiac fibroblasts were prepared and grown to confluence on elastin-coated Flex I platesTM (Flexcell International, Chapel Hill, NC, USA) as previously described [8]. Upon reaching visual confluence (3–4 days), cells were incubated for 24 h in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 50 µg/ml ascorbic acid, 0.2 mM proline, 1 mg/ml bovine serum albumin (BSA), and 1 µg/ml transferrin. The medium was then replaced with DMEM/10% foetal calf serum (FCS) for 24 h and, subsequently, the cells were subjected to mechanical load. Cells were cyclically loaded for up to 24 h at a 1.5-Hz frequency and 20% maximum elongation, using the Flexercell FX3000 apparatus. Rigid control cultures on elastin-coated Flex I plates were grown in parallel. Cells were used between passages 4 and 8.

2.3 Northern analysis of type I procollagen gene expression
Total RNA, pooled from the cell layers of six wells, was extracted using 1 ml of TRIzol reagent as per manufacturers' instructions (GIBCO-BRL). RNA loading buffer, containing ethidium bromide (EtBr), was added to 5 µg of RNA, which was then fractionated on a 1% agarose gel. Integrity of the RNA and uniformity of loading were confirmed by quantitation of the EtBr-stained 28S ribosomal RNA band, visualised and photographed under UV light. RNA was transferred to a nylon membrane and hybridised overnight with the {alpha}1(I) procollagen probe Hf677 [American Type Culture Collection (ATCC)], labelled with [{alpha}32P]dCTP. Two bands were obtained following hybridisation, corresponding to 5.8 and 4.8 Kb of {alpha}1(I) procollagen mRNA transcripts. The signal generated by both bands was quantitated using a phosphorimager (Fuji) linked to Advanced Image Data Analysis (AIDA) software (Fujifilm) and normalised relative to the loading of total RNA in the same sample analysing the ethidium bromide-stained 28S ribosomal RNA band with the AIDA software.

2.4 Electrophoresis and immunoblotting
Confluent cultures of cells were lysed on ice with lysis buffer [62.5 mM Tris–HCl (pH 6.8, 25 °C), 2% wt/vol sodium dodecyl sulfate (SDS), 10% glycerol, 50 mM DTT, and 0.1% bromophenol blue]. The cellular extracts, pooled from two wells, and molecular mass standards were electrophoresed in 10% (wt/vol) SDS polyacrylamide gels and transferred to nitrocellulose membranes. Even transfer and equal loading were verified by Ponceau S staining of the membranes. The blots were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% (wt/vol) Tween 20, and incubated with primary antibody overnight at 4 °C. After washing, the membranes were incubated for 1 h with secondary antibody at room temperature. Immunoreactive bands were visualised by enhanced chemiluminescence and quantitated by densitometric scanning.

2.5 Immunoassay for p46/54JNK activity
The procedure followed was that described in the instructions for the kit provided by New England Biolabs. Cells were grown on elastin-coated Flex I plates as described above. Briefly, cell lysis was accomplished by adding the lysis buffer provided. Protein concentration was determined by the BCA protein assay according to the manufacturer's instructions. Three hundred micrograms of protein was incubated with 2 µg of GST-c-Jun (1–89) fusion protein bound to glutathione Sepharose beads to selectively precipitate JNKs. The kinase reaction was performed by the addition of 100 µM ATP for 30 min at 30 °C. c-Jun phosphorylation was selectively measured using a phospho-specific c-Jun antibody and immune complexes on nitrocellulose membrane were visualised by enhanced chemiluminescence as described previously for ERK 1/2 and p38MAPK.

2.6 Expression vectors and reporter constructs
Expression vectors for MKK6 (Ala) (a dominant negative mutant of MKK6 in which lysine 82 is replaced with alanine) and MKK6 (Glu) (a constitutively active mutant of MKK6 in which serine 207 and threonine 211 are replaced with glutamic acid) were kind gifts from Dr. R. Davis and have been described elsewhere [21]. pColCAT3.6/1.6 (also known as B16) was a gift from Drs. A. Lichtler and D. Rowe (University of Connecticut Health Center, CT, USA). This plasmid construct is identical to the B15 described by Breault et al. [22], except that an EcoRV site in the polylinker has been converted to a ClaI site. The construct contains sequences between –3518 and +1594 (relative to the transcription start site) of the rat COL1A1 gene, which includes the upstream promoter region, the first exon (the translational start site, ATG, has been converted to a NotI site) and most of the first intron.

2.7 Transient transfections and promoter activity assays
Rat cardiac fibroblasts were transfected using the calcium phosphate ProFection Mammalian Transfection System according to the manufacturer's recommendations. Cells were seeded onto 12-well plates 2 days before transfection. Three hours after the addition of fresh serum containing medium, transfections were performed in triplicate, with 0.5 µg of DNA for pColCAT 3.6/1.6 and 0.5 µg of DNA for MKK6(Ala)/MKK6(Glu) or empty vector (pcDNA3). After 16 h, cells were rinsed twice with phosphate-buffered saline (PBS) and incubated in serum-free DMEM containing serum replacement 1, HEPES buffer, glutamine, ascorbic acid, and proline, as described above. Twenty-four hours later, transfected cells were switched to medium containing 2% foetal calf serum, and were incubated for a further 24 h. Finally, plates were washed with PBS and lysed, and CAT protein levels were measured by ELISA.

2.8 Pharmacological inhibitors
All inhibitors used were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution and stored at –20 °C. The required concentrations of U0126 and SB203580 were added to the cells 0.5–1 h prior to the application of mechanical load. Vehicle (DMSO only) was added to control cultures.

2.9 Statistical analysis
All numerical results are presented as mean±S.E.M. The variation between data sets was tested with ANOVA, and the significance between two data sets was tested with unpaired t tests. Differences were considered significant when p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Mechanical load upregulates procollagen {alpha}1(I) gene expression by foetal rat cardiac fibroblasts
Confluent foetal rat cardiac fibroblasts were subjected to continuous cyclic mechanical load and procollagen {alpha}1(I) gene expression was assessed by Northern analysis (Fig. 1). Procollagen {alpha}1(I) mRNA exists as two molecular weight species, 4.8 and 5.8 kb, arising from two different polyadenylation sites (Fig. 1A). Fig. 1B shows that cyclic mechanical loading resulted in a doubling in steady-state procollagen {alpha}1(I) mRNA levels compared with static controls. Similar results were seen in six separate experiments, with steady-state procollagen {alpha}1(I) mRNA levels increased between 65% and 140% in response to mechanical load.


Figure 1
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  		Fig. 1 Effect of cyclic mechanical load on procollagen {alpha}1(I) mRNA levels in foetal rat cardiac fibroblasts. Foetal rat cardiac fibroblasts were grown on FlexTM I plates as described in Materials and Methods. Cells were then maintained rigid or subjected to continuous cyclic mechanical load for 24 h. (A) Northern blot analysis of 5 µg of total RNA using a cDNA probe for the {alpha}1(I) procollagen. The 28S ethidium bromide-stained band is also shown. (B) Densitometric analysis of procollagen {alpha}1(I) mRNA levels normalised to the 28S ethidium bromide-stained band. Values are expressed as percentage change from rigid control levels. Data were pooled from six independent experiments and expressed as mean±S.E.M. *p<0.01 versus rigid control.

 
3.2 The ERK 1/2 cascade positively regulates procollagen {alpha}1(I) gene expression in cardiac fibroblasts
The effect of mechanical load on MAPKs activation in foetal rat cardiac fibroblasts is shown in Fig. 2. ERK 1/2 phosphorylation was markedly enhanced after 10 min of mechanical load and returned to baseline levels by 120 min. Phosphorylation was shown to remain at basal levels after 6 h of continuous cyclic mechanical load (data not shown). p38MAPK phosphorylation was also increased after 10 min and returned to basal levels by 60 min. p46/54JNK activity was detectable, as c-Jun phosphorylation, after 10 min of loading, reached maximal levels after 20 min and returned to basal levels by 2 h of continuous cyclic mechanical load.


Figure 2
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  		Fig. 2 Kinetics of activation of ERK 1/2, p38MAPK, and p46/54JNK in foetal rat cardiac fibroblasts subjected to cyclic mechanical load. Cells were grown as described in Fig. 1 and either maintained rigid or subjected to mechanical load for the indicated times. Cell extracts were prepared as described in Materials and Methods. Equal amounts of lysates were separated by SDS polyacrylamide gel electrophoresis (PAGE) in 10% gels, followed by immunoblotting with anti-phospho-ERK 1/2 (A), anti-phospho-p38 (B). For JNK activity, cell lysates were incubated with c-Jun fusion protein for 16 h at 4 °C. Immunoprecipitates were then collected by centrifugation and incubated with 100 µM ATP. Twenty-five microliters of the reaction mixture was subjected to SDS-PAGE and immunoblotting with anti-phospho-c-Jun (C).

 
Next we examined the role of the ERK 1/2 cascade in mediating load-induced procollagen {alpha}1(I) gene expression using the selective inhibitor of MEK activation, U0126 [23]. Fig. 3A shows that mechanical load resulted in an increase in procollagen {alpha}1(I) mRNA levels of 67±10% (p<0.01), which was markedly reduced at both concentrations of U0126 examined: 2.5 and 5 µM. Fig. 3B shows that the effect of U0126 in blocking load-induced ERK 1/2 activation occurs without any effects on the activation of p46/54JNK or p38MAPK.


Figure 3
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  		Fig. 3 Effects of U0126 and SB203580 inhibitors on mechanical load-induced procollagen {alpha}1(I) gene expression and phosphorylation of ERK 1/2, p46/54JNK, and p38MAPK. Fibroblasts were maintained rigid or mechanically loaded for 24 h either in the absence or presence of the indicated concentrations of U0126 (A) and SB203580 (C) added 30 min prior to the start of the mechanical loading. Total RNA (5 µg) was subjected to Northern blot analysis using a cDNA probe for the {alpha}1(I) procollagen. A representative autoradiogram is shown above, and the results of the densitometric analysis of {alpha}1(I) procollagen mRNA levels, normalised to the 28S ethidium bromide-stained band, are shown below (percent change from rigid media control). Values are means of four independent experiments ±S.E.M. for U0126 and six independent experiments ±S.E.M. for SB203580. *p<0.01 versus rigid control, §p<0.01 versus loaded control. Cells were mechanically loaded for 10 min (20 min for c-jun) in the presence or absence of the indicated concentrations of U0126 (B) or SB203580 (D). The inhibitors were added 1 h prior to mechanical loading. Cell extracts were prepared and equal amounts were separated by SDS-PAGE, followed by immunoblotting with anti-phospho ERK 1/2, anti-phospho-p38MAPK, and anti-phospho-c-jun for p46/54JNK, as described in Fig 2. Representative immunoblots from at least three separate experiments are shown.

 
3.3 The p38MAPK cascade negatively regulates procollagen {alpha}1(I) gene expression in cardiac fibroblasts
Fig. 3C shows the effect of SB203580, a potent p38MAPK kinase inhibitor, on the load-induced increase in procollagen type I gene expression. Treatment with this agent resulted in an increase in procollagen mRNA levels above those observed in both rigid control cells and mechanically loaded cells. Basal levels of procollagen mRNA increased by approximately 50–70% in treated cells and there was a threefold increase in the stretch response in the presence of the inhibitor. Examination of mechanical load-induced p38MAPK phosphorylation in the presence of SB203580 revealed that in foetal rat cardiac fibroblasts, this compound inhibits phosphorylation of this kinase (Fig. 3D).

To further investigate the role of the p38MAPK pathway in the regulation of procollagen type I gene expression, we used previously characterised expression constructs to modulate endogenous p38MAPK activity. Procollagen {alpha}1(I) gene expression was monitored by transfecting cardiac fibroblasts with a fragment of the rat COL1A1 promoter fused to the CAT reporter gene (pColCAT3.6/1.6). Expression of the constitutively activated MKK6(Glu) reduced CAT protein levels at least threefold, when compared with CAT protein levels in control cells transfected with the empty vector (Fig. 4). Expression of the dominant negative regulatory MKK6(Ala) construct had no effect on CAT protein levels compared with control cells. These results suggested that activation of p38MAPK by itself, via its upstream activator MKK6, is sufficient to suppress procollagen type I gene transcription.


Figure 4
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  		Fig. 4 Activation of the p38MAPK pathway interferes with procollagen {alpha}1(I) promoter activity. Cardiac fibroblasts were cotransfected with the pColCAT 3.6/1.6 reporter and expression vectors encoding MKK6(Ala), MKK6(Glu), or empty pcDNA3 vector (vector). Following transfection, CAT protein levels were determined as described in Materials and Methods and normalised to the protein content of each extract. CAT protein levels in cells transfected with empty vector were given an arbitrary value of 1. The results are presented as mean±S.E.M. and are representative of three independent experiments performed in triplicate.

 
3.4 SB203580-induced procollagen {alpha}1(I) gene expression is mediated by the ERK 1/2 cascade
Since activation of the ERK 1/2 cascade was shown to play an important role in the induction of procollagen type I gene expression, and cross-talk between these two signalling pathways has recently been reported, we determined whether the SB203580 compound has an effect on activation of ERK 1/2. We therefore performed an extended time course examining the effect of SB203580 on ERK 1/2 phosphorylation. SB203580 treatment resulted in a transient activation of ERK 1/2, which peaked at 20 min and returned to near basal levels by 2 h (Fig. 5A). SB203580 also increased ERK 1/2 activation above control levels in cells mechanically loaded over the same time period (Fig. 5B). To further elucidate the role of this pathway in SB203580-induced procollagen {alpha}1(I) gene expression, we used the U0126 inhibitor. U0126, at both concentrations examined, inhibited SB203580-induced procollagen {alpha}1(I) gene expression, (Fig. 5C), suggesting that the induction of procollagen type I gene expression is mediated by the ERK 1/2 pathway.


Figure 5
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  		Fig. 5 SB203580-induced procollagen {alpha}1(I) gene expression is mediated by the ERK 1/2 pathway. (A and B) Kinetics of ERK 1/2 phosphorylation by SB203580 in rigid and mechanically loaded cells. Cells were maintained rigid or mechanically loaded as described in Materials and Methods. At the start of the experiment, cells were either left untreated or treated with SB203580 (5 µM), or U0126 (2.5 and 5 µM). After the indicated times, equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted with phospho-specific ERK 1/2 antibody. (C) Cardiac fibroblasts were grown to confluence in six-well plates as described in Materials and Methods and were either untreated or treated with SB203580 (5 µM) for 24 h either in the absence or presence of indicated concentrations of U0126 that was added 30 min prior to SB203580 addition. Total RNA (5 µg) was subjected to Northern blot analysis using a cDNA probe for the {alpha}1(I) procollagen. A representative autoradiogram is shown above, and the results of the densitometric analysis of {alpha}1(I) procollagen mRNA levels, normalised to the 28S ethidium bromide-stained band, are shown below (percent change from media control). Results shown are representative of one experiment.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Mechanical stimuli have been shown to be potent regulators of gene expression in the cardiovascular system. Although studies have been carried out to determine the mechano-signalling pathways that regulate hypertrophy in isolated myocytes, few studies have examined the pathways that are important in the load response in cardiac fibroblasts.

Cyclic mechanical load was shown to activate the three major MAPK signalling pathways (ERK 1/2, p46/54JNK, and p38MAPK) in foetal rat cardiac fibroblasts. Activation of ERK 1/2 has previously been demonstrated in various cell types in response to mechanical stimuli [12,24–27]. ERK 1/2 and JNK1 have also been shown to be activated by static stretch in adult cardiac fibroblasts [14]. This study is the first to demonstrate p38MAPK kinase activation by cyclic mechanical load in cardiac fibroblasts.

As the three MAP kinases that we have examined have distinct downstream targets, this simultaneous activation may be a reflection of the multiple effects of mechanical load on cellular function. In this study, we have focused on the effect of mechanical load on procollagen gene expression. We demonstrate that, first, cyclic mechanical load leads to an activation of ERK 1/2 in a time-dependent manner (Fig. 2); and, second, U0126, a specific inhibitor of MEK activity, inhibits both ERK 1/2 activation and procollagen {alpha}1(I) gene expression induced by mechanical load, without affecting phosphorylation levels of p46/54JNK and p38MAPK (Fig. 3A and B).

The precise mechanism by which ERK 1/2 regulates procollagen gene expression is presently unclear. We found that activation of ERK 1/2 occurs within minutes of the application of mechanical load, yet a significant upregulation of procollagen {alpha}1(I) mRNA levels was not observed until 24 h later. Since active ERK 1/2 translocates to the nucleus, it may act directly on the procollagen gene. Transcription factors of the Egr, Ets, and AP-1 families have been suggested as possible downstream ERK 1/2 substrates [28–33]. The {alpha}1(I) procollagen gene contains both AP-1 and Ets binding sites present in its 5'-UTR and first intron [34–40], and therefore these sequences may represent downstream targets of ERK 1/2. The delayed increase in mRNA levels could then be due to a slow rate of transcription of the procollagen gene. Another possibility is that transcription factors activated by ERK 1/2 do not act directly on the procollagen promoter, but on genes coding for other downstream proteins necessary to cause the increase in procollagen mRNA levels (e.g., transcription factors, signalling molecules, or growth factors). These mechanisms are currently under investigation.

We also investigated the role of the p38MAPK pathway in the regulation of procollagen gene expression. Classically, the p38MAPK cascade has been shown to be involved in apoptosis and inflammatory reactions in response to cellular stresses [41]. In the cardiovascular system, activation of the p38MAPK pathway has been linked to both hypertrophy and apoptosis in cardiomyocytes [42]. Recently, a study by Chin et al. [43] has implicated the p38MAPK cascade in the induction of procollagen type I gene expression in response to TGF-β1 in mesangial cells.

The results presented in this study suggest that the p38MAPK signalling cascade exerts a negative regulatory effect on procollagen type I expression. Using a specific p38MAPK inhibitor, SB203580 [44], we observed that inhibition of this kinase had the opposite effect to inhibition of ERK 1/2. Treatment with SB203580 resulted in superinduction of procollagen type I expression in both rigid and mechanically loaded cells (Fig. 3B). Although the SB203580 compound is commonly used as a specific inhibitor of this kinase, reports have suggested that it may act on other enzymes such as cyclooxygenase-1 and cyclooxygenase-2 (COX-1/COX-2) and thromboxane A2 [45]. Cyclooxygenases are regulatory enzymes in the prostaglandin biosynthetic pathway, and prostaglandins of the E-type have been shown to be potent inhibitors of procollagen metabolism [46]. At this stage, we cannot rule out that the observed effect of SB203580 on procollagen type I gene expression in our system may be due to inhibition of COX-1/COX-2. However, our observation that expression of MKK6(Glu), a constitutive activator of p38MAPK, significantly suppressed procollagen type I promoter activity (Fig. 4) suggests that the effect of SB203580 on procollagen type I gene expression is through its inhibition of the p38MAPK signalling cascade.

The exact mechanism(s) by which the p38MAPK signalling cascade may exert its effects on procollagen type I gene expression remains to be elucidated. Our results demonstrate cross-talk between the p38MAPK and ERK 1/2 pathways, previously undescribed with relation to its role in the regulation of procollagen {alpha}1(I) gene expression. Treatment with SB203580 alone under static conditions (i.e., inhibition of the p38MAPK pathway) resulted in transient but delayed (as compared to load) activation of the ERK 1/2 pathway. Maximal activation was observed at 20 min, and was sustained for up to 30 min following SB203580 treatment. Examination of the effect of SB203580 on load-induced ERK 1/2 activation, over the same time period, also revealed increased ERK 1/2 activation observed up to 2 h of continuous loading (Fig. 5B). The existence of cross-talk between the two pathways was further supported by the observation that inhibition of ERK 1/2 phosphorylation by U0126 attenuated SB203580-induced procollagen type I expression. Taken together, our results suggest that the p38MAPK pathway represses activation of the ERK 1/2 pathway.

Cross-talk has been shown between different signalling cascades and in a variety of cell types [47–50]. At this point, the exact mechanism underlying this cross-talk is not clear. Recent evidence, however, suggests that p38{alpha}MAPK can physically interact with ERK 1/2. This interaction is enhanced upon phosphorylation of p38{alpha}MAPK and is correlated with inhibition of ERK 1/2 activity [51]. These observations provide a possible mechanism for the regulation of procollagen type I gene expression by the p38MAPK pathway. Our results suggest that the p38MAPK cascade indirectly regulates procollagen type I gene expression through its negative regulation of the ERK 1/2 cascade. This latter effect may be due to a direct effect of a p38MAPK isoform on ERK 1/2 phosphorylation.

In summary, we have shown that ERK 1/2 plays an essential role in the induction of procollagen {alpha}1(I) gene transcription by mechanical load, thus establishing a link between MAPK-mediated intracellular signalling and regulation of procollagen gene expression. As all three MAPK pathways appear to be activated by mechanical load, a dynamic balance may be critical for determining/regulating the final biological outcome(s). Understanding the cytoplasmic signalling involved in the regulation of such key structural and disease related genes could lead to the identification of potential therapeutic targets.


   Acknowledgements
 
We are grateful to R. Davies for the MKK6 Ala and Glu plasmids. This work was supported by the University College London Medical School and the Wellcome Trust.


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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