Clara Dees

Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis

The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

Treatment with imatinib prevents fibrosis in different preclinical models of systemic sclerosis and induces regression of established fibrosis

OBJECTIVE Imatinib is a small-molecule tyrosine kinase inhibitor capable of selective, dual inhibition of the transforming growth factor beta and platelet-derived growth factor (PDGF) pathways. Imatinib has previously been shown to prevent the development of inflammation-driven experimental fibrosis when treatment was initiated before administration of the profibrotic stimulus. The aim of this study was to confirm the efficacy of imatinib in a murine model of systemic sclerosis (SSc) that is less driven by inflammation and to investigate whether imatinib is also effective for the treatment of established fibrosis. METHODS The tight skin 1 (TSK-1) mouse model of SSc was used to evaluate the antifibrotic effects of imatinib in a genetic model of the later stages of SSc. In addition, the efficacy of imatinib for the treatment of preestablished fibrosis was analyzed in a modified model of bleomycin-induced dermal fibrosis in which the application of bleomycin was prolonged and the onset of treatment was late. RESULTS Treatment with imatinib reduced dermal and hypodermal thickening in TSK-1 mice and prevented the differentiation of resting fibroblasts into myofibroblasts. In the model of preestablished dermal fibrosis, imatinib not only stopped further progression of fibrosis but also induced regression of preexisting dermal fibrosis, with a reduction in dermal thickness below pretreatment levels. CONCLUSION These results indicate that combined inhibition of the tyrosine kinase c-Abl and PDGF receptor might be effective in the later, less inflammatory stages of SSc and for the treatment of established fibrosis. Thus, imatinib might be an interesting candidate for clinical trials in patients with longstanding disease and preexisting tissue fibrosis.

Platelet-derived serotonin links vascular disease and tissue fibrosis

Blocking 5-HT2B receptor provides a therapeutic target for fibrotic diseases caused by activated platelet release of serotonin during vascular damage.

Dual inhibition of c-abl and PDGF receptor signaling by dasatinib and nilotinib for the treatment of dermal fibrosis.

Abelson kinase (c‐abl) and platelet‐derived growth factor (PDGF) are key players in the pathogenesis of systemic sclerosis (SSc). The aim of the present study was to evaluate the antifibrotic potential of dasatinib and nilotinib, 2 novel inhibitors of c‐abl and PDGF, which are well tolerated and have recently been approved. Dasatinib and nilotinib dose‐dependently reduced the mRNA and protein levels of extracellular matrix proteins in human stimulated dermal fibroblasts from SSc patients (IC50 of 0.5–2.0 nM for dasatinib and 0.8–2.5 nM for nilotinib). In a mouse model of bleomycin‐induced dermal fibrosis, dasatinib and nilotinib potently reduced the dermal thickness, the number of myofibroblasts, and the collagen content of the skin in a dose‐dependent manner at well‐tolerated doses. These data indicate that dasatinib and nilotinib potently inhibit the synthesis of extracellular matrix in vitro and in vivo at biologically relevant concentrations. Thus, we provide the first evidence that dasatinib and nilotinib might be promising drugs for the treatment of patients with SSc.—Akhmetshina, A., Dees, C., Pileckyte, M., Maurer, B., Axmann, R., Jüngel, A., Zwerina, J., Gay, S., Schett, G., Distler, O., Distler, J. H. W. Dual inhibition of c‐abl and PDGF receptor signaling by dasatinib and nilotinib for the treatment of dermal fibrosis. FASEB J. 22, 2214–2222 (2008)

Autophagy regulates TNFα-mediated joint destruction in experimental arthritis

Objectives Autophagy is a homeostatic process to recycle dispensable and damaged cell organelles. Dysregulation of autophagic pathways has recently been implicated in the pathogenesis of various diseases. Here, we investigated the role of autophagy during joint destruction in arthritis. Methods Autophagy in osteoclasts was analysed in vitro and ex vivo by transmission electron microscopy, Western blotting and immunohistochemistry for Beclin1 and Atg7. Small molecule inhibitors, LysMCre-mediated knockout of Atg7 and lentiviral overexpression of Beclin1 were used to modulate autophagy in vitro and in vivo. Osteoclast differentiation markers were quantified by real-time PCR. The extent of bone and cartilage destruction was analysed in human tumour necrosis factor α transgenic (hTNFα tg) mice after adoptive transfer with myeloid specific Atg7-deficient bone marrow. Results Autophagy was activated in osteoclasts of human rheumatoid arthritis (RA) showing increased expression of Beclin1 and Atg7. TNFα potently induced the expression of autophagy-related genes and activated autophagy in vitro and in vivo. Activation of autophagy by overexpression of Beclin1-induced osteoclastogenesis and enhanced the resorptive capacity of cultured osteoclasts, whereas pharmacologic or genetic inactivation of autophagy prevented osteoclast differentiation. Arthritic hTNFα tg mice transplanted with Atg7fl/fl×LysMCre+ bone marrow cells (BMC) showed reduced numbers of osteoclasts and were protected from TNFα-induced bone erosion, proteoglycan loss and chondrocyte death. Conclusions These findings demonstrate that autophagy is activated in RA in a TNFα-dependent manner and regulates osteoclast differentiation and bone resorption. We thus provide evidence for a central role of autophagy in joint destruction in RA.

β-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis

Objectives Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). β-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of β-catenin in fibroblasts for the development of SSc dermal fibrosis. Methods Nuclear accumulation of β-catenin in fibroblasts was assessed by triple staining for β-catenin, prolyl-4-hydroxylase-β and 4′,6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of β-catenin in fibrosis. Results The auhors found significantly increased nuclear levels of β-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of β-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of β-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of β-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of β-catenin significantly reduced bleomycin-induced dermal fibrosis. Conclusions The present study findings identify β-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of β-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of β-catenin/Wnt signaling have recently entered clinical trials.

The Wnt antagonists DKK1 and SFRP1 are downregulated by promoter hypermethylation in systemic sclerosis

Objectives Activated Wnt signalling with decreased expression of endogenous inhibitors has recently been characterised as a central pathomechanism in systemic sclerosis (SSc). Aberrant epigenetic modifications also contribute to the persistent activation of SSc fibroblasts. We investigated whether increased Wnt signalling and epigenetic changes in SSc are causally linked via promoter hypermethylation-induced silencing of Wnt antagonists. Methods The methylation status of endogenous Wnt antagonists in leucocytes and fibroblasts was evaluated by methylation-specific PCR. 5-aza-2′-deoxycytidine was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and in the mouse model of bleomycin-induced skin fibrosis. Activation of Wnt signalling was assessed by analysing Axin2 mRNA levels and by staining for β-catenin. Results The promoters of DKK1 and SFRP1 were hypermethylated in fibroblasts and peripheral blood mononuclear cells of patients with SSc. Promoter hypermethylation resulted in impaired transcription and decreased expression of DKK1 and SFRP1 in SSc. Treatment of SSc fibroblasts or bleomycin-challenged mice with 5-aza prevented promoter methylation-induced silencing and increased the expression of both genes to normal levels. Reactivation of DKK1 and SFRP1 transcription by 5-aza inhibited canonical Wnt signalling in vitro and in vivo and effectively ameliorated experimental fibrosis. Conclusions We demonstrate that hypermethylation of the promoters of DKK1 and SFRP1 contributes to aberrant Wnt signalling in SSc and that Dnmt inhibition effectively reduces Wnt signalling. These data provide a novel link between epigenetic alterations and increased Wnt signalling in SSc and also have translational implications because Dnmt inhibitors are already approved for clinical use.

Orphan nuclear receptor NR4A1 regulates transforming growth factor-β signaling and fibrosis

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

Rho-Associated Kinases Are Crucial for Myofibroblast Differentiation and Production of Extracellular Matrix in Scleroderma Fibroblasts

OBJECTIVE Rho-associated kinases (Rock) are the major cellular mediators of Rho GTPases and play an important role in the organization of the actin cytoskeleton. Inhibitors of Rock are currently being evaluated for the treatment of pulmonary arterial hypertension. This study was undertaken to analyze the role of Rock in the activation of fibroblasts in systemic sclerosis (SSc). METHODS Rock signaling was inhibited using chemical inhibitors and small interfering RNA. The expression of extracellular matrix (ECM) proteins and alpha-smooth muscle actin was analyzed by real-time polymerase chain reaction, Western blotting, and SirCol assay. Metabolic activity was quantified by MTT assay. Cell viability was assessed by staining with annexin V and propidium iodide. The role of MAP kinases was investigated using selective inhibitors and Western blotting. RESULTS Inhibition of Rock strongly reduced the synthesis of the major ECM proteins at the messenger RNA level as well as the protein level. Counterregulatory changes in the expression of tissue inhibitors of metalloproteinases and matrix metalloproteinases were not observed. Inhibition of Rock prevented myofibroblast differentiation. Transforming growth factor beta activated ERK in a Rock-dependent manner, and ERK mediated in part the stimulatory effects of Rock on myofibroblast differentiation. Toxic adverse effects of the inhibition of Rock were not observed. CONCLUSION Our findings demonstrate that Rock potently stimulates the differentiation of resting fibroblasts into myofibroblasts and the production of ECM at biologically relevant concentrations without cell toxicity. These findings, along with the beneficial effects of Rock inhibition on vascular disease, indicate that inhibition of Rock might be an interesting novel therapeutic approach for the treatment of SSc.

Hedgehog signaling controls fibroblast activation and tissue fibrosis in systemic sclerosis

OBJECTIVE Hedgehog signaling not only plays crucial roles during human development but also has been implicated in the pathogenesis of several diseases in adults. The aim of the present study was to investigate the role of the hedgehog pathway in fibroblast activation in systemic sclerosis (SSc). METHODS Activation of the hedgehog pathway was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). The effects of sonic hedgehog (SHH) on collagen synthesis were analyzed by reporter assays, real-time PCR, and Sircol assays. Myofibroblast differentiation was assessed by quantification of α-smooth muscle actin and stress fiber staining. The role of hedgehog signaling in vivo was analyzed by adenoviral overexpression of SHH and using mice lacking 1 allele of the gene for inhibitory receptor Patched homolog 1 (Ptch(+/-) mice). RESULTS SHH was overexpressed and resulted in activation of hedgehog signaling in patients with SSc, with accumulation of the transcription factors Gli-1 and Gli-2 and increased transcription of hedgehog target genes. Activation of hedgehog signaling induced an activated phenotype in cultured fibroblasts, with differentiation of resting fibroblasts into myofibroblasts and increased release of collagen. Adenoviral overexpression of SHH in the skin of mice was sufficient to induce skin fibrosis. Moreover, Ptch(+/-) mice with increased hedgehog signaling were more sensitive to bleomycin-induced dermal fibrosis. CONCLUSION We demonstrated that the hedgehog pathway is activated in patients with SSc. Hedgehog signaling potently stimulates the release of collagen and myofibroblast differentiation in vitro and is sufficient to induce fibrosis in vivo. These findings identify the hedgehog cascade as a profibrotic pathway in SSc.