Abstract:Objectives Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis.Methods JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. ResultsThe expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycininduced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. ConclusionWe present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.
Background:Pathologic activation of fibroblasts is a central feature of fibrotic tissue disease in Systemic Sclerosis (SSc). Although individual key signaling pathways of fibroblast activation such as transforming growth factor β (TGFβ) and WNT/β-catenin signaling have been identified, the consequences of the concomitant upregulation of these pathways and their crosstalk are incompletely characterized. Given the high medical need, the identification of mutual activation and amplification loops of profibrotic signals is essential to identify novel candidates for antifibrotic therapies. XIAP (X-linked inhibitor of apoptosis protein) is a ubiquitously expressed member of the IAP protein family which are implicated in the regulation of various cellular functions and tissue turnover. XIAP was recently described to be implicated in WNT/β-catenin signaling and TGFβ signaling.Objectives:The aim of this study is to characterize the role of XIAP in fibrotic disease.Methods:XIAP-expression was analyzed by qPCR, IF and Western blot. XIAP was targeted pharmacologically and with siRNA. The activation of WNT/β-catenin signaling was assessed by analyses of WNT target genes, by TOPflash/FOPflash luciferase reporter assay and in reporter mice.In vivo,XIAP inhibition was analysed in two different models of fibrosis.Results:The expression of XIAP is increased in the skin of SSc patients compared to matched healthy individuals with a particular prominent expression in fibroblasts. The overexpression of XIAP is more pronounced in SSc patients with diffuse and active skin fibrosis compared to SSc patients with limited and inactive disease. The overexpression of XIAP is also reflected in several experimental fibrosis models: the model of sclerodermatous graft versus host disease, the model of bleomycin induced skin fibrosis and Topoisomerase I induced fibrosis (TopoI) mice. TGFβ induces the expression of XIAP in vitro and in vivo and treatment with the TGFβ1 receptor antagonist SD208 reverses the TGFβ induced expression of XIAP. Inhibition of XIAP with embelin or siRNA reduces the TGFβ induced activation of fibroblasts with reduced collagen release and reduced expression of myofibroblast markers. In addition, XIAP inhibition reverted the activated fibroblast phenotype in SSc fibroblasts with reduced expression of stress fibers and αSMA. The antifibrotic effects of XIAP inhibition occurred in non-toxic doses as demonstrated by MTT and by TUNEL staining. In vivo, inhibition of XIAP reduced skin fibrosis in the models of bleomycin induced skin fibrosis and in TopoI-induced skin and lung fibrosis as demonstrated by analysis of dermal thickening, dermal hydroxyproline content and by analysis of myofibroblast differentiation. Mechanistically, XIAP inhibition reduced the activation of WNT/β-catenin signaling as demonstrated by TOPflash reporter assays and by the analysis of WNT target genes.Conclusion:XIAP is upregulated in SSc fibroblasts and murine SSc models in a TGFβ-dependent manner and promotes fibroblast activation by fostering canonical WNT signaling. Our data suggest that XIAP mediates an amplification loop between TGFβ and WNT/β-catenin signaling. Inhibition of XIAP may thus be a novel approach to target aberrant WNT/β-catenin signaling in fibrotic diseases.Disclosure of Interests:Christina Bergmann: None declared, Ludwig Hallenberger: None declared, Benita Merlevede: None declared, Clara Dees: None declared, Chih-Wei Chen: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim
ObjectiveX-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc).MethodsThe expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice.ResultsThe expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-β) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/β-catenin signalling. Inactivation of XIAP reduces binding of β-catenin to TCF to in a TLE-dependent manner to block WNT/β-catenin-dependent transcription.ConclusionsOur data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-β/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/β-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.
BackgroundAberrant activation of profibrotic pathways is a key feature of systemic sclerosis (SSc). Extensive evidence characterises TGFβ- and canonical WNT-signalling as key drivers of fibroblast activation. The crosstalk between those pathways, however, remains largely unknown. A better understanding of the interplay of different profibrotic pathways may be the key to the development of effective targeted therapies. XIAP (X-linked inhibitor of apoptosis protein) is an ubiquitously expressed member of the IAP protein family with important functions in tissue turnover. XIAP was recently described to be implicated in canonical Wnt signalling and TGFβ signalling.ObjectivesThe aim of this study is to characterise the role of XIAP in fibrotic disease.MethodsXIAP-expression was analysed by qPCR, IF and Western blot. XIAP was targeted pharmacologically with Embelin. The activation of the canonical Wnt pathway was assessed by analyses of Wnt target genes and by TOPflash/FOPflash luciferase reporter assay. In vivo, XIAP inhibition was analysed in two different models of fibrosis.ResultsThe expression of XIAP is increased in the skin of SSc patients compared to matched healthy individuals with a particularly prominent expression in fibroblasts. The overexpression of XIAP is more pronounced in SSc patients with diffuse and active skin fibrosis compared to SSc patients with limited and inactive disease. The overexpression of XIAP is also reflected in several experimental fibrosis models: the model of sclerodermatous graft versus host disease, the model of bleomycin induced skin fibrosis and in Wnt10b transgenic mice. Stimulation with either recombinant Wnt1 or TGFβ cytokine induces the expression of XIAP in cultured fibroblasts. Inhibition of XIAP reduced the Wnt1- and TGFβ induced activation of fibroblasts with reduced collagen release and expression of myofibroblast markers. In addition, XIAP inhibition reverted the activated fibroblast phenotype in SSc fibroblasts with reduced expression of stress fibres and αSMA. The antifibrotic effects of XIAP inhibition occurred in non-toxic doses. Mechanistically, XIAP inhibition reduced the activation of canonical Wnt signalling as demonstrated by TOPflash reporter assays and by the analysis of canonical Wnt target genes. XIAP inhibition also reduced the expression of canonical Wnt target genes in Wnt10b transgenic mice. To analyse the effects of XIAP inhibition in vivo, two mouse models were used: the model of bleomycin-induced dermal fibrosis and Wnt10b-transgenic mice. In both models, the pharmacological inhibition of XIAP exerted anti-fibrotic effects with reduced dermal thickening, reduced myofibroblast counts and reduced hydroxyproline contents.ConclusionsXIAP is upregulated in SSc fibroblasts in a TGFβ-dependent manner and promotes fibroblast activation by fostering canonical WNT signalling. Our data suggest that XIAP mediates an amplification loop between TGF-β and canonical Wnt signalling. Inhibition of XIAP may thus be a novel approach to target aberrant canonical WNT sign...
BackgroundThe pathologic activation of fibroblasts is a key feature of fibrotic disorders such as Systemic Sclerosis (SSc). Deregulation of TGFβ- and Hedgehog signalling has been shown to be critical for the persistent, uncontrolled activation of fibroblasts in SSc.1–3 However, the consequences of the concomitant upregulation of multiple profibrotic pathways are unknown and cross-talk between individual pathways in fibrotic diseases is currently poorly characterised. Mutual activation and amplification of profibrotic signals might be central for the persistent activation of fibroblasts.ObjectivesThe aim of this study is to characterise the crosstalk between AP1- and hedgehog signalling in fibrotic tissue disease in SSc.MethodsCo-localization of cJUN and GLI2 in fibrotic skin was analysed by confocal microscopy and interaction was shown by Co-IP. cJUN/AP1 signalling and GLI2 signalling were inhibited in vitro and in vivo using the pharmacological inhibitors T5224 and GANT61. Hedgehog signalling was activated in mice by fibroblast-specific overexpression of constitutively active Smoothend (SmoACT mice).ResultsExpression profiling of all AP1 family members revealed most pronounced differences for cJUN in SmoACT mice. The expression of cJUN colocalized with the hedgehog transcription factor GLI2. Overexpression and colocalization of cJUN and GLI2 were also observed in fibroblasts in the skin of SSc patients. The number of GLI2- and cJUN double-positive fibroblasts was strongly increased in involved SSc skin compared to healthy controls and was particularly high in samples of SSc patients with diffuse and progressive disease. Based on the upregulation of both cJun and GLI2 and their colocalization, we hypothesised that cJUN and GLI2 might interact with each other to amplify fibroblast activation and tissue fibrosis. Stimulation of resting fibroblasts with TGFβ induces both cJUN and GLI2 mRNA and protein in a time-dependent manner. cJUN and GLI2 are also induced upon stimulation with Sonic hedgehog (SHH), demonstrating that TGFβ and SHH are both capable to activate cJUN and GLI2-dependent transcription. This crosstalk occurs by direct interaction of cJUN and GLI2, which amplifies the transactivation potential of both transcription factors. Co-immunoprecipitation demonstrated that stimulation of fibroblasts with TGFβ or SHH induces direct interaction of cJUN and GLI2. Overexpression of cJun and Gli2 resulted in activation in both AP1 and Hedgheog target genes. The central role of the crosstalk between cJUN and GLI2 for tissue fibrosis was further highlighted by the finding that hedgehog-induced fibrosis was strongly reduced by AP1 inhibition. SmoACT mice developed extensive skin fibrosis. Treatment with a cJUN/AP1 inhibitor T5224, however, strongly ameliorated hedgehog-induced fibrosis in SmoACT mice.ConclusionsWe demonstrate in the present study that the concomitant activation of AP1- and hedgehog signalling amplifies signalling through both cascades to promote excessive fibroblast activation and tissue fibrosis. This f...
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