SAT0292 INTEGRATIVE TRANSCRIPTOMIC AND FUNCTIONAL ANALYSIS REVEALS A ROLE OF DIMETHYL-Α-KETOGLUTARATE IN TGFΒ-DRIVEN CYTOSKELETON REGULATION AND MYOFIBROBLAST DIFFERENTIATION

Abstract

Background:Myofibroblasts are the orchestrators of aberrant extracellular matrix (ECM) remodelling in fibrosis. Actin cytoskeleton is a central hub that integrates mechanical signals to promote myofibroblast differentiation and ECM remodelling. Targeting these pathways could represent a novel antifibrotic strategy. We have recently shown that metabolic intermediate dimethyl-α-ketoglutarate (dm-αKG) blocks TGFβ-driven myofibroblast differentiation in dermal fibroblasts (DF).Objectives:To investigate the mechanisms by which dm-αKG regulates TGFβ-driven myofibroblast differentiation and inflammatory responses in DF.Methods:DF from healthy controls and patients with systemic sclerosis (SSc) were treated with TGFβ (10 ng/ml) and/or dm-αKG (6 mM) for 24h, 48h and 72h. RNA sequencing (Ilumina 2000, n=3 per experimental group) was followed by the analysis of differentially expressed genes (DeSEQ2, log2 fold ≥ |0.5|, padj< 0.01), pathway enrichment analysis (GO terms) and supervised PCA analysis (ClustVis). Protein amounts (fibronectin, αSMA, IL-6), cell contraction and apoptosis were measured with Western blot (n=6), ELISA (n=4), collagen gel contraction assay (n=4) and real time Annexin V assay (n=6). Significance (p<0.05) was determined by one-sample t-test or ANOVA with Tukey’s correction for multiple comparisons.Results:TGFβ (24h) altered the expression of 4076 genes in DF as determined by RNA-seq, among which 1864 genes were upregulated. The upregulated genes were enriched in GO biological processes/molecular functions/cellular compartments related to ECM organization (p=1e-07), Wnt signalling (p=5e-06), actin binding (p=3e-07), focal adhesion (p=1e-10), stress fibers (p=3e-07) and actin cytoskeleton (p= 3e-06). Dm-αKG altered the expression of 589 genes in TGFβ-treated DF compared to TGFβ only. The most downregulated pathways in DF treated with dm-αKG + TGFβ compared to TGFβ only included actin binding (p=5e-05), muscle contraction (p=0.001), ECM organization (p=0.008), focal adhesion (p=0.01), Z disk (p=0.01) and stress fibers (p=0.03). Specifically, dm-aKG significantly (p<0.01, log2>-0.5) decreased the expression of many TGFβ-induced genes involved in actin organization and focal adhesion (NEXN, FRMD5, ANTXR1, ACTC1, LIMCH1, SORBS2, TGM2, CSRP2, CAP2, LMO7, FZD2), muscle contraction (SNTB1, LMOD1, ANKRD1, SULF1, JPH2, CAVIN4, OXTR, DYSF, FBXO32) and ECM organization (COL10A1, COL11A1, HAPLN1, MMP14, MMP3, SPINT2, GREM1, MATN3, ADAMTS4). The PCA analysis revealed that the experimental treatment (PC1, Fig 1A) accounted for 61% variability in the expression of these genes, while 19% was attributed to interdonor variability (PC2). Dm-αKG diminished TGFβ-induced production of αSMA protein (72h, p=0.02, mean O.D. ± SD in TGFβ + dm-αKG vs. TGFβ: 0.34 ± 0.38 vs. 3.1 ± 2.3) and repressed TGFβ-driven secretion of fibronectin protein (72h, p=0.047, 0.5 ± 0.1 vs. 1.2 ± 0.6). Dm-αKG reduced the contractile capacity of TGFβ-stimulated DF in collagen gel contraction assay (p=0.003, 0 vs. 67.1 ± 5.4%). Additionally, dm-αKG decreased TGFβ-driven production of IL-6 transcripts (24h, p=0.05, 2.9 ± 0.6 vs 1.9 ± 0.3) and protein (24h, p=0.0005, 5.9 ± 1.2 vs 3 ± 0.7, Fig 1B), but did not increase the apoptosis of DF (24h, 48h, 72h).Fig 1.A Supervised PCA analysis of RNA-seq data. B. IL-6 secretion (ELISA).Conclusion:Dm-αKG counteracted TGFβ-induced myofibroblast differentiation by regulating the cytoskeleton organization and ECM dynamics in DF and blocked the TGFβ-induced IL-6 production. This closely links metabolism to inflammatory and pro-fibrotic responses in DF. Therefore, regulating intracellular αKG might offer a novel strategy in combating the inflammatory and fibrotic stages of skin fibrosis in SSc.Acknowledgments:This work was supported by a research grant from FOREUM Foundation for Research in Rheumatology.Disclosure of Interests:Blaž Burja: None declared, Gabriela Kania: None declared, Matija Tomsic: None declared, Snežna Sodin-Šemrl: 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, Katja Lakota: None declared, Mojca Frank-Bertoncelj: None declared