Koukouritaki S B, Gravanis A, Stournaras C
Department of Biochemistry, School of Medicine, University of Crete, Heraklion, Greece.
Mol Med. 1999 Nov;5(11):731-42.
We have previously shown that dexamethasone (DEX) stimulates rapid polymerization of actin and stabilization of microfilaments in human endometrial adenocarcinoma cells. As the content of total cellular actin and the concentration of the actin transcript did not change, we concluded that polymerization of actin by glucocorticoids involves nongenomic mechanisms. However, the signaling events by which the latter is achieved remain unknown. In the present study we evaluated whether tyrosine phosphorylation is required for the rapid, nongenomic DEX effect on actin assembly. In cells preincubated with the tyrosine kinase inhibitors, genistein or erbstatin analogue (EA), before adding DEX the G-/total actin ratio remained unchanged, whereas DEX in the absence of both inhibitors reduced the ratio by 25%. In addition, when cells were preincubated with the protein tyrosine phosphatase inhibitor pervanadate and subsequently incubated with DEX, the G-/total actin ratio was dramatically reduced by 65%. Furthermore, DEX increased transiently the levels of tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin within 2 to 15 min, without a change in their expression levels. Pervanadate mimicked this effect of DEX and enhanced tyrosine phosphorylation of both proteins. In addition, when cells were exposed to the anticytoskeletal agent cytochalasin B, the basal levels of tyrosine phosphorylation of both proteins were reduced. This effect was reversed by DEX, indicating that actin cytoskeleton integrity is required for the effect of DEX on tyrosine phosphorylation of FAK and paxillin. Finally, we documented enhanced expression of the Ras-related GTP-binding protein Rho-B after long-term (12- and 24-hr) treatment with DEX, whereas Rho-B levels remained unchanged after short-term (3- and 6-hr) treatment. Our observations demonstrate a novel mechanism through which the rapid nongenomic effect of DEX on actin assembly requires tyrosine phosphorylation of the cytoskeleton-associated proteins FAK and paxillin. We also propose that the DEX-induced actin polymerization may constitute a mechanism for transduction of signals resulting in tyrosine phosphorylation of FAK and paxillin. Moreover, the enhanced Rho-B levels observed after long-term treatment with DEX imply a mechanism for the well-described, long-term effects of glucocorticoids on actin cytoskeleton.
我们之前已经表明，地塞米松（DEX）可刺激人子宫内膜腺癌细胞中肌动蛋白的快速聚合以及微丝的稳定。由于总细胞肌动蛋白含量和肌动蛋白转录本浓度未发生变化，我们得出结论，糖皮质激素诱导的肌动蛋白聚合涉及非基因组机制。然而，实现后者的信号转导事件仍不清楚。在本研究中，我们评估了酪氨酸磷酸化是否是DEX对肌动蛋白组装的快速非基因组效应所必需的。在用酪氨酸激酶抑制剂染料木黄酮或厄波他汀类似物（EA）预孵育的细胞中，在添加DEX之前，G-肌动蛋白/总肌动蛋白比值保持不变，而在没有这两种抑制剂的情况下，DEX可使该比值降低25%。此外，当细胞用蛋白酪氨酸磷酸酶抑制剂过钒酸钠预孵育，随后再与DEX一起孵育时，G-肌动蛋白/总肌动蛋白比值显著降低了65%。此外，DEX在2至15分钟内短暂增加了粘着斑激酶（FAK）和桩蛋白的酪氨酸磷酸化水平，而它们的表达水平没有变化。过钒酸钠模拟了DEX的这种效应，并增强了这两种蛋白的酪氨酸磷酸化。此外，当细胞暴露于抗细胞骨架药物细胞松弛素B时，这两种蛋白的酪氨酸磷酸化基础水平降低。这种效应被DEX逆转，表明肌动蛋白细胞骨架的完整性是DEX对FAK和桩蛋白酪氨酸磷酸化效应所必需的。最后，我们记录到，在用DEX进行长期（12小时和24小时）处理后，Ras相关GTP结合蛋白Rho-B的表达增强，而在短期（3小时和6小时）处理后，Rho-B水平保持不变。我们的观察结果证明了一种新机制，通过该机制，DEX对肌动蛋白组装的快速非基因组效应需要细胞骨架相关蛋白FAK和桩蛋白的酪氨酸磷酸化。我们还提出，DEX诱导的肌动蛋白聚合可能构成一种信号转导机制，导致FAK和桩蛋白的酪氨酸磷酸化。此外，长期用DEX处理后观察到的Rho-B水平升高，意味着糖皮质激素对肌动蛋白细胞骨架的长期效应存在一种机制。
