Usui T, Takase M, Kaji Y, Suzuki K, Ishida K, Tsuru T, Miyata K, Kawabata M, Yamashita H
Department of Ophthalmology, Faculty of Medicine, University of Tokyo, Japan.
Invest Ophthalmol Vis Sci. 1998 Oct;39(11):1981-9.
Production of extracellular matrix (ECM) by corneal endothelial cells is related to physiologic functions and pathologic conditions and is regulated by many cytokines, including transforming growth factor-beta (TGF-beta). In this study, the molecular mechanism of ECM production regulation by TGF-beta was investigated in cultured corneal endothelial cells.
The production of ECM components (laminin and fibronectin) was detected in cultured corneal endothelial cells by western blot analysis. To determine the signal transduction pathways, mutant TGF-beta type I receptor (TbetaR-I) and/or Smad protein family members (intracellular signal transducers in TGF-beta signaling) were overexpressed by transfecting their cDNA into the cultured cells, and the effects on ECM production were observed.
The production of laminin and fibronectin was stimulated by treatment with TGF-beta1 or TGF-beta2. After transient transfection of cDNA of the constitutively active (CA) mutant of TbetaR-I, the production of laminin and fibronectin was stimulated even in the absence of TGF-beta. The transfection of the dominant negative mutant of TbetaR-I counteracted the effects of TGF-beta. These results confirm that TGF-beta directly stimulates ECM production from corneal endothelial cells through TbetaR-I. The ECM production stimulation by TGF-beta or CA TbetaR-I was accelerated by the overexpression of Smad2, Smad3, and/or Smad4 and inhibited by that of Smad7. These results show that TGF-beta signals connected to ECM production are regulated by Smad family members, located downstream of TbetaR-I.
The results of this study show that TGF-beta stimulates ECM production from corneal endothelial cells through TbetaR-I and Smad family transducers.
角膜内皮细胞产生细胞外基质（ECM）与生理功能和病理状况相关，且受多种细胞因子调节，包括转化生长因子-β（TGF-β）。本研究在培养的角膜内皮细胞中探讨了TGF-β调节ECM产生的分子机制。
通过蛋白质免疫印迹分析检测培养的角膜内皮细胞中ECM成分（层粘连蛋白和纤连蛋白）的产生。为确定信号转导途径，将突变型TGF-βⅠ型受体（TβR-Ⅰ）和/或Smad蛋白家族成员（TGF-β信号传导中的细胞内信号转导分子）的cDNA转染到培养细胞中使其过表达，观察其对ECM产生的影响。
用TGF-β1或TGF-β2处理可刺激层粘连蛋白和纤连蛋白的产生。短暂转染组成型激活（CA）突变体TβR-Ⅰ的cDNA后，即使在无TGF-β的情况下，层粘连蛋白和纤连蛋白的产生也受到刺激。转染显性负性突变体TβR-Ⅰ可抵消TGF-β的作用。这些结果证实TGF-β通过TβR-Ⅰ直接刺激角膜内皮细胞产生ECM。TGF-β或CA TβR-Ⅰ对ECM产生的刺激作用在Smad2、Smad3和/或Smad4过表达时加速，而在Smad7过表达时受到抑制。这些结果表明，与ECM产生相关的TGF-β信号由位于TβR-Ⅰ下游的Smad家族成员调节。
本研究结果表明，TGF-β通过TβR-Ⅰ和Smad家族转导分子刺激角膜内皮细胞产生ECM。
