Objectives Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed 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.
Methods The 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. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I.
Results TGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis.
Conclusions Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.
- Systemic Sclerosis
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Handling editor Tore K Kvien
Contributors RL and BŠ contributed equally to the article. RL BŠ, CC and JHWD designed the study. RL, BS, CC, TM, YZ and TW were involved in acquisition of data. RL, BS, CC, KP, CD, AR, OD, LŠ and JHWD were involved in interpretation of data. RL, BS, OD, GS and JHWD were involved in manuscript preparation. DK and MMJ provided essential samples.
Funding Grants DI 1537/5-1, DI 1537/7-1, DI 1537/8-1, DI 1537/9-1, DI 1537/11-1, DE 2414/2-1, RA 2506/3-1 and AK 144/2-1 of the Deutsche Forschungsgemeinschaft, grants A57, J40 and A64 of the IZKF in Erlangen, grant 2013.056.1 of the Wilhelm-Sander-Foundation, grants 2014_A47, 2014_A248 and 2014_A184 of the Else-Kröner-Fresenius-Foundation, grant 2013.056.1 of the Wilhelm Sander Foundation and a Career Support Award of Medicine of the Ernst Jung Foundation.
Competing interests OD has consulted for, or has received research funding from, 4D Science, Actelion, Active Biotech, Bayer-Schering, Biogen, Biovitrium, BMS, Boehringer, EpiPharm, Ergonex, GSK, Inventiva, Medac, Novartis, Pfizer, Roche/Genentech, Sanofi/Genzyme, Serodapharm, Sinoxa and United BioSource Corporation; JHWD has consultancy relationships and/or has received research funding from Actelion, BMS, Celgene, Bayer Pharma, Boehringer Ingelheim, JB Therapeutics, Sanofi-Aventis, Novartis, UCB, GSK, Array Biopharma and Active Biotech in the area of potential treatments of SSc and is stock owner of 4D Science GmbH.
Ethics approval Ethical committee of the University of Erlangen-Nuremberg.
Provenance and peer review Not commissioned; externally peer reviewed.
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