POS0423 NCOA3 AMPLIFIES PROFIBROTIC TRANSCRIPTIONAL PROGRAMS IN SYSTEMIC SCLEROSIS

Abstract

Background:Excessive activation of fibroblasts with a TGFβ-biased gene signature and deposition of extracellular matrix are key features of fibrotic diseases. The mechanisms underlying these transcriptional changes remain poorly understood. Deregulation, mutations and malfunctions of transcriptional co-regulators, which can interact with multiple transcription factors and enable a broad-spectrum regulation of transcriptional networks, have been implicated as driving factors in a large number of diseases and pathologies.Objectives:In the present study, we aimed to analyze the role of the co-regulator Nuclear Receptor Co-Activator 3 (NCOA3) in fibroblast activation and tissue fibrosis, and to evaluate a potential interaction of NCOA3 with fibrosis-relevant transcription factors.Methods:NCOA3 was inhibited genetically by siRNA transfection and pharmacologically by the SRC3 inhibitor-2 (SI-2). We performed bulk RNASeq of human dermal fibroblasts and in silico transcription factor binding site screening of differentially expressed genes (DEGs). The interaction of NCOA3 and TGFβ-SMAD signaling was analyzed by reporter and CoIP assays.Results:The expression of NCOA3 in skin biopsies of SSc patients compared to normal controls demonstrated that SSc fibroblasts express modestly, but significantly reduced levels of NCOA3, which persisted in cultured SSc fibroblasts. Stimulation of normal fibroblasts with chronically high levels of TGFβ as they also occur in fibrotic tissue remodeling strongly decreased NCOA3 expression to a similar extent as in SSc fibroblasts. Furthermore, NCOA3 expression is also deregulated in different murine models of skin fibrosis. To investigate the functional effects of decreased NCOA3 levels, we targeted the expression of NCOA3 in normal fibroblasts. SiRNA-mediated knockdown of NCOA3 ameliorated TGFβ-induced gene expression, collagen release, myofibroblast differentiation and cell proliferation. In contrast, knockdown of NCOA3 had no effects on collagen release, expression of contractile proteins or gene expression in unstimulated fibroblasts, suggesting that NCOA3 is not required for cellular homeostasis. To characterize the molecular mechanisms, we performed RNASeq upon NCOA3 knockdown. We identified 343 significant differentially expressed genes (220 downregulated and 123 upregulated with a Benjamini-Hochberg false discovery rate FDR < 0.25 and fold change > 1.5) between TGFβ-stimulated fibroblasts with and without NCOA3 knockdown (NCOA3-DEGs) including the fibrosis-relevant genes EDNRB, COL5A3, HES1, IL11 or IL33. Functional analysis of the NCOA3-DEGs showed enrichment of pathway terms such as collagen binding and extracellular matrix organization. In silico screening of the promoters of the NCOA3-DEGs for potential transcription factor binding motifs revealed binding motifs of core transcription factors of fibroblast activation and tissue fibrosis such as SMAD2/3/4, RBPJ, ZEB1, TCF4, REL, and SNAIL2 amongst the downregulated NCOA3-DEGs. Experimental validation of our biostatistical results using SMAD3 as example demonstrated a higher percentage of NCOA3-pSMAD3 double-positive fibroblasts in skin sections of SSc patients compared to healthy controls. In addition, knockdown of NCOA3 reduced TGFβ-induced SMAD-reporter activity. Furthermore, stimulation with TGFβ increased the interaction of NCOA3 with SMAD3 as analyzed by co-immunoprecipitation. Simultaneous knockdown of NCOA3 and SMAD3 showed no additional reductions compared to the single knockdowns, suggesting that NCOA3 controls SMAD3-dependent gene transcription under fibrotic conditions. Finally, inhibition of NCOA3 showed anti-fibrotic effects in different murine models of experimental skin and lung fibrosis.Conclusion:Our findings characterize NCOA3 as regulator of multiple pro-fibrotic transcription programs. Pharmaceutical inhibition of NCOA3 might be a strategy to interfere simultaneously with several core pro-fibrotic mediators in fibrotic diseases such as SSc.Acknowledgements:We thank Lena Summa, Vladyslav Fedorchenko, Wolfgang Espach and Regina Kleinlein for excellent technical assistance.The study was funded by grants DI 1537/7-1, DI 1537/8-1, DI 1537/9-1 and -2, DI 1537/11-1, DI 1537/12-1, DI 1537/13-1, DI 1537/14-1, DI 1537/17-1, DE 2414/2-1, DE 2414/4-1, and RA 2506/3-1 of the German Research Foundation, SFB CRC1181 (project C01) and SFB TR221/ project number 324392634 (B04) of the German Research Foundation, grants J39, 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 14-12-17-1-Bergmann of the ELAN-Foundation Erlangen, BMBF (Era-Net grant 01KT1801), MASCARA program, TP 2 and a Career Support Award of Medicine of the Ernst Jung Foundation.Disclosure of Interests:Clara Dees: None declared, Sebastian Poetter: None declared, Maximilian Fuchs: None declared, Christina Bergmann: None declared, Alexandru-Emil Matei: None declared, Andrea-Hermina Györfi: None declared, Alina Soare: None declared, Andreas Ramming: None declared, Paolo Ceppi: None declared, Georg Schett: None declared, Meik Kunz: 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, ARXX, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB