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

Abstract

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.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

“…Twenty four h later, mice (three per group) were euthanized, and the injected skin was removed and processed for histological examination (18) or immunohistochemical analysis (19). Sections were incubated with antibodies to Egr-1 (C-19) (Santa Cruz Biotechnology) at a final dilution of 1:50.…”

Thyroid hormones inhibit TGF-β signaling and attenuate fibrotic responses

“…10A) induced by repeated local injections of bleomycin in the dorsal skin (46), in which SMAD-dependent signaling plays a crucial role (9,17,19). Thyroid hormone treatment caused the expected increase of circulating T3, reduction of T4, and induction of hepatic Dio1 mRNA levels (SI Appendix, Fig.…”

“…Negative feedback is provided by SMADinduced expression of the inhibitory SMAD7, which leads to polyubiquitylation and degradation of the TGF-β receptors (15). SMAD factors have been shown to be essential mediators of the fibrogenic actions of TGF-β (16)(17)(18)(19)(20).…”

The Early Growth Response Gene Egr2 (Alias Krox20) Is a Novel Transcriptional Target of Transforming Growth Factor-β that Is Up-Regulated in Systemic Sclerosis and Mediates Profibrotic Responses

“…29 The animal protocols were institutionally approved by the Northwestern University Animal Care and Use Committee. Filter-sterilized bleomycin (20 g per mouse dissolved in PBS) (Mayne Pharma, Paramus, NJ) or PBS was administered by daily s.c. injections into the shaved backs of 6-to 8-week-old female BALB/c mice.…”