Sustained Activation of Fibroblast Transforming Growth Factor-β/Smad Signaling in a Murine Model of Scleroderma

Section of Rheumatology, University of Illinois at Chicago, 60607, USA.
Journal of Investigative Dermatology (Impact Factor: 7.22). 08/2003; 121(1):41-50. DOI: 10.1046/j.1523-1747.2003.12308.x
Source: PubMed


Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the possibility that Smads plays an important part in the pathogenesis of fibrosis, and may therefore represent targets for selective anti-fibrotic interventions.

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    • "Bleomycin (0.02 units /day) (Teva Parenteral Medicines, Irvine, CA) dissolved in saline or saline alone was administered to eight week old mice by daily subcutaneous injections. On day 28, mice were sacrificed and lesional skin was obtained for protein lysates, total RNA, and histology (Takagawa et al., 2003). Each group consisted of 10–15 mice. "
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    ABSTRACT: Osteopontin (OPN) is a matricellular protein with proinflammatory and profibrotic properties. Previous reports demonstrate a role for OPN in wound healing and pulmonary fibrosis. Here, we determined whether OPN levels are increased in a large cohort of patients with systemic sclerosis (SSc) and whether OPN contributes to the development of dermal fibrosis. The plasma OPN levels were increased in SSc patients, including patients with limited and diffuse disease, compared with healthy controls. Immunohistology demonstrated OPN on fibroblast-like and inflammatory cells in SSc skin and lesional skin from mice in the bleomycin (bleo)-induced dermal fibrosis model. OPN-deficient (OPN(-/-)) mice developed less dermal fibrosis compared with wild-type (WT) mice in the bleo-induced dermal fibrosis model. Additional in vivo studies have demonstrated that lesional skin from OPN(-/-)mice had fewer Mac-3-positive cells, fewer myofibroblasts, decreased transforming growth factor (TGF)-β and genes in the TGF-β pathway, and decreased numbers of cells expressing phosphorylated SMAD2 (pSMAD) and extracellular signal-regulated kinase. In vitro, OPN(-/-) dermal fibroblasts had decreased migratory capacity but similar phosphorylation of SMAD2 by TGF-β. Finally, TGF-β production by OPN-deficient macrophages was reduced compared with WT. These data demonstrate an important role for OPN in the development of dermal fibrosis and suggest that it may be a new therapeutic target in SSc.
    Preview · Article · Mar 2012 · Journal of Investigative Dermatology
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    • "Smad7 has been used in experimental models to block TGF-β effects in different disorders [17-19]. Dooley et al[20] and Tahashi et al[16] demonstrated that Smad7 expression was low in experimental liver fibrosis, and this event allows liver scar formation. "
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    ABSTRACT: Liver fibrosis is the result of continuous liver injury stemming from different etiological factors. Bile duct injury induces an altered expression of TGF-beta, which has an important role in liver fibrosis because this cytokine induces the expression of target genes such as collagens, PAI-1, TIMPs, and others that lead to extracellular matrix deposition. Smad7 is the principal inhibitor that regulates the target gene transcription of the TGF-beta signaling. The aim of the study was to determine whether Smad7 mRNA expression correlates with the gene expression of TGF-beta, Col I, Col III, Col IV, or PAI-1 in liver fibrosis secondary to bile duct injury (BDI). Serum TGF-beta concentration was higher in BDI patients (39 296 pg/ml) than in liver donors (9008 pg/ml). Morphometric analysis of liver sections showed 41.85% of tissue contained fibrotic deposits in BDI patients. mRNA expression of Smad7, Col I, and PAI-1 was also significantly higher (P < 0.05) in patients with BDI than in controls. Smad7 mRNA expression correlated significantly with TGF-beta concentration, Col I and Col III expression, and the amount of fibrosis. We found augmented serum concentration of TGF-beta and an increase in the percentage of fibrotic tissue in the liver of BDI patients. Contrary to expected results, the 6-fold increase in Smad7 expression did not inhibit the expression of TGF-beta, collagens, and PAI-1. We also observed greater expression of Col I and Col III mRNA in BDI patients and significant correlations between their expression and TGF-beta concentration and Smad7 mRNA expression.
    Full-text · Article · Oct 2009 · BMC Gastroenterology
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    • "P144- treated mice showed a significant reduction in the number of a-SMA-positive myofibroblasts compared with vehicletreated mice (Fig 3). We also observed an increase in the number of dermal fibroblasts displaying phosphorylated SMAD2/3 in a nuclear and cytoplasmic pattern in bleomycin-injected mice, confirming previous observations in this model (Takagawa et al, 2003). The number of phospho- SMAD2/3-positive fibroblasts was also significantly decreased in P144-treated mice compared with vehicle-treated mice (Fig 4). "
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    ABSTRACT: Transforming growth factor beta (TGF-β) plays a crucial role in the pathogenesis of skin fibrotic diseases by inducing extracellular matrix gene expression and sustaining fibroblast growth and differentiation. Systemic inhibition of TGF-β by different agents has been shown to effectively inhibit fibrosis in different animal models. However, systemic inhibition of TGF-β raises important safety issues because of the pleiotropic physiological effects of this factor. Targeting of downstream factors specifically involved in TGF-β profibrotic signaling or local targeting of TGF-β represents potential alternatives to systemic inhibitors. Topical application of a short peptide derived from TGF-β1 type III receptor is effective in preventing or ameliorating established fibrosis in a model of bleomycin-induced scleroderma, suggesting that topical application of small anti-TGF-β peptides is a feasible strategy to treat pathological skin scarring and skin fibrotic diseases.
    Full-text · Article · Jan 2008
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