Wojciechowski Magdalena C, Shu Daisy Y, Lovicu Frank J
a Discipline of Anatomy and Histology , Bosch Institute, University of Sydney , Sydney , Australia.
b Save Sight Institute , University of Sydney , Sydney , Australia.
Curr Eye Res. 2018 Aug;43(8):986-997. doi: 10.1080/02713683.2018.1464193. Epub 2018 Apr 23.
This study aims to highlight some of the genes that are differentially regulated by ERK1/2 signaling in TGFβ-induced EMT in lens, and their potential contribution to this pathological process.
Rat lens epithelial explants were cultured with or without TGFβ over a 3-day-culture period to induce EMT, in the presence or absence of UO126 (ERK1/2 signaling inhibitor), both prior to TGFβ-treatment, or 24 or 48 hours after TGFβ treatment. Smad2/3-nuclear immunolabeling was used to indicate active TGFβ signaling, and quantitative RT-PCR was used to analyze changes in the different treatment groups in expression of the following representative genes: TGFβ signaling (Smad7, Smurf1, and Rnf111), epithelial markers (Pax6, Cdh1, Zeb1, and Zeb2), cell survival/death regulators (Bcl2, Bax, and Bad) and lens mesenchymal markers (Mmp9, Fn1, and Col1a1), over the 3 days of culture.
ERK1/2 was found to regulate the expression of Smurf1, Smad7, Rnf11, Cdh1, Pax6, Zeb1, Bcl2, Bax, and Bad genes in lens cells. TGFβ signaling was evident by nuclear localization of Smad2/3 and this was effectively blocked by pre-treatment with UO126, but not by post-treatment with this ERK1/2 signaling inhibitor. TGFβ induced the expression of its signaling partners (Smad7, Smurf1, and Rnf111), as well as lens mesenchymal genes (Mmp9, Fn1, and Col1a1), consistent with its role in inducing an EMT. These TGFβ-responsive signaling genes, as well as the mesenchymal markers, were all positively regulated by ERK1/2-activity. The expression levels of the lens epithelial genes we examined, and genes that were associated with cell death/survival, were not directly impacted by TGFβ.
TGFβ-mediated ERK1/2 signaling positively modulates the expression of mesenchymal genes in lens epithelial explants undergoing EMT, in addition to regulating TGFβ-mediated regulatory genes. Independent of TGFβ, ERK1/2 activity can also regulate the expression of endogenous lens epithelial genes, highlighting its potential key role in regulation of both normal and pathological lens cellular processes.
本研究旨在突出一些在转化生长因子β（TGFβ）诱导的晶状体上皮-间质转化（EMT）过程中受细胞外信号调节激酶1/2（ERK1/2）信号通路差异调节的基因，以及它们对这一病理过程的潜在作用。
将大鼠晶状体上皮外植体在有或无TGFβ的条件下培养3天以诱导EMT，同时在TGFβ处理前、处理后24小时或48小时，分别在有或无UO126（ERK1/2信号通路抑制剂）的情况下进行培养。采用Smad2/3核免疫标记来指示活跃的TGFβ信号通路，并使用定量逆转录聚合酶链反应（RT-PCR）分析不同处理组中以下代表性基因表达的变化：TGFβ信号通路相关基因（Smad7、Smurf1和Rnf111）、上皮标志物（Pax6、Cdh1、Zeb1和Zeb2）、细胞存活/死亡调节因子（Bcl2、Bax和Bad）以及晶状体间充质标志物（Mmp9、Fn1和Col1a1），培养时间为3天。
发现ERK1/2可调节晶状体细胞中Smurf1、Smad7、Rnf11、Cdh1、Pax6、Zeb1、Bcl2、Bax和Bad基因的表达。Smad2/3的核定位表明TGFβ信号通路激活，而在TGFβ处理前用UO126预处理可有效阻断该信号通路，但在TGFβ处理后用这种ERK1/2信号通路抑制剂处理则无效。TGFβ诱导其信号通路相关基因（Smad7、Smurf1和Rnf111）以及晶状体间充质基因（Mmp9、Fn1和Col1a1）的表达，这与其诱导EMT的作用一致。这些对TGFβ有反应的信号通路基因以及间充质标志物均受ERK1/2活性的正向调节。我们检测的晶状体上皮基因以及与细胞死亡/存活相关的基因的表达水平不受TGFβ的直接影响。
TGFβ介导的ERK1/2信号通路除了调节TGFβ介导的调节基因外，还正向调节正在经历EMT的晶状体上皮外植体中间充质基因的表达。独立于TGFβ，ERK1/2活性也可调节内源性晶状体上皮基因的表达，突出了其在调节正常和病理性晶状体细胞过程中的潜在关键作用。
