Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
The Wnt Signalling Inhibitor Gene, WIF1, Expression Is Downregulated in Fibroblasts Derived from Systemic Sclerosis Patients by a Persistent Oxidative Stress.
Gabrielli2, Armando, Avvedimento1, Enrico, Moroncini2, Gianluca, De Gennaro2, Lucia, Spadoni2, Tatiana, Grieco2, Antonella, Marrone3, Giusi
Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università Federico II, Napoli, Italy
Dipartimento di Scienze Mediche e Chirurgiche, Università Politecnica delle Marche, Ancona, Italy
Oncogenomic Center, NOGEC, CEINGE, Italy
Systemic sclerosis is an autoimmune disease characterized by extensive fibrosis and vascular lesions. Primary fibroblasts derived from systemic sclerosis (SSc) patients contain high level of cytoplasmatic and peri-nuclear free radicals (ROS) and breaks in the genomic DNA.
Stimulatory IgG auto-antibodies to the PDGF receptor were identified in sera from systemic sclerosis patients. These autoantibodies were capable of converting normal fibroblasts into SSc-like cells inducing excessive oxygen species (ROS) production by activating membrane NADPH oxidase complex. Wnt family constitutes a large group of highly conserved glycoproteins that are implicated in developmental processes and recently in carcinogenesis, aging and fibrosis. Wnt signaling is tightly controlled by several groups of negative regulators that interfere either with receptor-ligand binding or with intracellular signaling.
Wnt inhibitor factor 1, WIF1, is frequently silenced in human cancer by DNA methylation. Recently, its inhibition has been associated to ageing of mesenchymal stem cells and fibrosis, induced by unrestrained Wnt signaling. In order to identify specific markers of the disease, we have decided to analyze WIF1expression in cells derived from patients affected by systemic sclerosis and explore the mechanism of WNT signaling regulation.
Materials and Methods:
Human skin fibroblasts were obtained from punch biopsies taken from normal volunteers and from the involved skin of scleroderma patients. We have investigated WIF-1 expression by reverse transcription and quantitative real-time PCR. Total RNA isolation was performed with total RNA mini kit (BioRad) and reverse transcription PCR was performed using iScript cDNA synthesis kit from BioRad. Quantitative PCR was performed in triplicate with SYBR Green (Biorad).
WIF gene expression was significantly down-regulated in cells derived from systemic sclerosis patients. Moreover, the gene was not methylated, as in breast cancer cells, and its expression was reactivated by inhibiting histone de-acetylase enzymes, trichostain (TSA). PDGF in 24 hours robustly stimulated WIF-1 expression and this effect was prevented by inhibiting NADPH oxidase. Prolonged oxidative stress induced by IgG SSc silenced WIF1 by triggering DNA damage. Inhibition of ATM kinase by the specific inhibitor KU-55933 rescued WIF expression in fibroblasts exposed to long term oxidative stress.
These data indicate that in SSc fibroblasts a persistent oxidative stress, triggered by PDGF or anti PDGF receptor autoantibodies, induce WIF-1 downregulation and possibly altered WNT signaling. Our data indicate that the ultimate cause of silencing WIF-1 is DNA damage.
To cite this abstract, please use the following information:
Gabrielli, Armando, Avvedimento, Enrico, Moroncini, Gianluca, De Gennaro, Lucia, Spadoni, Tatiana, Grieco, Antonella, et al; The Wnt Signalling Inhibitor Gene, WIF1, Expression Is Downregulated in Fibroblasts Derived from Systemic Sclerosis Patients by a Persistent Oxidative Stress. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :2008