Targeted Disruption of TGF-β/Smad3 Signaling Modulates Skin Fibrosis in a Mouse Model of Scleroderma

Abstract

Transforming growth factor-beta (TGF-beta) is a potent stimulus of connective tissue accumulation, and is implicated in the pathogenesis of scleroderma and other fibrotic disorders. Smad3 functions as a key intracellular signal transducer for profibrotic TGF-beta responses in normal skin fibroblasts. The potential role of Smad3 in the pathogenesis of scleroderma was investigated in Smad3-null (Smad3(-/-)) mice using a model of skin fibrosis induced by subcutaneous injections of bleomycin. At early time points, bleomycin-induced macrophage infiltration in the dermis and local TGF-beta production were similar in Smad3(-/-) and wild-type mice. In contrast, at day 28, lesional skin from Smad3(-/-) mice showed attenuated fibrosis, lower synthesis and accumulation of collagen, and reduced collagen gene transcription in situ, compared to wild-type mice. Connective tissue growth factor and alpha-smooth muscle actin expression in lesional skin were also significantly attenuated. Electron microscopy revealed an absence of small diameter collagen fibrils in the dermis from bleomycin-treated Smad3(-/-) mice. Compared to fibroblasts derived from wild-type mice, Smad3(-/-) fibroblasts showed reduced in vitro proliferative and profibrotic responses elicited by TGF-beta. Together, these results indicate that ablation of Smad3 is associated with markedly altered fibroblast regulation in vivo and in vitro, and confers partial protection from bleomycin-induced scleroderma in mice. Reduced fibrosis is due to deregulated fibroblast function, as the inflammatory response induced by bleomycin was similar in wild-type and Smad3(-/-) mice.

Combinatorial activation of FAK and AKT by transforming growth factor-β1 confers an anoikis-resistant phenotype to myofibroblasts

“…Although studies by our group [12] and others [36] have shown the capacity of TGF-β1 to activate FAK in certain cell types, a role for SMAD3 in mediating this effect has not been previously demonstrated. The requirement for SMAD3 in FAK activation is consistent with a role for SMAD3 in myofibroblast differentiation both in-vitro [37,38] and in-vivo [39][40][41].…”

Regulation of scar formation by vascular endothelial growth factor

“…In contrast, scar tissue from control mice injected with IgG antibodies contained a high frequency of very small-diameter fibrils (less than 40 nm), similar to what is found in models of cutaneous fibrosis. [20][21][22] Scars from mice injected with anti-VEGF antibodies contained a lower frequency of the small-diameter fibrils (0-40 nm) and a higher frequency of fibrils within the normal range (41-80 nm) compared with control scars. The results suggest that neutralization of VEGF not only reduced the amount of scar tissue formed (Figure 6), but also improved the quality of the scar tissue that did form by shifting the collagen fibril distribution to a state more closely resembling normal skin (Figure 7).…”

“…Collagen fibril diameter measurements have been used previously to evaluate the degree of cutaneous fibrosis. [20][21][22] Three normal skin samples and six scar samples from each treatment group, all from different mice, were used for analysis. Each micrograph was divided into four parts for analysis and fibrils from four micrographs per sample were measured.…”

Fibroblast growth factor-2 mediates transforming growth factor-β action in prostate cancer reactive stroma

“…Together, these studies support the concept that the actions of TGF-b and specific signaling pathways, including downstream mediators expressed in reactive stroma, is an important area for further study. In addition, although the role of Smad3-mediated TGF-b signaling in tissue fibrosis or wounding has been studied (Roberts et al, 2003;Lakos et al, 2004), it is not known whether Smad3 mediates TGF-b induction of angiogenesis and reactive stroma during carcinoma progression. Accordingly, in order to more clearly define the complicated regulation of carcinogenesis by TGF-b action in stroma, the purposes of this study were to determine whether Smad3-mediated TGF-b signaling is a key pathway in cancerassociated stroma and to subsequently assess candidate downstream mediators of TGF-b/Smad3 biological action.…”