BackgroundHedgehog signalling plays a key-role in the pathogenesis of fibrosis in SSc. Besides the canonical hedgehog pathway with activation of Gli transcription factors via ligand-binding to the cell surface receptors, Gli can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways).ObjectivesThe aim of the present study was to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct Gli-inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.MethodsThe Gli-inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the smo-inhibitor vismodegib was used to selectively target canonical hedgehog signalling. The effects of inhibition of Gli2 on TGF-β signalling were analysed in cultured fibroblasts, in the mouse model of bleomycin-induced pulmonary fibrosis and in a model of fibrosis induced by overexpression of a constitutively active TGF-b receptor I (TBRI) in vivo.ResultsTGF-β upregulated the expression of Gli2 in cultured fibroblasts and in murine skin by direct transcriptional regulation. TGF-β as well as its downstream mediator phosphorylated Smad3 were co-expressed with Gli-2 in the skin of SSc patients. Consistent a novel role of Gli2 as a downstream mediator of TGF-β in fibroblasts, inhibition of Gli2 by GANT-61 ameliorated the stimulatory effects of TGF-β on fibroblasts in vitro and in vivo. Treatment with GANT-61 downregulated the mRNA levels of prototypical TGF-b targeted genes, whereas inhibition of canonical hedgehog signalling had no effects. Moreover, GANT-61 reduced the mRNA levels of col1a2 and the release of collagen protein. GANT-61 also blocked TGF-b-induced myofibroblast differentiation and decreased the levels of a-smooth muscle actin and the formation of stress fibres. Moreover, GANT-61 ameliorated experimental fibrosis of the skin and lungs more efficiently as compared to vismodegib. In the model of TBRI-induced fibrosis, mice treated with GANT-61 showed reduced dermal thickening, lower myofibroblast counts and decreased the hydroxyproline content. In contrast, vismodegib had no anti-fibrotic effect in this model. GANT-61 also exerted potent anti-fibrotic effects in the model of bleomycin-induced pulmonary fibrosis and induced regression of pre-established fibrosis. In both models, GANT-61 did not only reduce levels of the hedgehog target genes, but also the levels of the TGF-b target genes PAI-1, CTGF and Smad7, thus confirming inhibition of TGF-b signalling upon targeting of Gli2.ConclusionsWe characterize Gli2 as a novel intracellular mediator of the pro-fibrotic effects of TGF-b. Pharmacologic inhibition of Gli2 targets canonical as well as non-canonical hedgehog signalling and ameliorates the pro-fibrotic effects of TGF-b. The potent anti-fibrotic effects of Gli2 inhibitors on cultured fibroblasts, dermal and pulmonary fibrosis and availability of Gli2 inhibitors for clinical use encourage additional studies to further explore the ...
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