The nuclear hormone receptor , peroxisome proliferator-activated receptor (PPAR)-␥ , originally identified as a key mediator of adipogenesis , is expressed widely and implicated in diverse biological responses. Both natural and synthetic agonists of PPAR-␥ abrogated the stimulation of collagen synthesis and myofibroblast differentiation induced by transforming growth factor (TGF)-␤ in vitro. To characterize the role of PPAR-␥ in the fibrotic process in vivo, the synthetic agonist rosiglitazone was used in a mouse model of scleroderma. Rosiglitazone attenuated bleomycin-induced skin inflammation and dermal fibrosis as well as subcutaneous lipoatrophy and counteracted the up-regulation of collagen gene expression and myofibroblast accumulation in the lesioned skin. Rosiglitazone treatment reduced the induction of the early-immediate transcription factor Egr-1 in situ without also blocking the activation of Smad2/3. In both explanted fibroblasts and skin organ cultures, rosiglitazone prevented the stimulation of collagen gene transcription and cell migration elicited by TGF-␤. Rosiglitazone-driven adipogenic differentiation of both fibroblasts and preadipocytes was abrogated in the presence of TGF-␤; this effect was accompanied by the concomitant down-regulation of cellular PPAR-␥ mRNA expression. Collectively, these results indicate that rosiglitazone treatment attenuates inflammation, dermal fibrosis, and subcutaneous lipoatrophy via PPAR-␥ in a mouse model of scleroderma and suggest that pharmacological PPAR-␥ ligands, widely used as insulin sensitizers in the treatment of type-2 diabetes mellitus, may be potential therapies for scleroderma. (Am J Pathol

Because recent studies implicate Toll-like receptors (TLRs) in the pathogenesis of fibrosis, we sought to investigate the in vitro and in vivo role and mechanism of TLR4-mediated fibroblast responses in fibrogenesis. We found that TLR4 was constitutively expressed, and accumulation of endogenous TLR4 ligands significantly elevated, in lesional skin and lung tissues from patients with scleroderma. Activation of TLR4 signaling in explanted fibroblasts resulted in enhanced collagen synthesis and increased expression of multiple genes involved in tissue remodeling and extracellular matrix homeostasis. Moreover, TLR4 dramatically enhanced the sensitivity of fibroblasts to the stimulatory effect of transforming growth factor-β1. These profibrotic responses were abrogated by both genetic and pharmacological disruption of TLR4 signaling in vitro, and skin fibrosis induced by bleomycin in vivo was attenuated in mice harboring a mutated TLR4. Activation of TLR4 in fibroblasts augmented the intensity of canonical Smad signaling, and was accompanied by suppression of anti-fibrotic microRNA expression. Together, these results suggest a novel model to account for persistent fibrogenesis in scleroderma, in which activation of fibroblast TLR4 signaling, triggered by damage-associated endogenous TLR4 ligands, results in augmented transforming growth factor-β1 sensitivity with increased matrix production and progressive connective tissue remodeling. Under these conditions, fibroblast TLR4 serves as the switch for converting self-limited tissue repair into intractable fibrosis.

Objective Recent studies identify aberrant Wnt signaling in scleroderma and pulmonary fibrosis. The aim of present study was to investigate the effect of ectopic Wnt10b expression on skin homeostasis and differentiation in transgenic mice with FABP4-directed Wnt10b expression. Methods The expression of Wnt10b was analyzed by real-time qPCR in bleomycin-induced fibrosis. Dermal thickness and the myofibroblasts were determined in skin sections from FABP4-Wnt10b transgenic and wildtype mice. The expression of collagen was analyzed by Sircol assays, immunoblot and real-time qPCR. The effects of Wnt10b were examined in explanted fibroblasts and in adenovirus infected preadipocytes or fibroblasts. Results FABP4-Wnt10b transgenic mice showed progressive loss of adipose tissue in the skin. The dermis was fibrotic and showed increased collagen deposition, fibroblast activation and myofibroblast accumulation. Increased canonical Wnt signaling correlated with elevated collagen gene expression in these biopsies. Explanted fibroblasts from transgenic mice showed increased canonical Wnt signaling and elevated Type I collagen and α-smooth muscle actin gene expression in vitro. Infection of normal preadipocytes with Wnt10b resulted in blockade of adipogenesis, whereas in fibroblasts Wnt10b induced marked up-regulation of Type I collagen. Conclusion We demonstrate that ectopic Wnt10b expression in vivo causes progressive loss of adipose tissue accompanied by the spontaneous development of dermal. These findings suggest that ectopic Wnt10b drives a switch in mesenchymal cell fate toward myofibroblasts by induction of a fibrogenic program while suppressing adipogenesis. Therefore FABP4-Wnt10b transgenic mice represent a novel animal model to study the role and mechanisms of Wnt signaling in fibrosis.

The early growth response gene (Egr-1) codes for a zinc finger transcription factor that has important roles in the regulation of cell growth, differentiation, and survival. Aberrant Egr-1 expression is implicated in carcinogenesis, inflammation, atherosclerosis, and ischemic injury. We reported previously that normal fibroblasts stimulated by transforming growth factor-ß showed rapid and transient induction of Egr-1. Moreover, we observed that tissue expression of Egr-1 was elevated in patients with scleroderma, which suggests that Egr-1 may be involved in tissue repair and fibrosis. Here, we investigated matrix remodeling and wound healing in mice harboring gain of function or loss of function mutations of Egr-1. Using the model of bleomycin-induced scleroderma, we found that the early influx of inflammatory cells into the skin and lungs, and the subsequent development of fibrosis in these organs, were markedly attenuated in Egr-1 null mice. Furthermore, full-thickness incisional skin wound healing was impaired, and skin fibroblasts lacking Egr-1 showed reduced migration and myofibroblast transdifferentiation in vitro. In contrast, transgenic mice with fibroblast-specific Egr-1 overexpression showed exuberant tissue repair, with enhanced collagen accumulation and increased tensile strength of incisional wounds. Together, these results point to the fundamental role that Egr-1 plays in the regulation of transforming growth factor-ß-dependent physiological and pathological matrix

Fibrosis, the pathological hallmark of scleroderma and related conditions, is due to sustained activation of tissue fibroblasts. Accumulating evidence implicates cytokine networks in initiating, and propagating or terminating fibroblast activation, and the specific cytokine phenotype dictates evolution of the fibrotic response toward either resolution or scarring. In particular, cytokines that promote fibroblast proliferation and myofibroblast differentiation and extracellular matrix (ECM) accumulation functionally define a type 2 (Th2) immune response, whereas interferon-γ, which suppresses diverse fibroblast activities, defines a type 1 (Th1) immune response. It remains unclear what role the balance between Th1 and Th2 cytokines plays in the pathogenesis of fibrosis. Here we used bleomycin-induced skin fibrosis as a murine model for human scleroderma in order to study the fibrotic response in mice lacking T-bet, a transcription factor that is essential for initiating Th1 lineage development of CD4+ T lymphocytes. Spleen cells from T-bet null (T-bet^–/–) mice exhibited a typical Th2 cytokine profile ex vivo, with elevated production of interleukin-4 (IL-4), IL-5 and IL-13, and diminished production of interferon-γ. Bleomycin-induced early mast cells and eosinophil accumulation, and eosinophil degranulation, in the lesional tissue were greater in T-bet^–/– mice than in wild-type control mice. At a later time point, T-bet^–/– mice developed significantly more extensive dermal and especially hypodermal fibrosis. Elevated TGF-β expression and intracellular Smad activation were prominent in lesional skin. Infiltrating eosinophils appeared to be an important cellular source of TGF-β. These results demonstrate that in mice lacking T-bet bleomycin induced exaggerated skin fibrosis, suggesting that T-bet has an important physiologic role in regulation of tissue repair by promoting Th1 immune responses that prevent excessive ECM accumulation.