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AB0247 Alteration of WNT pathway in human dermal skin in response to bleomycin treatment
  1. T. Spadoni1,
  2. S. Svegliati1,
  3. M. Rovinelli1,
  4. G. Moroncini1,
  5. E. Avvedimento2,
  6. A. Gabrielli1
  1. 1Sc Cliniche e Molecolari, Univ Politecnica delle Marche, Ancona
  2. 2Biologia e Patologia cellulare e molecolare, Università Federico II, Napoli, Italy

Abstract

Background Dysregulation of Wnt signaling is common in a variety of human malignancies, especially in cancer, aging and fibrosis. Wnt signaling is tightly controlled by several negative regulators, such as WIF1, Wnt inhibitor factor 1. WIF1 is frequently silenced in human cancer by DNA methylation and, recently, its inhibition has been associated to aging of mesenchymal stem cells and fibrosis, attributed to unrestrained Wnt signaling. Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive fibrosis and vascular lesions. We have recently demonstrated that WIF1 expression was consistently downregulated in fibroblasts derived from SSc patients, through histone de-acetylation and not through DNA methylation, as occurs in cancer. Moreover, WIF1 expression in SSc fibroblasts is directly linked to oxidative stress and DNA damage.

Objectives The present study investigated the role of WIF1 in fibrogenesis of human dermal fibroblasts and its regulation to reactive oxygen species (ROS).

Methods Human dermal cells were incubated with bleomycin (20U/ml) in the presence or absence of selective inhibitors. Intracellular ROS production was determined fluorometrically using DCF-DA and DHE as fluorescent probes for peroxide and superoxide, respectively. 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-ΔΔCT method. Collagen content in cell culture supernatant was determined by Sircol assay (Biocolor), according to the manufacturer’s protocol. To analyzed protein expression, cells were lysed with RIPA buffer and subjected to western blot with specific antibodies. For immunocytochemistry, cells were fixed in PFA, permeabilized and labelled with specific antibodies to be evaluated through fluorescence microscopy.

Results The fibrogenic effects of bleomycin were dependent on ROS generation, particularly hydrogen peroxide and superoxide anion. Treatment with bleomycin resulted in downregulation of WIF1, characteristic of SSc. Inhibition of ROS generation by antioxidant, abrogated the fibrogenic effects of bleomycin as well as inhibition of WIF1. Pre-incubation of bleomycin-treated cells with a selective inhibitor of check-point ATM kinase or with a demethylating drug reverted all bleomycin-treatment effects.

Conclusions Our data confirm that bleomycin treatment is a valide model to study SSc. WIF1 expression is inhibited in cells incubated with bleomycin, due to histone de-acetylation of WIF1 promoter. WIF1 inhibition is linked to an increase of oxidative stress, DNA damage, and fibrotic process, as we showed in SSc.

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  3. Huber L.C. Arthritis Rheum 2007; 56: 2755–2764.

Disclosure of Interest None Declared

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