Down-regulation of 14-3-3 Zeta Inhibits TGF-β1-Induced Actomyosin Contraction in Human Trabecular Meshwork Cells Through RhoA Signaling Pathway.
Down-regulation of 14-3-3 Zeta Inhibits TGF-β1-Induced Actomyosin Contraction in Human Trabecular Meshwork Cells Through RhoA Signaling Pathway.
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DOI：
10.1167/iovs.15-17438
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发表时间：
2016-02
影响因子：
4.4
通讯作者：
Yiming Ye;Yangfan Yang;Xiaoxiao Cai;Liling Liu;Kaili Wu;Minbin Yu
中科院分区：
文献类型：
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作者：
Yiming Ye;Yangfan Yang;Xiaoxiao Cai;Liling Liu;Kaili Wu;Minbin Yu
目的本研究的目的是描述 14-3-3 zeta 在小梁网 (TM) 细胞中的表达和分布及其在肌动球蛋白系统中的调节作用。方法采用Western blot分析、RT-PCR和免疫荧光染色检测14-3-3 zeta的表达。 TGF-β1 用于诱导细胞收缩。使用蛋白质印迹分析测定 14-3-3 zeta、总 RhoA 水平以及肌球蛋白轻链 (MLC) 和丝切蛋白磷酸化的变化。使用免疫荧光测定 14-3-3 zeta 敲低对肌动蛋白细胞骨架和粘着斑的影响。使用定量 RT-PCR 检查纤连蛋白以及胶原蛋白 I 和 III 的 mRNA 水平。使用胶原凝胶收缩（CGC）测定法检测TM细胞的收缩。使用特定试剂盒分析 RhoA 途径的激活。结果 14-3-3 zeta 蛋白在 TM 细胞中高表达。 14-3-3 zeta 的下调会导致以下结果：MLC 和 cofilin 磷酸化减少、应力纤维和粘着斑形成减少、细胞外基质 (ECM) mRNA 组成改变以及 TGF-β1 诱导的细胞收缩受到抑制。此外，14-3-3 zeta 的沉默直接降低了 TM 细胞中的总 RhoA 水平。结论 总的来说，我们的数据表明 14-3-3 zeta 通过 RhoA 信号通路发挥作用，在调节 TM 细胞的细胞骨架结构、ECM 稳态和 TGF-β1 诱导的收缩中发挥着至关重要的作用。
PURPOSE The aim of this study was to describe the expression and distribution of 14-3-3 zeta in trabecular meshwork (TM) cells and its regulatory role in the actomyosin system. METHODS The expression of 14-3-3 zeta was detected using Western blot analysis, RT-PCR, and immunofluorescence staining. TGF-β1 was used to induce cell contraction. Changes in the levels of 14-3-3 zeta, total RhoA, and the phosphorylation of myosin light chain (MLC) and cofilin were determined using Western blot analysis. The effects of 14-3-3 zeta knockdown on the actin cytoskeleton and focal adhesion were determined using immunofluorescence. The mRNA levels of fibronectin and collagen I and III were examined using quantitative RT-PCR. The contraction of TM cells was detected using collagen gel contraction (CGC) assays. The activation of the RhoA pathway was analyzed using a specific kit. RESULTS The 14-3-3 zeta protein was highly expressed in TM cells. Down-regulation of 14-3-3 zeta resulted in the following: a decrease in the phosphorylation of both MLC and cofilin, a decrease in the formation of stress fibers and focal adhesion, alteration of the mRNA composition of the extracellular matrix (ECM), and the inhibition of TGF-β1-induced cell contraction. In addition, silencing of 14-3-3 zeta directly decreased total RhoA levels in TM cells. CONCLUSIONS Collectively, our data suggest that 14-3-3 zeta plays a crucial role in regulating cytoskeletal structures, ECM homeostasis, and TGF-β1-induced contraction in TM cells by acting through the RhoA signaling pathway.