通过 CTGF 结构域与 TGFβ2 及其受体 TGFβRII 的特异性相互作用调节纤连蛋白 EDA。
Regulation of fibronectin-EDA through CTGF domain-specific interactions with TGFβ2 and its receptor TGFβRII.
机构信息
Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.
出版信息
Invest Ophthalmol Vis Sci. 2011 Jul 7;52(8):5068-78. doi: 10.1167/iovs.11-7191.
PURPOSE
To investigate the role of fibronectin containing extra domain A (FN-EDA) in the pathogenesis of proliferative vitreoretinopathy (PVR) and the regulation of FN-EDA by transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) in retinal pigment epithelial (RPE) cells.
METHODS
Expression of FN-EDA in normal human retinas and PVR membranes was evaluated by immunohistochemistry. The effects of TGFβ and CTGF on FN-EDA mRNA and protein expression in primary cultures of human RPE cells were analyzed at different time points by real-time PCR and Western blot, respectively. The interaction of CTGF with TGFβ2 or with its type II receptor TGFβRII was examined by ELISA, immunoprecipitation, and solid-phase binding assays.
RESULTS
FN-EDA was abundantly expressed in PVR membranes but absent from the RPE monolayer in normal human retinas. Treatment of RPE cells with TGFβ2 induced FN-EDA expression in a time- and dose-dependent manner, but CTGF alone had no effect. However, CTGF, through its N-terminal half fragment, augmented TGFβ2-induced expression of FN-EDA at the protein level. This effect was blocked by antibodies against TGFβ2 or TGFβRII. Interaction of TGFβ2 or TGFβRII with CTGF was dose dependent and specific. CTGF directly bound TGFβ2 and TGFβRII at its N- and C-terminal domains, respectively.
CONCLUSIONS
These findings suggest that CTGF promotes the profibrotic activities of TGFβ acting as a cofactor through direct protein interactions and complex regulatory mechanisms.
目的
研究纤维连接蛋白含有额外结构域 A（FN-EDA）在增生性玻璃体视网膜病变（PVR）发病机制中的作用，以及转化生长因子（TGF）-β和结缔组织生长因子（CTGF）在视网膜色素上皮（RPE）细胞中对 FN-EDA 的调节作用。
方法
通过免疫组织化学法检测正常人和 PVR 膜中 FN-EDA 的表达。分别采用实时 PCR 和 Western blot 分析 TGFβ 和 CTGF 在原代培养的人 RPE 细胞中不同时间点对 FN-EDA mRNA 和蛋白表达的影响。采用 ELISA、免疫沉淀和固相结合测定法检测 CTGF 与 TGFβ2 或其 II 型受体 TGFβRII 的相互作用。
结果
FN-EDA 在 PVR 膜中大量表达，但在正常人类视网膜的 RPE 单层中不存在。TGFβ2 以时间和剂量依赖的方式诱导 RPE 细胞中 FN-EDA 的表达，但 CTGF 单独无作用。然而，CTGF 通过其 N 端半片段在蛋白水平上增强 TGFβ2 诱导的 FN-EDA 表达。该作用被针对 TGFβ2 或 TGFβRII 的抗体阻断。TGFβ2 或 TGFβRII 与 CTGF 的相互作用呈剂量依赖性和特异性。CTGF 分别直接结合 TGFβ2 和 TGFβRII 的 N 和 C 端结构域。
结论
这些发现表明，CTGF 通过直接的蛋白相互作用和复杂的调节机制作为辅因子促进 TGFβ 的促纤维化活性。
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