Results

The p38 MAPK inhibitors abolished the increase of type I collagen in normal dermal fibroblasts stimulated with TGF-

In order to investigate the possible role of p38 MAPK in TGF--stimulated type I collagen synthesis in normal dermal fibroblasts, we used the specific p38 MAPK inhibitors, SB202190 and SB203580. The condition medium was collected and clarified by centrifugation, and immunoblotting was performed with a goat monoclonal antibody (mAB) against human type I collagen. As shown in Figure 1, the p38 MAPK inhibitor SB202190 did not change type I collagen synthesis significantly (lanes 1–3), but decreased TGF- (3 ng per mL)-induced type I collagen synthesis in normal fibroblasts in a concentration-dependent manner (lanes 7, 9,10). Moreover, SB203580 also abolished TGF--induced type I collagen synthesis in normal fibroblasts in a concentration-dependent manner (lanes 7, 11, 12). But, PD98059, a specific inhibitor of the mitogen-activated protein kinase kinase (MEK)1 and MEK2 activation (^{Alessi et al, 1995;Dudly et al, 1995}), did not abolish TGF--induced type I collagen synthesis in normal fibroblasts (Figure 1, lanes 7,8). PD98059 upregulated the basal expression of type I collagen protein. Cell viability was determined with trypan blue stain, which demonstrated that the addition of these concentrations of the inhibitors tested did not have cytotoxic effects.

Figure 1.

The p38 mitogen-activated protein kinase (MAPK) inhibitors abolished the increase of type I collagen synthesis in normal dermal fibroblasts stimulated with transforming growth factor- (TGF-). Synthesis of type I collagen in normal fibroblasts was determined by immunoblotting of tissue culture medium using antibodies to type I collagen. Comparisons of the reactivities were determined using a densitometer. Normal dermal fibroblasts (1 10⁵ cells) were seeded in six-well plates in minimum essential medium (MEM) with 10% fetal calf serum and grown to confluency. Cells were placed in 0.5 mL of MEM and 0.1% bovine serum albumin for 24 h prior to the TGF-1 treatment. The indicated reagents were added 1 h before the TGF-1 treatment. After incubation with or without 3 ng per mL TGF-1 for 24 h, the conditioning medium was collected and clarified by centrifugation, and the cells remaining in the dishes were treated with trypsin and counted electronically. The samples were normalized for cell number. The addition of the selective p38 MAPK inhibitor SB202190 or SB203580 abolished the TGF--mediated induction of type I collagen synthesis in a concentration-dependent manner, whereas PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK)1 and MEK2 activation, did not change TGF--mediated induction of type I collagen synthesis. Antibodies against -actin were also used as a loading control. Cell viability was determined with trypan blue stain, which demonstrated that the addition of these concentrations of the inhibitors tested did not have cytotoxic effects. Representative result of five separate experiments is shown.

Full figure and legend (56K)

Northern blot analysis was performed to investigate the effects of the p38 MAPK inhibitors on the expression of the human 2(I) collagen gene in normal fibroblasts. As shown in Figure 2, p38 MAPK inhibitors did not change basal expression of the 2(I) collagen gene, but inhibited the upregulated expression of the 2(I) collagen gene by TGF- (3 ng per mL) in normal dermal fibroblasts. These results strongly suggested that p38 MAPK is involved in TGF--induced type I collagen expression in normal dermal fibroblasts.

Figure 2.

Northern blot analysis of the 2(I) collagen gene expression in normal dermal fibroblasts stimulated with transforming growth factor- (TGF-). (A) Expression of the 1(I) and 2(I) collagen messenger RNA (mRNA) in normal fibroblasts was investigated by northern blot analysis in the presence or absence of the specific p38 mitogen-activated protein kinase inhibitors (10 M). Fibroblasts were treated or untreated with 3 ng per mL TGF-1 in the presence or absence of the indicated inhibitors. (B) The meansSE for the results of four separate experiments are shown. Values indicate the band density relative to untreated fibroblasts, which was set at 100. Comparison of each mRNA expression was made between untreated and TGF-1-treated normal fibroblasts under the same conditions (^*p<0.001).

Full figure and legend (101K)

TGF--induced p38 MAPK activation in normal dermal fibroblasts

Since the inhibition of p38 MAPK significantly decreased TGF--induced type I collagen expression in normal dermal fibroblasts, we determined whether TGF- activates p38 MAPK in normal dermal fibroblasts by immunoblotting using antibodies specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182). Immunoblotting of whole-cell extracts revealed that p38 MAPK Thr¹⁸⁰/Tyr¹⁸² phosphorylation occurred as early as 15 min after treatment with TGF-1. The maximal activation (4.4-fold) was noted at 30 min and a gradual decrease of p38 MAPK phosphorylation was seen (Figure 3a). The phosphorylation of p38 MAPK was shown to be very rapid and transit, as compared with the previous study (^{Sato et al, 2002}).

Figure 3.

Transforming growth factor- (TGF-) induced p38 mitogen-activated protein kinase (MAPK) activation in normal dermal fibroblasts. (A) Phosphorylation of p38 MAPK in normal fibroblasts was determined by immunoblotting using antibodies specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182). Immunoblotting of whole-cell extracts revealed that p38 MAPK Thr¹⁸⁰/Tyr¹⁸² phosphorylation occurred after treatment with 3 ng per mL TGF-1. TGF--induced phosphorylation of p38 MAPK reached the maximum level after 30 min. Antibodies against p38 MAPK were also used to confirm that the protein concentrations of p38 MAPK were maintained with or without TGF-1 stimulation. Representative result of three separate experiments is shown. (B) p38 MAPK was collected by immunoprecipitation, and subjected to an in vitro kinase assay in the presence of ATF-2 as described in Materials and Methods. In the p38 MAPK assay, TGF-1 stimulation (3 ng per mL) increased phosphorylation of ATF-2. The levels of phosphorylated ATF-2 were maximal 30 min after stimulation with TGF-. SB203580 (10 M) entirely abolished the TGF-1-induced (3 ng per mL, for 30 min) activation of ATF-2, whereas PD98059 (30 M) did not change TGF-1-mediated induction of phosphorylated ATF-2. Antibodies against -actin were also used as a loading control. Cell viability was determined with trypan blue stain, which demonstrated that the addition of these concentrations of the inhibitors tested did not have cytotoxic effects. Representative result of three separate experiments is shown.

Full figure and legend (77K)

The p38 MAPK assay showed that TGF-1 stimulation increased the p38 MAPK activity (Figure 3b). The level of phosphorylated activating transcription factor (ATF)-2 was maximal at 30 min and 1 h (5.8-fold) of incubation with TGF-1 (Figure 3b). In addition, SB203580, a specific p38 MAPK inhibitor, considerably inhibited the activation of ATF-2, whereas PD98059 did not affect the TGF-1-mediated induction of phosphorylated ATF-2. These results suggest that treatment with TGF-1 results in the activation of p38 MAPK in normal fibroblasts.

Effects of the p38 MAPK inhibitors on the TGF--induced transcriptional activity of the human 2(I) collagen gene in normal fibroblasts

Previous studies on cultured fibroblasts have shown that TGF- stimulates type I collagen synthesis in normal fibroblasts by acting mostly at the transcriptional level (^{Ignotz et al, 1987;Kähäri et al, 1990}). Therefore, we examined the effects of p38 MAPK inhibitors on the promoter activity of the human 2(I) collagen gene in normal fibroblasts. As seen in Figure 4, PD98059 (30 M) did not change basal promoter activity or TGF--induced promoter activity of the human 2(I) collagen gene. The addition of either SB202190 or SB203580 did not significantly change basal promoter activity of the human 2(I) collagen gene. On the other hand, the addition of either SB202190 or SB203580 abolished TGF--induced promoter activity of the human 2(I) collagen gene in normal fibroblasts.

Figure 4.

Effects of the p38 mitogen-activated protein kinase inhibitors on the transforming growth factor- (TGF-)-induced transcriptional activity of the human 2(I) collagen gene in normal fibroblasts. The plasmid carrying a -3.5 kb fragment of the human 2(I) collagen promoter was used in transient transfections of normal fibroblasts in the absence or presence of the indicated inhibitors. These inhibitors were added 1 h prior to TGF-1 stimulation (3 ng per mL, for 24 h). Values indicate the 2(I) collagen promoter activities relative to untreated fibroblasts, which were set at 100. The meansSE for four separate experiments are shown. Comparison of each promoter activity was made between untreated and TGF-1-treated normal fibroblasts under the same conditions (^*p<0.001). Cell viability was determined with trypan blue stain, which demonstrated that the addition of these concentrations of the inhibitors tested did not have cytotoxic effects.

Full figure and legend (16K)

The expression of the dominant-negative mutant p38 MAPK repressed the TGF--induced transcriptional activity of the human 2(I) collagen gene in normal fibroblasts

To further confirm the role of p38 MAPK in TGF--induced transcriptional activity of the human 2(I) collagen gene in normal dermal fibroblasts, transient transfection of the dominant-negative mutant p38 MAPK into normal fibroblasts was performed. As shown in Figure 5, transient transfection of either the dominant-negative mutant p38 MAPK or the dominant-negative mutant p38 MAPK did not significantly change basal promoter activity of the human 2(I) collagen gene in normal fibroblasts. Transient transfection of either the dominant-negative mutant p38 MAPK or the dominant-negative mutant p38 MAPK, however, abolished TGF--induced promoter activity of the human 2(I) collagen gene in a concentration-dependent manner. Cell viability was determined by trypan blue stain, which demonstrated that transient transfection of these amounts of dominant-negative mutant p38 MAPK did not have cytotoxic effects. On the other hand, transient transfection of the dominant-negative mutant extracellular signal-regulated kinase (ERK)2 in normal fibroblasts did not abolish TGF--induced promoter activity of the human 2(I) collagen gene, which indicates that the expression of the dominant-negative mutant p38 MAPK specifically represses the TGF--induced transcriptional activity of the human 2(I) collagen gene in normal fibroblasts.

Figure 5.

Effects of the dominant-negative mutants of p38 mitogen-activated protein kinase on the transforming growth factor- (TGF-)-induced transcriptional activity of the human 2(I) collagen gene in normal fibroblasts. The plasmid carrying a -3.5 kb fragment of the human 2(I) collagen promoter was used in transient transfections of normal fibroblasts in the absence or presence of the indicated dominant-negative mutants or the vector. Then fibroblasts were treated with TGF-1 (3 ng per mL) for 24 h. Values indicate the 2(I) collagen promoter activities relative to the normal fibroblasts untreated and transfected with the vector, which was set at 100. The meansSE for four separate experiments are shown. Comparison of each promoter activity was made between untreated and TGF-1-treated normal fibroblasts under the same conditions (^*p<0.01). Cell viability was determined with trypan blue stain, which demonstrated that transient transfection of the plasmids did not have cytotoxic effects.

Full figure and legend (15K)

Constitutive phosphorylation and activation of p38 MAPK in SSc fibroblasts

Next, to determine whether p38 MAPK is activated in SSc fibroblasts, the constitutive phosphorylation of p38 MAPK in SSc and normal fibroblasts was investigated using serum-deprived SSc and normal dermal fibroblasts. Detection of the phosphorylated form of p38 MAPK was performed by immunoblotting using antibodies specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182). Immunoblotting of whole-cell extracts revealed that constitutive p38 MAPK Thr¹⁸⁰/Tyr¹⁸² phosphorylation occurred in SSc fibroblasts (Figure 6a, lanes 6–8 and Figure 6b). Antibodies against p38 MAPK were also used to determine the protein concentrations of p38 MAPK in SSc and normal fibroblasts, which demonstrated that SSc and normal fibroblasts had an equivalent amount of p38 MAPK (Figure 6a). We next examined whether p38 MAPK phosphorylation detected in SSc fibroblasts was associated with increased p38 MAPK activity using the p38 MAP kinase assay. Increased p38 MAPK activity was also detected in SSc fibroblasts (Figure 6c, lanes 6–8 and Figure 6d).

Figure 6.

Constitutive phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in systemic sclerosis (SSc) fibroblasts. (A) Phosphorylation of p38 MAPK in normal and SSc fibroblasts was determined by immunoblotting using antibodies specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182). Immunoblotting of whole-cell extracts revealed that constitutive p38 MAPK Thr¹⁸⁰/Tyr¹⁸² phosphorylation occurred in SSc fibroblasts. Antibodies against p38 MAPK were also used to confirm that the protein concentrations of p38 MAPK were maintained between normal and SSc fibroblasts. Representative result of four separate experiments is shown. (B) Quantitative analysis of levels of p38 MAPK phosphorylation. The meansSE for the results of four separate experiments are shown. Values indicate the band density relative to normal fibroblasts, which was set at 100. Comparison of constitutive levels of p38 MAPK phosphorylation was made between normal and SSc fibroblasts under the same conditions (^*p<0.001). (C) p38 MAPK was collected by immunoprecipitation, and subjected to an in vitro kinase assay in the presence of ATF-2 as described in Materials and Methods. In the p38 MAPK assay, constitutive activated p38 MAPK increased phosphorylation of ATF-2 in SSc fibroblasts. Antibodies against ATF-2 were also used as a loading control. Representative result of four separate experiments is shown. (D) Quantitative analysis of the p38 MAPK activities. The meansSE for the results of four separate experiments are shown. Values indicate the band density relative to normal fibroblasts, which was set at 100. Comparison of each p38 MAPK activity was made between normal and SSc fibroblasts under the same conditions (^*p<0.001). (E) Expression levels of type I collagen and fibronectin were determined in normal and SSc fibroblasts using immunoblotting. The levels of p38 MAPK phosphorylation in SSc fibroblasts were correlated with the expression levels of type I collagen and fibronectin proteins. Antibodies against -actin were also used as a loading control. Representative results of four separate experiments is shown. (F) Quantitative analysis of type I collagen and fibronectin protein levels. The meansSE for the results of four separate experiments are shown. Values indicate the band density relative to normal fibroblasts, which was set at 100. Comparison of each protein expression was made between normal and SSc fibroblasts under the same conditions (^*p<0.001).

Full figure and legend (32K)

Constitutive phosphorylation of p38 MAPK was correlated with the increased synthesis of type I collagen and fibronectin in SSc dermal fibroblasts

Next, we investigated the significance of constitutive phosphorylation and activation of p38 MAPK in SSc fibroblasts. Expression levels of type I collagen and fibronectin were determined in normal and SSc fibroblasts using immunoblotting. The levels of p38 MAPK phosphorylation in SSc fibroblasts were correlated with the expression levels of type I collagen and fibronectin proteins (Figure 6e,f). Note that SSc fibroblasts in which constitutive p38 MAPK phosphorylation was detected produced greater amounts of type I collagen and fibronectin (Figure 6e, lanes 6–8).

Involvement of the p38 MAPK in the upregulated expression of type I collagen and fibronectin in SSc fibroblasts

Next, we investigated whether the constitutive phosphorylation and activation of p38 MAPK in SSc fibroblasts is involved in the upregulated ECM expression in SSc fibroblasts. Expression levels of type I collagen and fibronectin were determined in normal and SSc fibroblasts in the presence and absence of p38 MAPK inhibitors. The addition of p38 MAPK inhibitors abolished the upregulated expression of type I collagen as well as fibronectin in SSc fibroblasts in a concentration-dependent manner (Figure 7).

Figure 7.

Effects of the p38 mitogen-activated protein kinase (MAPK) inhibitors on the upregulated expression of type I collagen and fibronectin in systemic sclerosis (SSc) fibroblasts. Expression levels of type I collagen (A, B) and fibronectin (C, D) in normal and SSc fibroblasts were determined by immunoblotting of tissue culture medium using antibodies to type I collagen or fibronectin. The addition of the selective p38 MAPK inhibitor SB202190 or SB203580 abolished the upregulated expression of type I collagen and fibronectin in SSc fibroblasts in a concentration-dependent manner, whereas PD98059, a specific inhibitor of mitogen-activated kinase kinase (MEK)1 and MEK2 activation, did not change the expression levels of type I collagen. Cell viability was determined with trypan blue stain, which demonstrated that the addition of these concentrations of the inhibitors tested did not have cytotoxic effects. Antibodies against -actin were also used as a loading control. Representative result of four separate experiments is shown (A, C). Quantitative analysis of type I collagen (B) and fibronectin (D) proteins. The meansSE for the results of four separate experiments are shown. Values indicate the band density relative to normal fibroblasts, which was set at 100. Comparison of each p38 MAPK activity was made between normal and SSc fibroblasts under the same conditions (^*p<0.001).

Full figure and legend (38K)

SSc fibroblasts were shown to exhibit elevated activity of the human 2(I) collagen promoter (^{Ihn et al, 2001b}). Therefore, we examined the effects of p38 MAPK inhibitors on the promoter activity of the human 2(I) collagen gene. As seen in Figure 8a, normal fibroblasts treated with p38 MAPK inhibitors showed little reduction in the promoter activity of the human 2(I) collagen gene. The treatment with PD98059 had little effect on the promoter activity of the human 2(I) collagen gene in SSc fibroblasts. SSc fibroblasts treated with p38 MAPK inhibitors, however, showed concentration-dependent reduction in this activity (Figure 8a).

Figure 8.

Involvement of p38 mitogen-acitvated protein kinase in the upregulated transcriptional activity of the human 2(I) collagen gene in systemic sclerosis (SSc) fibroblasts. (A) The plasmid carrying a -3.5 kb fragment of the human 2(I) collagen promoter was used in transient transfections of normal and SSc fibroblasts in the absence or presence of the indicated inhibitors. Values indicate the 2(I) collagen promoter activities relative to untreated normal fibroblasts, which was set at 100. The meansSE for four separate experiments are shown. Comparison of each promoter activity was made with untreated normal fibroblasts (^*p<0.001). Cell viability was determined with trypan blue stain, which demonstrated that the addition of these concentrations of the inhibitors tested did not have cytotoxic effects. (B) The plasmid carrying a -3.5 kb fragment of the human 2(I) collagen promoter was used in transient transfections of normal and SSc fibroblasts in the absence or presence of the indicated dominant-negative mutants or the vector. Values indicate the 2(I) collagen promoter activities relative to the normal fibroblasts transfected with the vector, which was set at 100. The meansSE for four separate experiments are shown. Comparison of each promoter activity was made with normal fibroblasts transfected with the vector (^*p<0.01). Cell viability was determined with trypan blue stain, which demonstrated that transient transfection of the plasmids did not have cytotoxic effects.

Full figure and legend (33K)

To further confirm the role of p38 MAPK in the upregulated transcriptional activity of the human 2(I) collagen gene in SSc fibroblasts, transient transfection of the dominant-negative mutant p38 MAPK into SSc fibroblasts was performed. As shown in Figure 8b, transient transfection of either the dominant-negative mutant p38 MAPK or the dominant-negative mutant p38 MAPK did not significantly change the basal promoter activity of the human 2(I) collagen gene in normal fibroblasts. But transient transfection of either the dominant-negative mutant p38 MAPK or the dominant-negative mutant p38 MAPK abolished the upregulated promoter activity of the human 2(I) collagen gene in SSc fibroblasts in a concentration-dependent manner. Cell viability was determined by trypan blue stain, which demonstrated that transient transfection of these amounts of dominant-negative mutant p38 MAPK did not have cytotoxic effects. On the other hand, transient transfection of the dominant-negative mutant ERK2 in SSc fibroblasts did not abolish the upregulated promoter activity of the human 2(I) collagen gene in SSc fibroblasts, which indicates that the expression of the dominant-negative mutant p38 MAPK specifically represses the upregulated transcriptional activity of the human 2(I) collagen gene in SSc fibroblasts.

Discussion

Excessive ECM deposition in the skin, lungs, or other organs is a hallmark of SSc. The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that activation of lesional fibroblasts contributes to the fibrotic process (^{Jelaska et al, 1996;Ihn et al, 2001b}). The mechanism of dermal fibroblast activation in SSc is presently unknown; however, many of the characteristics of SSc fibroblasts resemble those of normal fibroblasts stimulated by TGF- (^{LeRoy et al, 1989;Ihn et al, 2001b}), suggesting that the dermal fibroblast activation in SSc may be a result of stimulation by autocrine TGF-. This notion is supported by our recent findings that SSc fibroblasts express elevated expression levels of TGF- receptor types I and II and that this correlates with elevated expression levels of type I collagen (^{Ihn et al, 2001b;Yamane et al, 2002}).

This study demonstrated constitutive phosphorylation and activation of p38 MAPK in SSc fibroblasts (Figure 6a–d), which was correlated with increased expression of type I collagen and fibronectin (Figure 6e and Figure 6f). During the preparation of this paper, Sato et al reported that some SSc fibroblasts showed increased phosphorylation of p38 MAPK (^{Sato et al, 2002}), which is consistent with our results. They concluded, however, that the phosphorylation of p38 MAPK was not correlated with collagen production, which is not consistent with our results. At this point, the reason for this discrepancy is not clear, but it may be partially because of the different patient populations, and further studies are required to address this point. But the inhibition of p38 MAPK by p38 MAPK inhibitors or dominant-negative mutant p38 MAPK abolished the upregulated expression of type I collagen or fibronectin in SSc fibroblasts (Figure 7 and Figure 8), which suggests the crucial role of p38 MAPK in upregulated expression of ECM in SSc fibroblasts. Basal activation of p38 MAPK was not detected in all SSc fibroblasts lines. This may suggest that SSc fibroblasts have heterogeneity and that p38 MAPK plays a role in some parts of SSc patients.

TGF- is a multifunctional peptide that regulates cell growth and differentiation (^{Roberts and Sporn, 1993}). It is important in the developmental process, regulating the immune response, and plays a fundamental role in ECM formation (^{Roberts and Sporn, 1993}). TGF- has been shown to induce many tissue repair factors, and its pathogenic role in the development of fibrosis is becoming increasingly clear (^{Border and Noble, 1994}). Recent studies have identified Smad proteins as major downstream targets of TGF- receptor kinases (^{Kretzschmar and Massaguë, 1998}). As for the collagen gene, the synergistic cooperation between Sp1 and Smad3/Smad4 was shown to be required for TGF- response of the type I collagen gene (^{Zhang et al, 2000;Poncelet and Schnaper, 2001}), and the cooperation of p300/CBP with Smad was demonstrated in TGF- response of the type I collagen gene (^{Chen et al, 1999;Ghosh et al, 2000}). Other signaling pathways besides the Smad proteins have also been shown to mediate TGF- signaling, and these pathways include the p38 MAP pathway (^{Kyriakis and Avruch, 1996}). In human dermal fibroblasts, collagen-dependent induction of matrix metalloproteinase (MMP)-13 requires p38 MAPK activity (^{Ravanti et al, 1999a}). In human gingival fibroblasts, TGF-1 induces MMP-13 via p38 MAPK (^{Ravanti et al, 1999b}). In this study, we showed that the stimulation of human normal dermal fibroblasts by TGF-1 resulted in the phosphorylation and activation of p38 MAPK. The TGF-1-mediated induction of 2(I) collagen gene expression was significantly inhibited by selective p38 MAPK inhibitors, but not by a selective MEK1/2 inhibitor PD98059 in normal fibroblasts. TGF-1-mediated transcriptional activation of the 2(I) collagen gene was significantly inhibited by selective p38 MAPK inhibitors or transfection of the dominant-negative mutant p38 MAPK in normal fibroblasts. These results suggest that the p38 MAPK pathway is involved in the regulation of the collagen gene by TGF-1 in normal fibroblasts. As described above, the dermal fibroblast activation in SSc may be a result of stimulation by autocrine TGF-. Therefore, there was a possibility that constitutive phosphorylation and activation of p38 MAPK in SSc fibroblasts may be a result of stimulation by autocrine TGF-, which is consistent with these results.

In conclusion, this study demonstrated constitutive phosphorylation and activation of p38 MAPK in SSc fibroblasts and the contribution of p38 MAPK signaling both to the TGF--mediated regulation of the human 2(I) collagen gene in normal fibroblasts and to the constitutive upregulated ECM expression in SSc fibroblasts. Our results raise the possibility of the development of a therapy for fibrosis, especially for SSc, using an approach that inhibits the p38 MAPK pathway.

Materials and Methods

Cytokines and other materials

Recombinant human TGF-1 was purchased from R&D Systems Inc. (Minneapolis, Minnesota). SB203580, SB202190, and PD98059, which were obtained from Calbiochem Corp. (La Jolla, California), were dissolved in dimethyl sulfoxide (DMSO). Controls were incubated with an equal concentration of DMSO. The p38 MAPK rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California). The phospho-p38 MAPK (Thr180/Tyr182) rabbit polyclonal antibody and ATF-2 fusion protein were obtained from New England Biolabs (Beverly, Massachusetts). Protein G sepharose was obtained from Zymed Laboratories Inc. (San Francisco, California).

Cell cultures

Human adult skin fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of seven randomly selected patients with diffuse cutaneous SSc of less than 2 y' duration. Control fibroblasts were obtained from skin biopsy specimens from seven healthy donors. Institutional Review Board approval and written informed consent were obtained according to the Declaration of Helsinki. The controls were matched with each SSc patient for age, sex, and biopsy site, and their specimens were processed in parallel. Primary explant cultures were established in 25 cm² culture flasks in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 50 g per mL amphotericin (^{Ihn et al, 1997;Ihn and Tamaki, 2000b}). Fibroblast cultures independently isolated from different individuals were maintained as monolayers at 37°C in 95% air, 5% CO₂, and studied between the third and sixth subpassages. Cell viability was determined by trypan blue stain (^{Ihn and Tamaki, 2000c}).

Western blot analysis

Dermal fibroblasts (1 10⁵ cells) were seeded in six-well plates in MEM with 10% FCS and grown to confluency. For the preparation of media collection from dermal fibroblasts, cells were placed in 0.5 mL of MEM and 0.1% bovine serum albumin (BSA) for 24 h prior to the cytokine treatment. After incubation with the indicated reagent for 24 h, the condition medium was collected and clarified by centrifugation, and the cells remaining in the dishes were treated with trypsin and counted electronically. Immunoblotting was performed as described previously (^{Ihn and Trojanowska, 1997;Ihn and Tamaki, 2000a, c}). The samples (10 L) were normalized for cell number, subjected to electrophoresis on 7.5% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred onto nitrocellulose filters. The nitrocellulose filters were then incubated with a goat mAB against human type I collagen (Southern Biotechnology Associates Inc., Birmingham, Alabama) or a mouse mAB against human fibronectin (Santa Cruz Biotechnology). Then, the filters were incubated overnight with horseradish peroxidase-conjugated secondary antibodies and immunoreactive bands were visualized using an enhanced chemilunescent system (ECL, Amersham Corporation Piscataway, New Jersey) according to the manufacturer's recommendations. The densities of bands were measured using a densitometer.

RNA preparation and northern blot analysis

Fibroblasts were grown to confluence in MEM supplemented with 10% FCS and incubated for 24 h in serum-free medium (MEM plus 0.1% BSA) before addition of the indicated reagent. Total RNA was extracted and analyzed by northern blotting as described previously (^{Ihn et al, 1997;Ihn and Tamaki, 2000a}). Filters were sequentially hybridized with probes for 2(I) procollagen, and glyceraldehyde-3-phosphate dehydrogenase. The filters were scanned with a densitometer.

Plasmid construction

Generation of a -3500 COL1A2/CAT construct consisting of the human collagen 2(I) gene fragment (+58 to -3500 bp relative to the transcription start site) linked to the chloramphenicol acetyltransferase reporter gene was previously described (^{Ihn et al, 1996, 1997;Ihn and Trojanowska, 1997;Ihn and Tamaki, 2000b}). Dominant-negative mutants (TGY–AGF) of p38 (dn p38 ) and p38 (dn p38) MAPK were generously provided by Dr Jiahuai Hans (The Scripps Research Institute, La Jolla, California) (^{Wang et al, 1998}). The plasmids used encode the ERK2 (p42 MAPK) cDNA in which Thr¹⁸³ and Tyr¹⁸⁵, which must be phosphorylated for activity were replaced with either glutamic acid or alanine and phenylalanine, thus rendering the protein inactive (^{Ihn and Tamaki, 2000c}). Plasmids used in transient transfection assays were twice purified on CsCl gradients. At least two different plasmid preparations were used for each experiment.

Transient transfection and chloramphenicol acetyltransferase assay

Human fibroblasts were grown to 80% confluence in 100-mm dishes in Dulbecco's MEM containing 10% FCS. Monolayers were washed, and cells were transfected by the Lipofectin technique (FuGene 6 Transfectin Reagent, Roche Diagnostic, Indianapolis, Indiana) (^{Ihn et al, 2001a, b, 2002}) with 5 g of -3500 COL1A2/ chloramphenicol acetyltransferase constructs. pSV--galactosidase control vector (Promega, Madison, Wisconsin) was co-transfected to normalize for transfection efficiency (^{Ihn et al, 2001a, b, 2002}). After incubation overnight, the medium was replaced with MEM containing 0.1% BSA or with MEM containing various amounts of TGF-, and incubation was continued for 48 h. Cells were harvested in 0.25 M Tris-HCl (pH 8) and fractured by freeze-thawing. Extracts, normalized for protein content as measured by the Bio-Rad reagent, were incubated with butyryl-CoA and [¹⁴C]chloramphenicol for 90 min at 37°C. Butyrated chloramphenicol was extracted using an organic solvent (2:1 mixture of tetramethylpentadecane and xylene) and quantitated with scintillation counting. Each experiment was performed in duplicate. The Mann–Whitney U test was used to determine statistical significance.

Assays of p38 MAPK activation

The activation of p38 MAPK was determined by immunoblotting using antibodies specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182) (New England Biolabs) (^{Matsumoto et al, 1999}).

In both experiments, fibroblasts were serum-starved for 24 h and treated with cytokines for the indicated time. Then, the conditioned medium was removed and the cells were washed with ice-cold phosphate-buffered saline. The cells were lysed by scraping into solubilization buffer (50 mM Tris/Cl, pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, 10 g per mL aprotinin, 10 g per mL leupeptin, and 10 g per mL pepstatin). The lysate was incubated at 4°C for 30 min and then centrifuged for 5 min at 4°C. Protein concentrations of lysates were determined using a Bio-Rad (Hercules, California) Protein Assay, as recommended by the manufacturer.

Immunoblotting was performed as described above. Cell lysates (30 g) obtained from fibroblasts were subjected to electrophoresis on 10%/20% gradient SDS-polyacrylamide gels, and transferred onto nitrocellulose filters. The nitrocellulose filters were then incubated overnight with mAB specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182) (1:1000 dilution). Bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) and immunoreactive bands were visualized using an ECL system as described above. Antibodies against p38 MAPK (Santa Cruz Biotechnology Inc.) were also used to confirm that the protein concentrations of p38 MAPK were maintained.

In p38 MAP kinase assay (^{Matsumoto et al, 1999}), 200 g of the lysates were incubated with an immobilized phospho-p38 MAPK (Thr180/Tyr182) mAB overnight at 4°C for immunoprecipitation. For kinase assays, the beads were incubated with 200 M adenosine triphosphate and 2 g ATF-2 fusion protein as a substrate for p38 MAPK at 30°C for 30 min. The reaction was terminated with 25 L of an SDS sample buffer. The samples were then boiled and subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated overnight with a phospho-ATF-2 (Thr71) antibody (New England Biolabs) overnight at 4°C. The membranes were washed, and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG, and immunoreactive bands were visualized with ECL.

Statistical analysis

Statistical analysis was carried out using the Mann–Whitney test for the comparisons of means. p values less than 0.05 were considered significant.

References

1. Alessi, DR, Cuenda, A, Cohen, P, Dudley, DT, Saltiel, AR: PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 1995 270:27489–27494,  | Article | PubMed | ISI | ChemPort |
2. Border, WA, Noble, NA: Transforming growth factor in tissue fibrosis. N Engl J Med 1994 331:1286–1292,  | Article | PubMed | ISI | ChemPort |
3. Bornstein, P, Sage, H: Structurally distinct collagen types. Annu Rev Biochem 1980 49:957–1003,  | Article | PubMed | ISI | ChemPort |
4. Chang, E, Goldberg, H: Requirements for transforming growth factor- regulation of the pro 2(I) collagen and plasminogen activator inhibitor-1 promoters. J Biol Chem 1995 270:4473–4477,  | Article | PubMed | ISI | ChemPort |
5. Chen, S-J, Yuan, W, Mori, Y, Levenson, A, Trojanowska, M, Varga, J: Stimulation of type I collagen transcription in human skin fibroblasts by TGF-: Involvement of Smad 3. J Invest Dermatol 1999 112:49–57,  | Article | PubMed | ISI | ChemPort |
6. Chung, KY, Agarwal, A, Uitto, J, Mauviel, A: An AP-1 binding sequence is essential for regulation of the human 2(I) collagen promoter activity by transforming growth factor-. J Biol Chem 1996 271:3272–3278,  | Article | PubMed | ISI | ChemPort |
7. Dudly, DT, Pang, L, Decker, ST, Bridges, AJ, Saltier, AR: A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 1995 92:7686–7689,  | PubMed | ChemPort |
8. Ghosh, AK, Yuan, W, Mori, Y, Varga, J: Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF- involves functional cooperation with p300/CBP transcriptional coactivators. Oncogene 2000 19:3546–3455,  | Article | PubMed | ISI | ChemPort |
9. Greenwel, P, Inagaki, Y, Hu, W, Walsh, M, Rramirez, F: Sp1 is required for the early response of 2(I) collagen to transforming growth factor-1. J Biol Chem 1997 272:19738–19745,  | Article | PubMed | ISI | ChemPort |
10. Ignotz, RA, Endo, T, Massaguë, J: Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-. J Biol Chem 1987 262:6443–6446,  | PubMed | ISI | ChemPort |
11. Ihn, H, Ihn, Y, Trojanowska, M: Sp1 phosphorylation induced by serum stimulates the human 2(I) collagen gene expression. J Invest Dermatol 2001a 117:301–308,  | Article | ISI | ChemPort |
12. Ihn, H, LeRoy, EC, Trojanowska, M: Oncostatin M stimulates transcription of the human 2(I) collagen gene via the Sp1/Sp3-binding site. J Biol Chem 1997 272:24666–24672,  | Article | PubMed | ISI | ChemPort |
13. Ihn, H, Ohnishi, K, Tamaki, T, LeRoy, EC, Trojanowska, M: Transcriptional regulation of the human 2(I) collagen gene: Combined action of upstream stimulatory and inhibitory cis-acting elements. J Biol Chem 1996 271:26717–26723,  | Article | PubMed | ISI | ChemPort |
14. Ihn, H, Tamaki, K: Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts: Association with increased expression of the type I collagen gene. Arthritis Rheum 2000a 43:2240–2247,  | Article | ISI | ChemPort |
15. Ihn, H, Tamaki, K: Competition analysis of the human 2(I) collagen promoter using synthetic oligonucleotides. J Invest Dermatol 2000b 114:1011–1016,  | Article | ISI | ChemPort |
16. Ihn, H, Tamaki, K: Oncostatin M stimulates the growth of dermal fibroblasts via a mitogen-activated protein kinase-dependent pathway. J Immunol 2000c 165:2149–2155,  | PubMed | ISI | ChemPort |
17. Ihn, H, Trojanowska, M: Sp3 is a transcriptional activator of the human 2(I) collagen gene. Nucleic Acids Res 1997 25:3712–3717,  | Article | PubMed | ISI | ChemPort |
18. Ihn, H, Yamane, K, Asano, Y, Kubo, M, Tamaki, K: Interleukin-4 up-regulates the expression of tissue inhibitor of metalloproteinase-2 in dermal fibroblasts via the p38 mitogen-activated protein kinase-dependent pathway. J Immunol 2002 168:1895–1902,  | PubMed | ISI | ChemPort |
19. Ihn, H, Yamane, K, Kubo, M, Tamaki, K: Blockade of endogenous transforming growth factor signaling prevents up-regulated collagen synthesis in scleroderma fibroblasts. Association with increased expression of transforming growth factor receptors. Arthritis Rheum 2001b 44:474–480,  | Article | PubMed | ISI | ChemPort |
20. Inagaki, Y, Truter, S, Ramirez, F: Transforming growth factor- stimulates 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol Chem 1994 269:14828–14834,  | PubMed | ISI | ChemPort |
21. Inagaki, Y, Truter, S, Tanaka, S, DiLiberto, M, Ramirez, F: Overlapping pathways mediate the opposing actions of tumor necrosis- and transforming growth factor- on 2(I) collagen gene transcription. J Biol Chem 1995 270:3353–3358,  | PubMed | ISI | ChemPort |
22. Jelaska, A, Arakawa, M, Broketa, G, Korn, JH: Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts: Increased proportion of high collagen-producing cells in systemic sclerosis fibroblasts. Arthritis Rheum 1996 39:1338–1346,  | PubMed | ISI | ChemPort |
23. Kähäri, VM, Chen, YQ, Su, MW, Ramirez, F, Uitto, J: Tumor necrosis factor- and interferon- suppress the activation of human type I collagen gene expression by transforming growth factor-1. J Clin Invest 1990 86:1489–1495,  | PubMed | ISI | ChemPort |
24. Kawabata, M, Inoue, H, Hanyu, A, Imamura, T, Miyazono, K: Smads proteins exist as monomers in vivo and undergo homo- and hetro-oligomerization upon activation by serine/threonine kinase receptor. EMBO J 1998 17:4056–4065,  | Article | PubMed | ISI | ChemPort |
25. Kretzschmar, M, Massaguë, J: SMADs: Mediators and regulators of TGF- signaling. Curr Opin Genet Dev 1998 8:103–111,  | Article | PubMed | ISI | ChemPort |
26. Kyriakis, JM, Avruch, J: Sounding the alarm: Protein kinase cascades activated by stress and inflammation. J Biol Chem 1996 271:24313–24316,  | Article | PubMed | ISI | ChemPort |
27. LeRoy, EC, Smith, EA, Kahaleh, MB, Trojanowska, M, Silver, RM: A strategy for determining the pathogenesis of systemic sclerosis: Is transforming growth factor the answer? Arthritis Rheum 1989 32:817–825,  | PubMed | ISI | ChemPort |
28. Massaguë, J: The transforming growth factor- family. Annu Rev Cell Biol 1990 6:597–641,  | Article | PubMed | ISI | ChemPort |
29. Matsumoto, T, Yokote, K, Tamura, K, Takemoto, M, Ueno, H, Saito, Y, Mori, S: Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration. J Biol Chem 1999 274:13954–13960,  | Article | PubMed | ISI | ChemPort |
30. Poncelet, AC, Schnaper, HW: Sp1 and Smad proteins cooperate to mediate transforming growth factor-1-induced 2(I) collagen expression in human glomerular mesangimal cells. J Biol Chem 2001 276:6983–6992,  | Article | PubMed | ISI | ChemPort |
31. Ramirez, F, DiLiberto, M: Complex and diversified regulatory programs control the expression of vertebrate collagen genes. FASEB J 1990 4:16116–23,
32. Ravanti, L, Hakkinen, L, Larjava, H, Saarialho-Kere, U, Foschi, M, Han, J, Kahari, VM: Transforming growth factor- induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase. J Biol Chem 1999b 274:37292–37300,  | Article | PubMed | ISI | ChemPort |
33. Ravanti, L, Heino, J, Lopez-Otin, C, Kahari, VM: Induction of collagenase-3 (MMP-13) expression in human dermal fibroblasts by three dimensional collagen is mediated by p38 mitogen-activated protein kinase. J Biol Chem 1999a 274:2446–2455,  | Article | PubMed | ISI | ChemPort |
34. Roberts, AB, Heine, UI, Flanders, KC, Sporn, MB: Transforming growth factor-. Major role in regulation of extracellular matrix. Ann NY Acad Sci 1990 58:225–232,
35. Roberts, AB, Sporn, MB: Physiological actions and clinical applications of transforming growth factor- (TGF-). Growth Factors 1993 8:1–9,  | PubMed | ISI | ChemPort |
36. Rossi, P, Karsenty, G, Roberts, AB, Roche, NS, Sporn, MB, de Crombrugghe, B: A nuclear factor I binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-. Cell 1988 52:405–415,  | Article | PubMed | ISI | ChemPort |
37. Sato, M, Shegogue, D, Gore, EA, Smith, EA, Mcdermott, PJ, Trojanowska, M: Role of p38 MAPK in transforming growth factor stimulation of collagen production by scleroderma and healthy dermal fibroblasts. J Invest Dermatol 2002 118:704–711,  | Article | PubMed | ISI | ChemPort |
38. Vuorio, E, de Crombrugghe, B: The family of collagen genes. Annu Rev Biochem 1990 59:832–837,
39. Wang, Y, Huang, S, Sah, VP, Ross, J, Jr, Brown, JH, Han, J, Chien, KR: Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem 1998 273:2161–2168,  | Article | PubMed | ISI | ChemPort |
40. Yamane, K, Ihn, H, Kubo, M, Tamaki, K: Increased transcriptional activities of transforming growth factor- receptors in scleroderma fibroblasts. Arthritis Rheum 2002 46:2421–2428,  | Article | PubMed | ISI | ChemPort |
41. Zhang, W, Ou, J, Inagaki, Y, Greenwel, P, Ramirez, F: Synergistic cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor-1 stimulation of 2(I) collagen (Col1A2) transcription. J Biol Chem 2000 275:39237–39245,  | Article | PubMed | ISI | ChemPort |

Acknowledgments

This work was supported by a grant for scientific research from the Ministry of Education, Japan (10770391), and by the project research for progressive SSc from the Ministry of Health and Welfare, Japan.