Activation of p38 MAPK suppresses matrix metalloproteinase-1 gene expression induced by platelet-derived growth factor

p38 mitogen-activated protein kinase (MAPK) regulates matrix metalloproteinase-1 (MMP-1) gene expression bidirectionally depending on the induction. We sought to determine whether cytokines related to the regulation of extracellular matrix could activate p38 MAPK in dermal fibroblasts. We determined p38 MAPK phosphorylation/activation in dermal fibroblasts stimulated with platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-beta or interleukin-4. Induction of MMP-1 mRNA by PDGF-BB was enhanced in the presence of a specific inhibitor of p38 MAPK, suggesting that p38 MAPK would function as a negative regulator of the MMP-1 mRNA level. We then determined which isoforms of p38 MAPK expressed in dermal fibroblasts were responsible for the downregulation of the MMP-1 mRNA level. Overexpression of p38beta2, but not of p38alpha, significantly decreased PDGF-BB-induced MMP-1 promoter activity, although PDGF-BB activated signaling pathways to both p38alpha and p38beta2. Taken together, the results of this study indicate that p38beta2 can function as a negative regulator of MMP-1 induced by PDGF-BB in vitro, suggesting that activation of p38beta2 might contribute to the pathogenesis of cutaneous fibrosis.     

Halofuginone, an inhibitor of type-I collagen synthesis and skin sclerosis, blocks transforming-growth-factor-beta-mediated Smad3 activation in fibroblasts

Halofuginone is a drug that has been shown to have an antifibrotic property in vitro and in vivo. Whereas halofuginone shows promise as a therapeutic agent for a variety of diseases including scleroderma, liver cirrhosis, cystic fibrosis, and certain types of cancer, the mechanism of action remains unknown. Using the tight skin mouse (TSK) model for scleroderma, we evaluated the ability of halofuginone to inhibit spontaneous development of dermal fibrosis. We found that administration of a low dose of halofuginone both in adult and newborn animals for 60 d prevented the development of cutaneous hyperplasia (dermal fibrosis). In vitro halofuginone was found to reduce the amount of collagen synthesized by fibroblasts. This effect was due to a reduction in the promoter activity of the type-I collagen genes as treatment of fibroblast cultures with 10(-8) M halofuginone reduced the level of alpha2(I) collagen message detectible by northern blot and greatly reduced the activity of a reporter construct under control of the -3200 to +54 bp alpha2(I) collagen promoter. In addition, analysis of transforming growth factor beta signaling pathways in fibroblasts revealed that halofuginone inhibited transforming-growth-factor-beta-induced upregulation of collagen protein and activity of the alpha2(I) collagen promoter. Further we found that halofuginone blocked the phosphorylation and subsequent activation of Smad3 after transforming growth factor beta stimulation. Apparently the inhibitory property was specific to Smad3 as there was no inhibitory effect on the activation of Smad2 after stimulation with transforming growth factor beta. Our results demonstrate that halofuginone is a specific inhibitor of type-I collagen synthesis and may elicit its effect via interference with the transforming growth factor beta signaling pathway.     

Effects of hepatocyte growth factor on the expression of type I collagen and matrix metalloproteinase-1 in normal and scleroderma dermal fibroblasts

We investigated the direct effect of hepatocyte growth factor (HGF) on the expression of type I collagen in normal and scleroderma dermal fibroblasts, and analyzed the mechanisms underlying the effect in vitro. HGF did not change the protein expression of type I procollagen in the medium of normal human fibroblasts, whereas it reduced the expression in scleroderma fibroblasts. But mRNA levels and the promoter activity of alpha2(I) collagen gene were not significantly affected by HGF in either of the cells. On the other hand, matrix metalloproteinase-1 expression or activity was increased by HGF in both cells, but HGF had stronger effects in scleroderma fibroblasts than normal fibroblasts. Scleroderma fibroblasts overexpressed c-met protein, the receptor for HGF. The overexpression in scleroderma fibroblasts was abolished by the addition of antisense transforming growth factor (TGF)-beta1 oligonucleotide. Our study indicated that HGF may reduce type I collagen accumulation only in scleroderma fibroblasts by enhancing collagenolysis activity, probably because of the overexpression of c-met because of autocrine TGF-beta signaling. Thus, further investigation of the effects of HGF on collagen metabolism may contribute to the treatment of fibrosis in scleroderma.     

Evaluation of transforming growth factor beta and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization

Full thickness biopsies of affected skin and fascia from one patient with diffuse fasciitis and eosinophilia (DF), two patients with generalized morphea (GM), and five patients with progressive systemic sclerosis (PSS) of recent onset were examined for the expression of transforming growth factor beta 1 (TGF beta 1) and type I procollagen genes by in situ hybridization with human sequence-specific cDNA. An increased number of fibroblasts showing clearly detectable expression of pro alpha 1(I)collagen gene was found in all fibrotic lesions when compared with unaffected skin from the patient with DF and skin from two normal individuals examined in parallel. Expression of the TGF beta 1 gene was noted in a fibroblast subpopulation of the affected tissues from the patients with DF and GM. In contrast, the TGF beta 1 gene was not expressed at a detectable level in affected skin from the five patients with PSS. The results suggest that TGF beta 1 may play a role in the development of skin fibrosis in cases of DF and GM. However, from these studies, we cannot implicate TGF beta 1 in the pathogenesis of skin fibrosis in PSS.     

Constitutive activation of c‐Abl/protein kinase C‐δ/Fli1 pathway in dermal fibroblasts derived from patients with localized scleroderma

Background A noncanonical pathway of transforming growth factor‐β signalling, the c‐Abl/protein kinase C‐δ (PKC‐δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts. Objectives To investigate the significance of the c‐Abl/PKC‐δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc). Methods The activation status of the c‐Abl/PKC‐δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet‐derived growth factor receptor inhibitor AG1296 and gene silencing of c‐Abl on the expression levels of type I collagen were evaluated by immunoblotting. Results The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c‐Abl were elevated compared with cultured normal fibroblasts and PKC‐δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c‐Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts. Conclusions Constitutive activation of the c‐Abl/PKC‐δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.     

Differential regulation of transforming growth factor-β receptors type I and II by platelet-derived growth factor in human dermal fibroblasts

BACKGROUND: Elevated expression of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta have been observed in a number of fibrotic diseases, including systemic sclerosis (SSc). This suggests a possible interaction between these factors in establishing a profibrotic programme in dermal fibroblasts. OBJECTIVES: To examine the effects of PDGF isoforms on the expression of TGF-beta receptors in human dermal fibroblasts. METHODS: Steady-state mRNA levels of TGF-beta receptor I and II (TbetaR-I and TbetaR-II) were analysed by northern blot. TbetaR-I protein levels were analysed by immunoprecipitation of 35S metabolically labelled cells. TbetaR-II protein levels were analysed by western blot. RESULTS: Steady-state mRNA levels of TbetaR-I and TbetaR-II were induced in response to PDGF isoforms. PDGF-AA and PDGF-AB stimulated both receptors with similar potency, whereas PDGF-BB was less potent. The MEK1 (mitogen-activated protein kinase [MAPK] or extracellular signal regulated kinase) inhibitor, PD98059, abrogated the stimulatory effect of PDGF-AB. In contrast to mRNA levels, only TbetaR-II protein levels were elevated in response to PDGF. CONCLUSIONS: These data suggest that PDGF receptor alpha and MAPK mediate stimulation of TGF-beta receptors by PDGF. Furthermore, TGF-beta receptor protein levels are discordantly regulated by PDGF.     

Antagonism of the serotonin pathway – a novel antifibrotic approach?

The neurotransmitter serotonin (5‐Hydroxytryptamin, 5‐HT) has been implicated in the pathogenesis of scleroderma and scleroderma‐like skin changes for years. In particular, a role of serotonin has been suggested as an important vasoactive mediator of the Raynaud's phenomenon in patients with systemic sclerosis. Accordingly, anti‐serotoninergic therapies have been utilized clinically to treat Raynaud's phenomenon. There is also recent evidence that multiple cell types of the skin have the capacity to synthesize serotonin. The biological effects of serotonin are mediated by at least 15 different 5‐HT‐receptor subtypes. One of them is the 5‐HT3‐receptor, which binds the 5‐HT‐antagonist tropisetron. Here we investigated the effect of tropisetron on transforming growth factor‐beta1 (TGF‐beta1)‐mediated collagen synthesis in human dermal fibroblasts (HDF) in vitro. In addition, we elucidated the impact of tropisetron on activation of SMAD, a signaling transduction pathway implicated in TGF‐beta1‐mediated collagen synthesis. Tropisetron at non‐cytotoxic doses significantly suppressed TGF‐beta1‐mediated collagen type I (alpha1 and 2) and type III (alpha1) expression at RNA level in HDF as shown by real‐time PCR analysis. This effect of tropisetron was paralleled by reduced amounts of intracellular collagen type I protein as evidenced by Western immunoblotting. In addition, tropisetron significantly decreased secreted procollagen‐I‐C‐peptide as demostrated by ELISA. In contrast, tropisetron alone did not have any detectable effect on collagen expression. Analysing the SMAD pathway we could further show that tropisetron in contrasts to TGF‐beta1 neither affects SMAD3 phosphorylation, nuclear translocation of SMAD2/3 nor SMAD3/4‐dependent promoter activity in HDF. Our results for the first time demonstrate a potent suppressive effect of tropisetron on TGF‐beta1‐induced collagen synthesis in vitro. Regardless of the molecular mechanism of TGF‐beta1 antagonism by tropisetron, these findings should be of special relevance towards the exploitation of tropisetron and related agents as a novel treatment for patients with fibrotic diseases.     

Pseudoscleroderma associated with lung cancer: correlation of collagen type I and connective tissue growth factor gene expression

Pseudoscleroderma as a paraneoplastic syndrome is a rare disease. We report here a patient with lung cancer (undifferentiated squamous cell carcinoma), who developed acrosclerosis. Using in situ hybridization, marked expression of alpha1(I)-collagen and connective tissue growth factor (CTGF) mRNA was found in fibroblasts scattered throughout the dermis. However, transforming growth factor (TGF)-beta1 expression was not detected. The pattern of CTGF gene expression and collagen synthesis was similar to that in systemic scleroderma. The absence of TGF-beta1 mRNA could indicate that tumour-derived factors induce the expression of CTGF.     

Epidermal growth factor-induced matrix metalloproteinase-1 expression is negatively regulated by p38 MAPK in human skin fibroblasts

Background

Many extracellular stimuli, including epidermal growth factor (EGF), are known to induce MMP-1 expression. Recently, several reports have shown that ERK activity plays an important role in EGF-induced MMP-1 expression. However, EGF is also known to activate many signaling pathways in addition to the ERK pathway, but the roles of these pathways during the induction of MMP-1 by EGF are unclear.

Objective

We investigated the role of JNK, p38 MAPK, and PI3K/Akt pathways in EGF-induced MMP-1 expression in human skin fibroblasts. Then, we further explored the inhibitory effect of p38 MAPK pathway on EGF-induced MMP-1 expression and studied the molecular mechanisms involved in the processes.

Methods

Human skin fibroblasts were pretreated with various chemical inhibitors or small interfering RNA (siRNA) at the indicated concentrations and then treated with EGF, TNF-alpha, or IL-1beta for the indicated times. Protein and mRNA levels of various target molecules were assessed by Western blotting and quantitative real-time PCR, respectively.

Results

We found that EGF-induced MMP-1 expression was positively regulated by JNK as well as ERK but negatively regulated by p38 MAPK in human skin fibroblasts. On the other hand, the PI3K/Akt pathway did not significantly affect MMP-1 induction by EGF. Then we found that the inhibition of p38 MAPK pathway specifically increased the MMP-1 expression stimulated by EGF but not by TNF-alpha or IL-1beta, indicating that the effect of p38 MAPK on MMP-1 expression may be stimulus-type specific in human skin fibroblasts. In addition, the inhibitory effect of p38 MAPK on EGF-induced MMP-1 expression was shown to be mainly mediated by p38-alpha MAPK. Our further studies showed that the inhibition of p38 MAPK but not PI3K specifically increased EGF-induced ERK and JNK activations, and that the augmentation of EGF-induced MMP-1 expression by p38 MAPK inhibition was significantly attenuated by inhibiting the activities of ERK and/or JNK.

Conclusions

Our results indicate that EGF-induced MMP-1 expression is differentially regulated by the JNK, p38 MAPK, and PI3K/Akt pathways, and suggest that p38 MAPK negatively regulates EGF-induced MMP-1 expression by suppressing the activations of ERK and JNK.     

Cytokine modulation of type XV collagen gene expression in human dermal fibroblast cultures

The expression of type XV collagen was studied in cultured human dermal fibroblasts exposed to transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), cytokines which have been shown previously to alter the expression of several extracellular matrix genes. TGF-beta enhanced the expression of the type XV collagen gene (COL15A1) in a time-dependent manner, up to 4.3-fold after 24 h of incubation, whereas TNF-alpha and IL-1beta reduced the mRNA steady-state levels by 32 and 80%, respectively. When TGF-beta and TNF-alpha were added together to the cultures, the stimulatory effect of TGF-beta on type XV collagen gene expression was abrogated, indicating antagonistic modulation by these 2 cytokines. These data suggest that the cytokines tested in this study may contribute to the regulation of type XV collagen synthesis in a variety of tissues which have recently been shown to express this particular collagen gene.