Minghua Wu

Rosiglitazone Abrogates Bleomycin-Induced Scleroderma and Blocks Profibrotic Responses Through Peroxisome Proliferator-Activated Receptor-γ

The nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-gamma, originally identified as a key mediator of adipogenesis, is expressed widely and implicated in diverse biological responses. Both natural and synthetic agonists of PPAR-gamma abrogated the stimulation of collagen synthesis and myofibroblast differentiation induced by transforming growth factor (TGF)-beta in vitro. To characterize the role of PPAR-gamma 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-beta. Rosiglitazone-driven adipogenic differentiation of both fibroblasts and preadipocytes was abrogated in the presence of TGF-beta; this effect was accompanied by the concomitant down-regulation of cellular PPAR-gamma mRNA expression. Collectively, these results indicate that rosiglitazone treatment attenuates inflammation, dermal fibrosis, and subcutaneous lipoatrophy via PPAR-gamma in a mouse model of scleroderma and suggest that pharmacological PPAR-gamma ligands, widely used as insulin sensitizers in the treatment of type-2 diabetes mellitus, may be potential therapies for scleroderma.

Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1.

Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of alpha-smooth muscle actin (alpha-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of alpha-SMA and vimentin was prevented in mitochondria-deficient rho(0) cells, which are incapable of ROS production during hypoxia. CoCl(2) and dimethyloxaloylglycine, two compounds that stabilize hypoxia-inducible factor (HIF)-alpha under normoxia, failed to induce alpha-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1alpha did not induce alpha-SMA. However, loss of HIF-1alpha or HIF-2alpha abolished induction of alpha-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-beta1, and preincubation of AEC with SB431542, an inhibitor of the TGF-beta1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-beta1 dependent. Furthermore, both ROS and HIF-alpha were necessary for hypoxia-induced TGF-beta1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-beta1 signaling.

PPARγ Downregulation by TGFß in Fibroblast and Impaired Expression and Function in Systemic Sclerosis: A Novel Mechanism for Progressive Fibrogenesis

The nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) is expressed in multiple cell types in addition to adipocytes. Upon its activation by natural ligands such as fatty acids and eicosanoids, or by synthetic agonists such as rosiglitazone, PPAR-γ regulates adipogenesis, glucose uptake and inflammatory responses. Recent studies establish a novel role for PPAR-γ signaling as an endogenous mechanism for regulating transforming growth factor-ß (TGF-ß)-dependent fibrogenesis. Here, we sought to characterize PPAR-γ function in the prototypic fibrosing disorder systemic sclerosis (SSc), and delineate the factors governing PPAR-γ expression. We report that PPAR-γ levels were markedly diminished in skin and lung biopsies from patients with SSc, and in fibroblasts explanted from the lesional skin. In normal fibroblasts, treatment with TGF-ß resulted in a time- and dose-dependent down-regulation of PPAR-γ expression. Inhibition occurred at the transcriptional level and was mediated via canonical Smad signal transduction. Genome-wide expression profiling of SSc skin biopsies revealed a marked attenuation of PPAR-γ levels and transcriptional activity in a subset of patients with diffuse cutaneous SSc, which was correlated with the presence of a “TGF-ß responsive gene signature” in these biopsies. Together, these results demonstrate that the expression and function of PPAR-γ are impaired in SSc, and reveal the existence of a reciprocal inhibitory cross-talk between TGF-ß activation and PPAR-γ signaling in the context of fibrogenesis. In light of the potent anti-fibrotic effects attributed to PPAR-γ, these observations lead us to propose that excessive TGF-ß activity in SSc accounts for impaired PPAR-γ function, which in turn contributes to unchecked fibroblast activation and progressive fibrosis.

Cadherin-11 contributes to pulmonary fibrosis: potential role in TGF-β production and epithelial to mesenchymal transition

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin‐11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11‐deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild‐type mice. Furthermore, anti‐CDH11‐neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF‐β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11‐deficient mice. Mechanistic studies demonstrated that TGF‐β up‐regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF‐β‐induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.—Schneider, D. J., Wu, M., Le, T. T., Cho, S.‐H., Brenner, M. B., Blackburn, M. R., Agarwal, S. K. Cadherin‐11 contributes to pulmonary fibrosis: potential role in TGFβ production and epithelial to mesenchymal transition. FASEB J. 26, 503–512 (2012). www.fasebj.org

A non-Smad mechanism of fibroblast activation by transforming growth factor-β via c-Abl and Egr-1: selective modulation by imatinib mesylate

The nonreceptor protein tyrosine kinase c-Abl regulates cell proliferation and survival. Recent studies provide evidence that implicate c-Abl as a mediator for fibrotic responses induced by transforming growth factor-β (TGF-β), but the precise mechanisms underlying this novel oncogene function are unknown. Here, we report that when expressed in normal fibroblasts, a constitutively active mutant of Abl that causes chronic myelogenous leukemia (CML) stimulated the expression and transcriptional activity of the early growth response factor 1 (Egr-1). Mouse embryonic fibroblasts (MEFs), lacking c-Abl, were resistant to TGF-β stimulation. Responsiveness of these MEFs to TGF-β could be rescued by wild-type c-Abl, but not by a kinase-deficient mutant form of c-Abl. Furthermore, Abl kinase activity was necessary for the induction of Egr-1 by TGF-β in normal fibroblasts, and Egr-1 was required for stimulation of collagen by Bcr-Abl. Lesional skin fibroblasts in mice with bleomycin-induced fibrosis of skin displayed evidence of c-Abl activation in situ, and elevated phospho-c-Abl correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response, delineate a novel Egr-1-dependent intracellular signaling mechanism that underlies the involvement of c-Abl in certain TGF-β responses, and identify Egr-1 as a target of inhibition by imatinib. Furthermore, the findings show in situ activation of c-Abl paralleling the upregulated tissue expression of Egr-1 that accompanies fibrosis. Pharmacological targeting of c-Abl and its downstream effector pathways may, therefore, represent a novel therapeutic approach to blocking TGF-β-dependent fibrotic processes.

Toll-like receptor 3 upregulation by type I interferon in healthy and scleroderma dermal fibroblasts

IntroductionIncreased levels of genes in the type I interferon (IFN) pathway have been observed in patients with systemic sclerosis (SSc), or scleroderma. How type I IFN regulates the dermal fibroblast and its participation in the development of dermal fibrosis is not known. We hypothesized that one mechanism by which type I IFN may contribute to dermal fibrosis is through upregulation of specific Toll-like receptors (TLRs) on dermal fibroblasts. Therefore, we investigated the regulation of TLR expression on dermal fibroblasts by IFN.MethodsThe expression of TLRs was assessed in cultured dermal fibroblasts from control and SSc patients stimulated with IFNα2. The ability of IFNα2 to regulate TLR-induced interleukin (IL)-6 and CC chemokine ligand 2 production was also assessed. Immunohistochemical analyses were performed to determine whether TLR3 was expressed in skin biopsies in the bleomycin-induced skin fibrosis model and in patients with SSc.ResultsIFNα2 increased TLR3 expression on human dermal fibroblasts, which resulted in enhanced TLR3-induced IL-6 production. SSc fibroblasts have an augmented TLR3 response to IFNα2 relative to control fibroblasts. Pretreatment of fibroblasts with transforming growth factor (TGF)-β increased TLR3 induction by IFNα2, but coincubation of TGF-β did not alter TLR3 induction by IFN. Furthermore, IFNα2 inhibits but does not completely block the induction of connective tissue growth factor and collagen expression by TGF-βin fibroblasts. TLR3 expression was observed in dermal fibroblasts and inflammatory cells from skin biopsies from patients with SSc as well as in the bleomycin-induced skin fibrosis model.ConclusionsType I IFNs can increase the inflammatory potential of dermal fibroblasts through the upregulation of TLR3.

Essential Roles for Early Growth Response Transcription Factor Egr-1 in Tissue Fibrosis and Wound Healing

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 remodeling.

Smad-Independent Transforming Growth Factor-β Regulation of Early Growth Response-1 and Sustained Expression in Fibrosis: Implications for Scleroderma

Transforming growth factor-beta (TGF-beta) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-beta and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-beta causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-beta to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-beta, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-beta enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-beta signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.

Increased Bleomycin-Induced Skin Fibrosis in Mice Lacking the Th1-Specific Transcription Factor T-bet

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.

Proteasomal inhibition after injury prevents fibrosis by modulating TGF-β 1 signalling

Background The development of organ fibrosis after injury requires activation of transforming growth factor β1 which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been reported to prevent the development of fibrosis in the liver, kidney and bone marrow. It is hypothesised that proteasomal inhibition would prevent lung and skin fibrosis after injury by inhibiting TGF-β1-mediated transcription. Methods Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7 days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40 days, respectively. To examine the mechanism of this protection, bortezomib was administered to primary normal lung fibroblasts and primary lung and skin fibroblasts obtained from patients with idiopathic pulmonary fibrosis and scleroderma, respectively. Results Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7 days after the initiation of bleomycin. In primary human lung fibroblasts from normal individuals and patients with idiopathic pulmonary fibrosis and in skin fibroblasts from a patient with scleroderma, bortezomib inhibited TGF-β1-mediated target gene expression by inhibiting transcription induced by activated Smads. An increase in the abundance and activity of the nuclear hormone receptor PPARγ, a repressor of Smad-mediated transcription, contributed to this response. Conclusions Proteasomal inhibition prevents lung and skin fibrosis after injury in part by increasing the abundance and activity of PPARγ. Proteasomal inhibition may offer a novel therapeutic alternative in patients with dysregulated tissue repair and fibrosis.