Uncontrolled activation of TGFβ signaling is a common denominator of fibrotic tissue remodeling. Here we characterize the tyrosine phosphatase SHP2 as a molecular checkpoint for TGFβ-induced JAK2/STAT3 signaling and as a potential target for the treatment of fibrosis. TGFβ stimulates the phosphatase activity of SHP2, although this effect is in part counterbalanced by inhibitory effects on SHP2 expression. Stimulation with TGFβ promotes recruitment of SHP2 to JAK2 in fibroblasts with subsequent dephosphorylation of JAK2 at Y570 and activation of STAT3. The effects of SHP2 on STAT3 activation translate into major regulatory effects of SHP2 on fibroblast activation and tissue fibrosis. Genetic or pharmacologic inactivation of SHP2 promotes accumulation of JAK2 phosphorylated at Y570, reduces JAK2/STAT3 signaling, inhibits TGFβ-induced fibroblast activation and ameliorates dermal and pulmonary fibrosis. Given the availability of potent SHP2 inhibitors, SHP2 might thus be a potential target for the treatment of fibrosis.

Activation of fibroblasts is essential for physiological tissue repair. Uncontrolled activation of fibroblasts, however, may lead to tissue fibrosis with organ dysfunction. Although several pathways capable of promoting fibroblast activation and tissue repair have been identified, their interplay in the context of chronic fibrotic diseases remains incompletely understood. Here, we provide evidence that transforming growth factor-β (TGFβ) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFβ induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, which regulates the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFβ on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFβ-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFβ signaling to aberrant autophagy and deregulated epigenetics in fibrotic diseases and may contribute to the development of therapeutic interventions in fibrotic diseases.

The Notch signaling pathway is highly conserved in all animal metazoa: upon Notch receptor activation, transcription of Notch target genes is turned on by an activator complex that centers on the transcription factor CSL. In the absence of signal, CSL assembles transcriptional repression complexes that display remarkable evolutionary diversity. The major antagonist of Notch signaling in insects named Hairless was originally identified in Drosophila melanogaster. It binds to the Drosophila CSL homologue Suppressor of Hairless [Su(H)] and recruits the two general co-repressors, Groucho and C-terminal binding protein. Whereas the majority of Notch signaling components is conserved between insects and vertebrates, Hairless is found only in insects. Here, we present the analysis of the Hairless gene from Daphnia pulex and, hence, for the first time from a crustacean. Daphnia and Drosophila Hairless protein sequences are highly diverged. Known functional domains, however, the Su(H), Groucho and the C-terminal binding protein interactions domains, are well conserved. Moreover, direct binding of the Daphnia Hairless protein and the respective Drosophila interaction partners was detected, demonstrating the conservation at the molecular level. In addition, interaction between Daphnia Hairless and Drosophila Su(H) was demonstrated in vivo, as co-overexpression of the respective genes during Drosophila development resulted in the expected downregulation of Notch activity in the fly. Structural models show that the Hairless-Su(H) repressor complexes from Daphnia and Drosophila are almost indistinguishable from one another. Amino acid residues in direct contact within the Hairless-Su(H) complex are at absolutely identical positions in the two homologues.

ObjectivesSystemic sclerosis (SSc) is characterised by aberrant hedgehog signalling in fibrotic tissues. The hedgehog acyltransferase (HHAT) skinny hedgehog catalyses the attachment of palmitate onto sonic hedgehog (SHH). Palmitoylation of SHH is required for multimerisation of SHH proteins, which is thought to promote long-range, endocrine hedgehog signalling. The aim of this study was to evaluate the role of HHAT in the pathogenesis of SSc.MethodsExpression of HHAT was analysed by real-time polymerase chain reaction(RT-PCR), immunofluorescence and histomorphometry. The effects of HHAT knockdown were analysed by reporter assays, target gene studies and quantification of collagen release and myofibroblast differentiation in cultured human fibroblasts and in two mouse models.ResultsThe expression of HHAT was upregulated in dermal fibroblasts of patients with SSc in a transforming growth factor-β (TGFβ)/SMAD-dependent manner. Knockdown of HHAT reduced TGFβ-induced hedgehog signalling as well as myofibroblast differentiation and collagen release in human dermal fibroblasts. Knockdown of HHAT in the skin of mice ameliorated bleomycin-induced and topoisomerase-induced skin fibrosis.ConclusionHHAT is regulated in SSc in a TGFβ-dependent manner and in turn stimulates TGFβ-induced long-range hedgehog signalling to promote fibroblast activation and tissue fibrosis. Targeting of HHAT might be a novel approach to more selectively interfere with the profibrotic effects of long-range hedgehog signalling.

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.

BackgroundAutophagy is catabolic process allowing cells to degrade unnecessary or dysfunctional cellular organelles. Aberrant activation of autophagy has been also implicated into the pathogenesis of fibrotic diseases. Several stimuli present in fibrosis such as pro-fibrotic cytokines are known to activate autophagy.ObjectivesThe objective of this work is characterise the regulation of autophagy by TGFβ and analyse whether targeting of autophagy in fibroblasts may prevent their aberrant activation in fibrotic diseases.MethodsTo selectively disable autophagy in fibroblasts we generate Atg7fl/flx Col1a2;CreER mice. The role of the autophagy was investigated in the model of bleomycin- and TβRIact-induced dermal and pulmonary fibrosis. Overexpression of Myst1 was achieved by adenovirus encoding for Myst1. Collagen release and protein expression were measure by Western blot. Target genes were analysed by RT-PCR. Co-immunoprecipitation and reporter assay were performed to study physical and functional interactions between MYST1 and SMAD3. To monitor the autophagic flux in vitro and in vivo we generate an adenovirus encoding for tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3), defined as reliable autophagy maker.ResultsWe provide evidence that transforming growth factor-β (TGFβ) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFβ induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16-histoneacetlytransferase MYST1, which controls the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFβ on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFβ-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFβ signalling to aberrant autophagy and altered epigenetics in fibrotic diseases and may open new avenues for therapeutic intervention in fibrotic diseases.ConclusionsWe demonstrate that the epigenetic control of autophagy is disturbed by a TGFβ-dependent downregulation of MYST1. The increased activation of autophagy induces fibroblast-to-myofibroblast transition and promotes fibrotic tissue remodelling. Re-expression of MYST1 prevents aberrant autophagy, limits the profibrotic effects of TGFβ and ameliorates experimental fibrosis. Restoration of the epigenetic control of autophagy might thus be a novel approach to ameliorate fibrotic tissue remodelling.Disclosure of InterestA. Zehender: None declared, N.-Y. Lin: None declared, A. Stefanica: None declared, C.-W. Chen: None declared, A. Soare: None declared, T. Wohlfahrt: None declared, S. Rauber: None declared, C. Bergmann: None declared, A. Ramming: None declared, O. Distler Grant/research support from: Actelion, Pfizer...

BackgroundSHP2, encoded by the PTPN11 gene, is a ubiquitously expressed non-receptor tyrosine phosphatase (PTP). Altered expression and activity levels of SHP2 have also been implicated into the pathogenesis of autoimmune diseases.ObjectivesIn our study, we aim to characterise the role of SHP2 as a possible novel molecular checkpoint of transforming growth factor-β (TGFβ)-induced JAK2/STAT3 signalling in fibrotic diseases.MethodsMice carrying Shp2fl/flxCol1a2-CreER were generated to specifically knockout Shp2 in fibroblasts. SHP2 was inhibited using NSC-87877 inhibitor. The role of Shp2 was investigated in three different mouse models: Bleomycin- and TBRICA induced dermal fibrosis as well as TSK1 genetic model of skin fibrosis. The construct pCMV3-Flag-JAK2 was used for overexpression and for in vitro mutagenesis of human JAK2. Co-immunoprecipitation was performed to confirm the interaction between SHP2 and JAK2. STAT3 activation was analysed by reporter assay. Protein and mRNA expression levels were analysed by Western Blot and qPCR.ResultsOur data characterise SHP2 as an important regulator of TGFβ signalling in fibroblasts. Although SHP2 expression is decreased in systemic sclerosis (SSc), the moderate downregulation of SHP2 expression is not sufficient to counterbalance the hyper-activation of TGFβ signalling in SSc. However, Shp2-deficient murine fibroblasts are less responsive to TGFβ. An impaired response to the profibrotic effects of TGFβ was also observed in human SSc fibroblasts with siRNA-mediated knockdown of SHP2 and upon pharmacological inhibition of SHP2. We show that SHP2 can dephosphorylate JAK2 at the inhibitory Y570 site to promote TGFβ-dependent activation of JAK2 and its downstream mediator STAT3. In contrast, pharmacologic inhibition of SHP2, overexpression of a catalytically inactive SHP2 mutant or knockout of SHP2 inhibits dephosphorylation of pJAK2Y570 preventing phosphorylation of JAK2 at the activation site Y1007/Y1008 and subsequent activation of STAT3 in TGFβ-stimulated fibroblasts and in experimental fibrosis. Fibroblasts overexpressing SHP2 are more susceptible to the profibrotic effects of TGFβ, whereas the stimulatory effects of TGFβ on myofibroblast differentiation and collagen release are reduced in Shp2 knockout fibroblasts. In addition, fibroblast-specific inactivation of Shp2 in vivo protected from experimental fibrosis induced by overexpression of a constitutively active TGFβ receptor type I, by bleomycin injection and ameliorated fibrosis in TSK1 mice.ConclusionsThe present study describes for the first time a role of SHP2 in the pathogenesis of SSc. SHP2 is required for the activation of JAK2 and STAT3 by TGFβ. Inactivation of SHP2 prevents fibroblast activation and tissue fibrosis, providing evidence that SHP2 may be a potential target for the treatment of fibrosis.Disclosure of InterestA. Zehender: None declared, J. Huang: None declared, A.-H. Györfi: None declared, A.-E. Matei: None declared, C. Dees: None declared, C. Beyer: None declared, K. Gelse: None declared, A. Ra...

Background:Autophagy is catabolic process allowing cells to degrade unnecessary or dysfunctional cellular organelles. Failure of appropriate regulation of autophagy, however, can severely perturb tissue homeostasis. Recent studies demonstrate that autophagy is activated in several fibrotic diseases such as liver fibrosis, renal interstitial fibrosis, cardiac fibrosis.Objectives:The objective of this work was to characterize the activation of autophagy in systemic sclerosis (SSc) and to decipher its role in the pathogenesis of SSc.Methods:Activation of autophagy in skin samples of patients and murine models of SSc was assessed by co-staining of LC3B and P62 with the lysosomal marker LAMP2. The role of the autophagy was investigated in the model of bleomycin-induced dermal fibrosis. Beclin1 was overexpressed using adenovirus encoding for Beclin1. To knockdown Atg7 in vivo was achieved by subcutaneous injections of Atg7 siRNA or non-targeting siRNA. In vivo, 3-methyladenine (3-MA) was administered i.p. in a concentration of 15 mg/kg ones daily. Protein expression was measured by Western blot. Target genes were analyzed by qPCR. To monitor the autophagic flux, we generated adenoviral vectors encoding for tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3).Results:In the present study, we demonstrate that autophagy is activated in fibroblasts in SSc skin and also in experimental fibrosis models as compared to respective non-fibrotic control tissue with enhanced activity in in vivo and in vitro autophagy reporter studies. The aberrant activation of autophagy had profound stimulatory effects on fibroblasts. Activation of autophagy by forced expression of BECLIN1 promoted fibroblast-to-myofibroblast transition and stimulated the collagen release by cultured human fibroblasts and induced fibrosis in murine model. Nevertheless, inhibition of autophagy can deactivate myofibroblasts and induce regression of tissue fibrosis. Knockdown of ATG7 or BECLIN1 in human fibroblasts reduced the expression of αSMA and the number of stress fibers in myofibroblasts, indicating re-differentiation of myofibroblasts into resting fibroblasts upon inhibition of autophagy. Similar results were obtained with the autophagy inhibitors CQ and 3-MA. In vivo, siRNA mediated knockdown of Atg7 effectively prevented progression of fibrosis in a model of established bleomycin-induced skin fibrosis. Inactivation of autophagy decreased dermal thickness, myofibroblast counts and hydroxyproline content to below pretreatment levels, indicating regression of bleomycin-induced skin fibrosis. In addition, treatment of mice with the autophagy inhibitor 3-MA ameliorated bleomycin-induced skin fibrosis.Conclusion:We demonstrate that autophagy activity is enhanced in fibroblasts of SSc patients and in murine models of SSc. The increased activation of autophagy induces fibroblast-to-myofibroblast transition and promotes fibrotic tissue remodeling. However, inhibition of autophagy can deactivate myofibroblasts and induce regression of tissue fibrosis.References:[1]Wynn, T. Cellular and molecular mechanisms of fibrosis. J Pathol 214, 199-210 (2008).[2]Klionsky DJ, Abeliovich H, Agostinis P, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151-175 (2008).[3]Wang, CW & Klionsky, DJ. The molecular mechanism of autophagy. Mol Med 9, 65-76 (2003).[4]Hernández-Gea V, Ghiassi-Nejad Z, Rozenfeld R, et al. Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues. Gastroenterology 142, 938-946 (2012).Disclosure of Interests:Ariella Zehender: None declared, Yi-Nan Li: None declared, Neng-Yu Lin: None declared, Andrea-Hermina Györfi: None declared, Alina Soare: None declared, Christina Bergmann: None declared, Andreas Ramming: None declared, Georg Schett: None declared, Jörg H.W. Distler Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB., Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingel-heim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB., Employee of: stock owner of 4D Science and Scientific head of FibroCure