Attenuation of Fibroblast Activation and Fibrosis by Adropin in Systemic Sclerosis (SSc)
In this study, we aimed to characterize the role of Adropin/ENHO in fibroblast activation and fibrotic tissue remodeling in SSc as a prototypic multisystem fibrotic disorder. Machine learning based evaluation of bulk RNA-seq datasets identified ENHO as a signature gene in SSc. Furthermore, these machine learning-derived results have been corroborated by various approaches, including DEG analysis. Screening of two large external cohorts, the PRESS and the GENISOS (15, 16), as well as our internal cohort with 133 SSc patients and 92 controls in total, demonstrated downregulation of Adropin/ENHO in dermal fibroblasts of SSc patients. Screening of different profibrotic mediators revealed that the reduction of Adropin/ENHO was mediated by profibrotic cytokine TGFbeta, which was confirmed by overexpression of TbetaRICA or pharmacological inhibition of TGFbeta signaling by the TbetaRI selective inhibitor SD-208 in mice. Screening using inhibitors targeting TGFbeta mediated signaling pathways identified the suppression of ENHO expression was dependent on JNK signaling, which was also confirmed by siRNA mediated knockdown. Studies on cultured human dermal fibroblasts, 3D full-thickness human skin equivalents, complementary murine models with pre-established fibrosis and PCSS were used to demonstrate that administration of bioactive peptides Adropin34-76 ameliorated fibroblast-to-myofibroblast differentiation, collagen release and tissue fibrosis. Study designs and timeline diagrams were shown in Fig. 4A, 5A, 5D and 7A respectively. Transcriptional profiling was used to reveal that the antifibrotic effects of Adropin34-76 were functionally linked to Hedgehog signaling and GLI1 deactivation, which was experimentally confirmed in vitro, in vivo and ex vivo. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to further investigate the intricate interactions between TGFbeta and GLI1 signaling in response to Adropin treatment. Knockdown of GPR19, a putative receptor of Adropin, abrogated the antifibrotic effects of Adropin in fibroblasts.