Nitric oxide enhances keratinocyte cell migration by regulating Rho GTPase via cGMP-PKG signalling.
Nitric oxide enhances keratinocyte cell migration by regulating Rho GTPase via cGMP-PKG signalling.
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一氧化氮通过 cGMP-PKG 信号调节 Rho GTPase 来增强角质形成细胞的迁移。
DOI：
10.1371/journal.pone.0121551
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Luo G
中科院分区：
文献类型：
--
作者：
Zhan R;Yang S;He W;Wang F;Tan J;Zhou J;Yang S;Yao Z;Wu J;Luo G
一氧化氮（NO）已被证明可以改善伤口愈合，但这种功能的机制还没有很好地定义。本文探讨了NO对人角质形成细胞系HaCaT迁移的影响及其可能的机制。在细胞迁移试验中评估了在不同浓度的NO供体硝普钠（SNP）存在下NO对HaCaT细胞的影响。随后，用共聚焦激光扫描显微镜观察用罗丹明-鬼笔环肽染色的培养HaCaT细胞的细胞骨架重组。采用RT-PCR和pull-down法分别检测CDC 42、Rac 1和RhoA的mRNA表达和活性蛋白。此外，还观察了cGMP、PKG和CDC 42、Rac 1、RhoA的特异性抑制剂或激动剂在NO对HaCaT细胞迁移、F-actin应力纤维形成和Rho GT3表达的影响中的作用。还发现SNP以剂量依赖性方式增加HaCaT细胞迁移，而另外两种NO供体精胺NONOate或SNAP对HaCaT细胞迁移的影响几乎相同。SNP处理的HaCaT细胞中F-actin应力纤维的形成增加。SNP处理后，CDC 42、Rac 1和RhoA的mRNA表达和活性蛋白表达上调。在用cGMP或PKG激动剂处理细胞后观察到类似的效果。此外，SNP介导的mRNA表达和活性蛋白的CDC 42，Rac 1和RhoA的上调被cGMP或PKG的抑制剂抑制。此外，SNP介导的促进迁移和细胞骨架重组的作用被cGMP，PKG，CDC 42，Rac 1和RhoA的抑制剂分别抑制。我们的数据表明，NO对HaCaT细胞的细胞迁移的刺激作用是通过cGMP信号通路通过上调Rho-GTdR的表达，这可能会促进细胞骨架重组介导的。
Nitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism. The effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed. It was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively. Our data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization.
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