Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells.
Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells.
复制标题
蛋白激酶抑制剂可预防MDCK上皮细胞中低细胞外钙诱导的连接解离。
DOI：
10.1083/jcb.117.1.169
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发表时间：
1992-04
影响因子：
7.8
通讯作者：
Citi, S
中科院分区：
文献类型：
--
作者：
Citi, S
当上皮细胞培养物从具有正常细胞外钙浓度（1-2 mM）的培养基转移到具有低细胞外钙浓度（LC，小于50 μ M游离Ca 2+）的培养基时，细胞-细胞接触被破坏，并且紧密连接依赖性跨上皮电阻下降。在本研究中，我使用MDCK上皮细胞来研究LC对紧密连接蛋白扣带蛋白定位的影响，以及蛋白激酶在LC诱导的事件中的作用。免疫荧光分析表明，在15分钟内的融合单层在LC中的孵育，扣带蛋白标记从细胞周边错位，作为一个阵列的颗粒形成一个环状结构。在钙去除后的后期，扣带蛋白标记主要出现在细胞质中，呈弥漫性和颗粒状，细胞呈圆形和较小。这些事件不受血清缺乏或用10 mM叠氮化钠或6 mg/ml放线菌酮预孵育的影响。然而，当细胞用蛋白激酶抑制剂H-7（大于或等于30 μ M）预处理时，LC诱导的细胞-细胞接触的破坏、细胞形状的改变以及扣带蛋白和其他连接蛋白的重新分布被抑制。抑制剂H-8和在较小程度上的星形孢菌素也是有效的，而HA-1004和ML-7基本上没有显示出活性，这表明不同抑制剂的作用的特异性。跨上皮阻力的测量表明，可以防止连接解体的激酶抑制剂也可以减少LC引起的跨上皮阻力的下降。剂量-反应曲线表明，H-7是最有效的抑制剂，和跨上皮电阻的70%的控制后1小时，钙去除。这些结果表明，低细胞外钙调节连接的完整性和细胞骨架组织通过效应系统涉及蛋白激酶。
When epithelial cell cultures are transferred from a medium with a normal extracellular calcium concentration (1-2 mM) to a medium with a low extracellular calcium concentration (LC, less than 50 microM free Ca2+) cell-cell contacts are disrupted, and the tight junction- dependent transepithelial resistance drops. In this study, I used MDCK epithelial cells to investigate the effects of LC on the localization of the tight junction protein cingulin, and the role of protein kinases in the events induced by LC. Immunofluorescence analysis showed that within 15 min of incubation of confluent monolayers in LC, cingulin labeling was dislocated from the cell periphery, as an array of granules forming a ring-like structure. At later times after calcium removal, cingulin labeling appeared mostly cytoplasmic, in a diffuse and granular pattern, and cells appeared rounded and smaller. These events were not influenced by lack of serum, or by preincubation with 10 mM sodium azide or 6 mg/ml of cycloheximide. However, the disruption of cell-cell contacts, the cell shape changes, and the redistribution of cingulin and other junctional proteins induced by LC were inhibited when cells were pretreated with the protein kinase inhibitor H-7 (greater than or equal to 30 microM). The inhibitors H-8 and, to a lesser degree, staurosporine were also effective, whereas HA-1004 and ML-7 showed essentially no activity, suggesting a specificity of action of different inhibitors. Measurement of the transepithelial resistance showed that the kinase inhibitors that could prevent junction disassembly could also reduce the drop in transepithelial resistance induced by LC. Dose-response curves demonstrated that H-7 is the most effective among the inhibitors, and the transepithelial resistance was 70% of control up to 1 h after calcium removal. These results suggest that low extracellular calcium modulates junctional integrity and cytoskeletal organization through an effector system involving protein kinases.