HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells.
HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells.
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DOI：
10.1152/ajpcell.1999.277.6.c1032
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发表时间：
1999-12
期刊：
影响因子：
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通讯作者：
C. Schäfer;P. Clapp;M. Welsh;R. Benndorf;John A. Williams
中科院分区：
文献类型：
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作者：
C. Schäfer;P. Clapp;M. Welsh;R. Benndorf;John A. Williams
我们研究了热休克蛋白27（HSP 27）及其磷酸化参与胆囊收缩素（CCK）对肌动蛋白细胞骨架的作用，通过遗传操作的中国仓鼠卵巢（CHO）细胞稳定转染CCK-A受体。在这些细胞中，在大鼠腺泡，CCK激活p38丝裂原活化蛋白（MAP）激酶，并增加HSP 27的磷酸化。这种作用可以用p38 MAP激酶抑制剂SB-203580阻断。共聚焦显微镜检查与罗丹明鬼笔环肽染色的细胞表明，CCK剂量依赖性地诱导肌动蛋白细胞骨架的变化，包括细胞形状的变化，这是一致的肌动蛋白细胞骨架断裂和肌动蛋白丝补丁的形成在细胞中。为了进一步评估HSP 27的作用，用野生型（wt）或突变型（3A、3G和3D）人HSP 27的表达载体转染CHO-CCK-A细胞。wt-HSP 27和3D-HSP 27的过表达抑制了高剂量CCK刺激后对肌动蛋白细胞骨架的影响。与此相反，过度表达nonphosphorylatable突变体，3A-和3G-HSP 27，或抑制HSP 27的磷酸化的wt-HSP 27转染细胞与SB-203580预孵育没有保护肌动蛋白细胞骨架。这些结果表明，HSP 27的磷酸化是稳定肌动蛋白细胞骨架和保护细胞免受高浓度CCK影响所必需的。
We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.