FK506 inhibits the enhancing effects of transforming growth factor (TGF)-β1 on collagen expression and TGF-β/Smad signalling in keloid fibroblasts: implication for new therapeutic approach.

  Keloid is a unique proliferative disorder of fibroblasts resulting from derailment of the typical wound healing process. Due to lack of animal models for therapeutic testing, treatment of keloids remains a clinical challenge. Transforming growth factor (TGF)-β1-related signalling plays a key role in keloid formation. As tacrolimus (FK506) has been reported to inhibit the effects of TGF-β1 on cultured fibroblasts, we hypothesized that FK506 may be useful in treating keloids. To explore the effects of FK506 on TGF-β1-stimulated keloid fibroblasts (KFs) in terms of proliferation, migration and collagen production and to investigate the regulatory pathways involved. Fibroblasts derived from keloids were treated with TGF-β1 with or without FK506. Relevant assays including 5-bromo-2'-deoxyuridine incorporation assay, in vitro scratch assay, reverse transcription-polymerase chain reaction (PCR), quantitative PCR and Western blotting were performed. The proliferation and migration of KFs were significantly higher than those of normal fibroblasts. FK506 markedly inhibited KF proliferation, migration and collagen production enhanced by TGF-β1. The increase in TGF-β receptor I and II expression in TGF-β1-treated KFs was suppressed by FK506 treatment. TGF-β1 increased the phosphorylation of Smad2/3 and Smad4 in KFs, and this enhancing effect was abrogated by FK506. In addition, FK506 significantly increased the expression of Smad7 which was suppressed by TGF-β1 treatment. Our results demonstrate that FK506 effectively blocks the TGF-β/Smad signalling pathway in KFs by downregulation of TGF-β receptors and suggest that FK506 may be included in the armamentarium for treating keloids.

Wu CS ，Wu PH ，Fang AH ，Lan CC ... -  《-》

Compound Astragalus and Salvia miltiorrhiza extract inhibits cell proliferation, invasion and collagen synthesis in keloid fibroblasts by mediating transforming growth factor-β / Smad pathway.

The transforming growth factor (TGF)-β/Smad pathway plays a key role in keloid development. We have previously demonstrated that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits liver fibrosis and reduces invasion capacity of HepG2 cells by mediating the TGF-β/Smad pathway. We therefore hypothesize that CASE may also exert antifibrotic effects in keloids by mediating the TGF-β/Smad pathway. To investigate the effects of CASE on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and to explore the effects of CASE on the TGF-β/Smad signal pathway in order to elucidate its mechanisms of action. The inhibitory effects of CASE on keloid fibroblasts were evaluated. Cell proliferation was studied by MTT assay; cell invasion was observed utilizing Transwell invasion chambers; and collagen synthesis in keloid fibroblasts was measured by (3) H-proline incorporation assay. Expression of proteins induced by TGF-β1 and their intracellular localization in keloid fibroblasts were investigated by Western blot and immunofluorescence, respectively. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was measured by real-time reverse transcription-polymerase chain reaction. CASE significantly inhibited cell proliferation induced by newborn bovine serum as well as collagen synthesis and cell invasion induced by TGF-β1 in keloid fibroblasts, while it showed weak effects on normal fibroblasts. The phosphorylation of Smad2/3 was markedly reduced by CASE treatment, while CASE exhibited stronger inhibitory effects on linker region phosphorylation (pSmad2L and pSmad3L) compared with effects on C-terminal region phosphorylation (pSmad2C and pSmad3C). In addition, CASE blocked formation of Smad2/3/4 complexes and their nuclear translocation, but upregulated Smad7 expression in a dose-dependent manner. PAI-1 mRNA and protein levels were also suppressed by CASE treatment. These results suggest that CASE exhibits inhibitory effects on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and its mechanisms of action may involve the TGF-β/Smad pathway.

He S ，Yang Y ，Liu X ，Huang W ，Zhang X ，Yang S ，Zhang X ... -  《-》

The effect of TLR4/7 on the TGF-β-induced Smad signal transduction pathway in human keloid.

Keloid formation is closely related with transforming growth factor (TGF)-β-induced Smad signal transduction. Recent studies have shown that toll-like receptor4 (TLR4) may mediate liver and kidney fibrosis, and activation of TLR7 has anti-scarring effect. The role of TLR4/7 signalling in keloid formation, however, remains unknown. Our previous tests have found that mute Smad4 inhibited scar. We then speculated that keloid may be affected by TLR4/7 through TGF-β-induced Smad signal transduction. The aim of this study was to evaluate effects of TLR4/7 on the TGF-β-induced Smad signal transduction pathway in human keloid, and provide information for the mechanism and therapy of keloid. Normal scar samples with normal fibroblasts (NFs) served as control samples and keloid samples with keloid fibroblasts (KFs) served as experiment samples. Expression of collagen, connective tissue growth factor (CTGF), Smad4 and Smad7 and TLR4/7 were tested by immunohistochemistry, reverse transcription polymerase chain reaction (PCR) (RT-PCR) and Western blotting, respectively. Expression of collagen, CTGF, Smad4 and TLR4 increased significantly while expression of Smad7 and TLR7 decreased in KFs while compared to NFs in keloid scar group (KFs), which were decreased in the normal scar group (NFs). However, expression of Smad7 and TLR7 decreased in the keloid scar group (KFs) while compared to the NFs. TLRs participate in fibrosis of scar tissue through the TLRs-TGF-β-Smads signal pathway. Higher expression of TLR4 in keloid increased expression of TGF-β, CTGF and collagen through the Smad4 signal pathway. Activation of TLR7 or Smad7 may inhibit scar formation.

Chen J ，Zeng B ，Yao H ，Xu J ... -  《-》

Mechanisms of transforming growth factor beta(1)/Smad signalling mediated by mitogen-activated protein kinase pathways in keloid fibroblasts.

He S ，Liu X ，Yang Y ，Huang W ，Xu S ，Yang S ，Zhang X ，Roberts MS ... -  《-》

Tacrolimus abrogates TGF-β1-induced type I collagen production in normal human fibroblasts through suppressing p38MAPK signalling pathway: implications on treatment of chronic atopic dermatitis lesions.

Atopic dermatitis (AD) is a commonly encountered inflammatory skin disease. Although acute lesions of acute AD are characterized by intense inflammation, the hallmarks of chronic AD lesions include lichenified fibrosis and thickening of the upper dermis. The increased expression of transforming growth factor beta 1 (TGF-β1), a well-known fibrogenic cytokine, is observed in chronic AD lesions. Tacrolimus (FK506) ointment has been reported to be effective for treating AD as well as some TGF-β1-induced fibrotic diseases. To evaluate the effect of tacrolimus on TGF-β1-stimulated cultured normal human dermal fibroblasts and explore the potential signalling pathways involved. Fibroblasts cultured from healthy adult human foreskins were treated with TGF-β1 with or without tacrolimus. The impact on cell viability and proliferation were assessed by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay and BrdU incorporation assay respectively. Reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and western blotting were performed to evaluate the relevant expressions of mRNA or proteins in fibroblasts. Our results revealed that the increased expressions of transforming growth factor-β receptor I (TGF-βRI) and TGF-βRII in TGF-β1-treated fibroblasts were suppressed by tacrolimus treatment. In addition, tacrolimus significantly inhibited fibroblast proliferation enhanced by TGF-β1. TGF-β1 increased type I collagen production, and this enhancing effect was suppressed by tacrolimus. The down-regulation of MMP-1 and up-regulation of TIMP-1 induced by TGF-β1 were reversed by tacrolimus. The increase in phosphorylated p38 mitogen-activated protein kinase (p38MAPK) expression stimulated by TGF-β1 was down-regulated by tacrolimus. Moreover, the fibroblasts treated with p38MAPK inhibitor significantly reduced type I collagen expression induced by TGF-β1. The present results demonstrated that tacrolimus significantly inhibited physiological functions of fibroblasts enhanced by TGF-β1 in vitro. Clinically, we propose that topical tacrolimus may not only reduce AD recurrence but also ameliorate dermal fibrosis often seen in chronic AD lesions.

Lan CC ，Fang AH ，Wu PH ，Wu CS ... -  《-》