Hurst V I V, Goldberg P L, Minnear F L, Heimark R L, Vincent P A
Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208, USA.
Am J Physiol. 1999 Apr;276(4):L582-95. doi: 10.1152/ajplung.1999.276.4.L582.
The signal transduction pathways that lead to disruption of pulmonary endothelial monolayer integrity by transforming growth factor-beta1 (TGF-beta1) have not been elucidated. The purpose of this investigation was to determine whether disassembly of the adherens junction is temporally associated with the TGF-beta1-induced decrease in pulmonary endothelial monolayer integrity. Measurement of albumin clearance and electrical resistance showed that monolayer integrity started to decrease between 1 and 2 h post-TGF-beta1 treatment and continued to slowly decrease over the next 6 h. Immunofluorescence microscopy of monolayers between 2 and 3 h post-TGF-beta1 showed that beta-catenin, plakoglobin, alpha-catenin, and cadherin-5 were colocalized both at the cell periphery and in newly formed bands that are perpendicular to the cell-cell border. At 4 h post-TGF-beta1, cells began separating; however, beta- and alpha-catenin, plakoglobin, and cadherin-5 could still be found at the cell periphery at areas of cell separation and in strands between separated cells. By 8 h, these junctional proteins were no longer present at the cell periphery at areas of cell separation. The myosin light chain kinase inhibitor KT-5926 prevented the TGF-beta1-induced change in integrity but did not inhibit the formation of actin stress fibers or the formation of bands containing adherens junction proteins that were perpendicular to the cell-cell junction. Overall, these results suggest that adherens junction disassembly occurs after cell separation during TGF-beta1-induced decreases in pulmonary endothelial monolayer integrity and that the loss of integrity may be due to the activation of a myosin light chain kinase-dependent signaling cascade.
转化生长因子-β1（TGF-β1）导致肺内皮单层完整性破坏的信号转导途径尚未阐明。本研究的目的是确定黏附连接的解体是否与TGF-β1诱导的肺内皮单层完整性降低在时间上相关。白蛋白清除率和电阻的测量表明，TGF-β1处理后1至2小时之间单层完整性开始下降，并在接下来的6小时内持续缓慢下降。TGF-β1处理后2至3小时单层的免疫荧光显微镜检查显示，β-连环蛋白、桥粒斑蛋白、α-连环蛋白和钙黏蛋白-5在细胞周边以及垂直于细胞-细胞边界的新形成带中均共定位。TGF-β1处理后4小时，细胞开始分离；然而，在细胞分离区域的细胞周边以及分离细胞之间的条带中仍可发现β-连环蛋白、α-连环蛋白、桥粒斑蛋白和钙黏蛋白-5。到8小时时，这些连接蛋白在细胞分离区域的细胞周边不再存在。肌球蛋白轻链激酶抑制剂KT-5926可防止TGF-β1诱导的完整性变化，但不抑制肌动蛋白应激纤维的形成或垂直于细胞-细胞连接的包含黏附连接蛋白的带的形成。总体而言，这些结果表明，在TGF-β1诱导的肺内皮单层完整性降低过程中，黏附连接解体发生在细胞分离之后，并且完整性的丧失可能是由于肌球蛋白轻链激酶依赖性信号级联的激活。
