Effects of CCN3 on fibroblast proliferation, apoptosis and extracellular matrix production

Abstract

CCN2 and CCN3 belong to the CCN family of proteins, which show a high level of structural similarity. Previous studies have shown that CCN2 mediates the ability of transforming growth factor (TGF)-β to stimulate collagen synthesis, leading to keloid formation. CCN2 and CCN3 are opposing factors in regulating the promoter activity and secretion of this extracellular matrix (ECM) protein. Thus, we hypothesize that CCN3 possesses an anti-scarring effect. However, the exact mechanism of CCN3 in this anti-scarring effect remains unclear. The aim of this study was to investigate the mechanism of CCN3 in reducing scar formation. Palatal fibroblasts were obtained from the explants of the oral palatal mucosa of 8-week-old male Sprague-Dawley rats. CCN3 overexpression vector was constructed and then transfected into cells. The inhibitory effects of CCN3 on cell growth were detected via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit and flow cytometry. The expression levels of collagen I, collagen III and α-smooth muscle actin (α-SMA) were determined by western blot analysis and RT-PCR. Following treatment with TGF-β1, we detected the expression of CCN3 and Smad1 in the fibroblasts. CCN3 significantly inhibited the growth and induction of apoptosis of fibroblasts. The expression of collagen I, collagen III and α-SMA was lower in the CCN3-transfected group as compared to the control and vector groups. TGF-β1 stimulation efficiently suppressed the expression of CCN3 at the mRNA and protein levels, and CCN3 was required for TGF-β1-induced Smad1 phosphorylation. Results of this study demonstrated that CCN3 is involved in the proliferation and apoptosis of fibroblasts and the synthesis of ECM proteins. Therefore, CCN3 may play an important role in the develop ment of scar tissue, and may represent a novel therapeutic target for reducing scar formation.