Ren Zhanping, Hou Yuxia, Ma Siwei, Tao Yongwei, Li Jinfeng, Cao Huiqin, Ji Lingling
Department of Cranio‑Maxillofacial Trauma Plastic Surgery, Stomatology Hospital of Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi 710004, P.R. China.
Department of Orthodontics, Stomatology Hospital of Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi 710004, P.R. China.
Int J Mol Med. 2014 Jun;33(6):1607-12. doi: 10.3892/ijmm.2014.1735. Epub 2014 Apr 8.
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 development of scar tissue, and may represent a novel therapeutic target for reducing scar formation.
CCN2和CCN3属于CCN蛋白家族，它们具有高度的结构相似性。先前的研究表明，CCN2介导转化生长因子（TGF）-β刺激胶原蛋白合成的能力，从而导致瘢痕疙瘩形成。CCN2和CCN3在调节这种细胞外基质（ECM）蛋白的启动子活性和分泌方面是相反的因素。因此，我们假设CCN3具有抗瘢痕形成的作用。然而，CCN3在这种抗瘢痕形成作用中的确切机制仍不清楚。本研究的目的是探讨CCN3减少瘢痕形成的机制。从8周龄雄性Sprague-Dawley大鼠的腭部口腔黏膜外植体中获取腭成纤维细胞。构建CCN3过表达载体，然后转染到细胞中。通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）法检测CCN3对细胞生长的抑制作用。使用膜联蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）凋亡检测试剂盒和流式细胞术检测细胞凋亡。通过蛋白质印迹分析和逆转录-聚合酶链反应（RT-PCR）测定I型胶原蛋白、III型胶原蛋白和α-平滑肌肌动蛋白（α-SMA）的表达水平。用TGF-β1处理后，我们检测了成纤维细胞中CCN3和Smad1的表达。CCN3显著抑制成纤维细胞的生长并诱导其凋亡。与对照组和载体组相比，CCN3转染组中I型胶原蛋白、III型胶原蛋白和α-SMA的表达较低。TGF-β1刺激有效抑制了CCN3在mRNA和蛋白质水平的表达，并且CCN3是TGF-β1诱导的Smad1磷酸化所必需的。本研究结果表明，CCN3参与成纤维细胞的增殖和凋亡以及ECM蛋白的合成。因此，CCN3可能在瘢痕组织的形成中起重要作用，并且可能代表减少瘢痕形成的新治疗靶点。
