OP0226 WIF1, a WNT Pathway Inhibitor, is Silenced in Systemic Sclerosis by DNA Damage: A Mechanism Linking DNA Damage to WNT and Fibrosis
Authors: T. Spadoni, S. svegliati, A. pezone, G. marrone, A. grieco, G. moroncini, A. Jüngel, O. distler, E. avvedimento, A. gabrielli
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> Dysregulation of Wnt signaling is common in a variety of human malignancies, carcinogenesis, aging and fibrosis. Wnt signaling is tightly controlled by several negative regulators, such as WIF1 (Wnt inhibitor factor 1). Activation of canonical Wnt signaling has been recently found in fibrotic diseases included Systemic Sclerosis (SSc). <h3>Objectives</h3> The objective of the present work is to identify the mechanism responsible for the silencing of WIF1 in SSc. <h3>Methods</h3> Skin fibroblasts from SSc patients and normal controls were treated with bleomycin or ATM-HDAC inhibitors. Cells were transiently transfected with the siRNA against c-jun and ATF-3 with Lipofectamine (Invitrogen). Total RNA was isolated and reverse-transcribed, according to the manufacturer’s instructions (Bio-Rad). Quantitative real-time PCR reactions were performed using SYBR-Green PCR Master Mix (Bio-Rad). The relative expression levels were calculated using the 2<sup>-ΔΔCT</sup> method. To analyzed protein expression, cells were lysed with RIPA buffer and subjected to western blot with specific antibodies. <h3>Results</h3> Our data indicate that WIF1 is silenced by DNA damage and the check point kinase, ATM. Cell derived from SSc patients reactivate WIF1 expression if exposed to ATM or HDACI-III inhibitors. ROS and SSc immunoglobulins silence WIF1 expression via PDGF receptor, stimulate b-catenin accumulation by inducing ROS-dependent DNA damage. Bleomycin, a drug widely used to induce local skin fibrosis in vivo, silences WIF1 and stimulates Wnt signaling and its effects are suppressed by ATM or HDAC inhibitors. Silencing of WIF1 in normal cells amplifies Wnt signaling and increases collagen expression. As molecular actors that silence WIF1 in DNA damaged cells, we report that the knocking down of the expression of transcription factors ATF-3 and c-jun relieves WIF1 inhibition and dowregulates collagen expression in SSc cells. Bleomycin profibrotic phenotype is caused by activation of ATF-3 which silences WIF1 and amplifies Wnt signaling and by c-jun which cooperates with Wnt and stimulates collagen expression. <h3>Conclusions</h3> These results explain Wnt signaling hypertrophy in fibrotic disease, unveil a direct link between DNA damage and Wnt, and pave a novel route to treat fibrosis. <h3>References</h3> Herr P, Hausmann G, Basler K. WNT secretion and signalling in human disease. Trends Mol Med 2012, 18: 483-493. Lam AP, Gottardi CJ. β-catenin signaling: a novel mediator of fibrosis and potential therapeutic target. Curr Opin Rheumatol. 2011, 23(6): 562–567. Beyer, C, Distler, O, Distler, JHW. Innovative antifibrotic therapies in systemic sclerosis<i>.</i> Curr Opinion Rheumatol 2012<i>,</i> 24<i>:</i> 274–280<i>.</i> Bhattacharyya S, Wei J, Varga J. Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities. Nat Rev Rheumatol 2012, 8: 42-54. <h3>Disclosure of Interest</h3> T. Spadoni: None Declared, S. svegliati: None Declared, A. pezone: None Declared, G. marrone: None Declared, A. grieco: None Declared, G. moroncini: None Declared, A. Jüngel: None Declared, O. distler Grant/research support from: Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, Roche/Genentech, medac, Biovitrium, Boehringer Ingelheim Pharma, Novartis, 4 D Science, Active Biotec and Sinoxa, Consultant for: Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, Roche/Genentech, medac, Biovitrium, Boehringer Ingelheim Pharma, Novartis, 4 D Science, Active Biotec and Sinoxa, E. avvedimento: None Declared, A. gabrielli Grant/research support from: Roche, Actelion