Transforming growth factor(beta)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly.
Transforming growth factor(beta)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly.
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发表时间：
1999-08
影响因子：
4.4
通讯作者：
J. Jester;J. Jester;Jiying Huang;P. Barry-Lane;W. Kao;W. Petroll;H. D. Cavanagh
中科院分区：
文献类型：
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作者：
J. Jester;J. Jester;Jiying Huang;P. Barry-Lane;W. Kao;W. Petroll;H. D. Cavanagh
目的 最近的研究表明，转化生长因子 (TGF)β 是角膜肌成纤维细胞分化和平滑肌特异性 α-肌动蛋白 (α-SMA) 表达的有效诱导剂。尽管已知 TGFbeta 可以增强细胞外基质蛋白和受体的合成，但人们对其如何调节非肌肉细胞中平滑肌蛋白的表达知之甚少。本研究的目的是确定精氨酸-甘氨酸-天冬氨酸 (RGD) 依赖性酪氨酸磷酸化在调节 α-SMA 基因表达和最终肌成纤维细胞发育中的作用。方法 由于含血清培养基中的细胞培养模拟肌成纤维细胞转化，因此所有实验均在铺在成分确定的无血清培养基中的新鲜分离的兔角膜细胞上进行。将细胞暴露于 TGFbeta (1 ng/ml)、Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP，50 microM)、Gly-Arg-AL-Asp-Ser-Pro (GRADSP；100 microM) 或除草霉素 A (0.1-10 nM) 24 小时（稀疏）或 7 天（汇合）。通过免疫细胞化学评估细胞，收集蛋白质和 RNA，使用 α-SMA、纤连蛋白、粘着斑蛋白和磷酸酪氨酸（克隆 4G10 和 PY20）特异的抗体进行蛋白质印迹和北方印迹分析；以及针对兔α-SMA的探针。所有实验至少重复三次。结果暴露于TGFbeta的角膜细胞显示出α-SMA的表达，这与肌动蛋白细胞骨架的细胞内重组和纤连蛋白原纤维的细胞外组装相一致。添加含有RGD但不含对照肽的RGD可阻断细胞内肌动蛋白、细胞外纤连蛋白以及α-SMA蛋白和mRNA的组织。用 4G10 或 PY20 对细胞蛋白进行免疫沉淀，鉴定出 TGFbeta 相关的桩蛋白、pp125fak、p130、PLCgamma 和张力蛋白的酪氨酸磷酸化，而添加 GRGDdSP 可以阻断该磷酸化。将除草霉素 A 添加到暴露于 TGFbeta 的角膜细胞中，显示出 α-SMA 蛋白和 mRNA 的剂量依赖性损失，这与酪氨酸磷酸化的损失、肌动蛋白重组和纤连蛋白组装的缺失相关。结论 数据表明，TGFbeta 介导的 α-SMA 基因表达导致肌成纤维细胞转化可能涉及 RGD 依赖性磷酸酪氨酸信号转导途径。
PURPOSE Recent studies indicate that transforming growth factor (TGF)beta is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, alpha-actin (alpha-SMA). Although TGFbeta is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating alpha-SMA gene expression and ultimately myofibroblast development. METHODS Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGFbeta (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 microM), Gly-Arg-AL-Asp-Ser-Pro (GRADSP; 100 microM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for alpha-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit alpha-SMA. All experiments were repeated at least three times. RESULTS Keratocytes exposed to TGFbeta showed expression of alpha-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and alpha-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGFbeta-associated tyrosine phosphorylation of paxillin, pp125fak, p130, PLCgamma, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGFbeta showed a dose-dependent loss of alpha-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly. CONCLUSIONS The data suggest that TGFbeta-mediated alpha-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.