Zhongshan Hospital Qingpu Branch, Fudan University, Shanghai 201700, P.R. China.
Int J Mol Med. 2017 Oct;40(4):1165-1171. doi: 10.3892/ijmm.2017.3100. Epub 2017 Aug 16.
Transforming growth factor-β (TGF-β) induces epithelial-mesenchymal transition (EMT) primarily via a Smad‑dependent mechanism. However, there are few studies available on TGF-β-induced EMT through the activation of non‑canonical pathways. In this study, the Cdc42-interacting protein-4 (CIP4)/partitioning-defective protein 6 (Par6) pathway was investigated in TGF-β1‑stimulated NRK-52E cells. Rat NRK-52E cells were obtained and stimulated with TGF-β1. The expression levels of E-cadherin, α-smooth muscle actin (α-SMA) and CIP4 were then examined by western blot analyses. Rat NRK-52E cells were transfected with Par6 or CIP4 small interfering RNA (siRNA), and scrambled siRNA as controls. The cells were incubated with 20 ng/ml of TGF-β1 for 72 h in order to observe the effects of Par6 and CIP4 silencing. Confocal fluorescence microscopy was also applied to reveal the expression and distribution of E-cadherin, α-SMA, Par6 and CIP4. The results demonstrated that E-cadherin expression was decreased, and α-SMA expression was increased in the TGF-β1‑stimulated cells. Simultaneously, the increased expression of CIP4 and p-Par6 was confirmed by western blot analyses. The results of confocal fluorescence microscopy revealed that rat CIP4 exhibited cluster formations located adjacent to the cell periphery; however, as for the protein expression and distribution of Par6, there was no obvious difference between the control cells and cells exposed to TGF-β1. siRNA molecules capable of CIP4 and Par6 knockdown were used to demonstrate reversed TGF-β1‑induced EMT. Moreover, CIP4 loss of function reversed the increase in p-Par6 protein expression in the TGF-β1‑stimulated NRK-52E cells. A similar result was observed with the decreased CIP4 protein expression due to Par6 loss of function. Our data thus suggest that the CIP4/Par6 complex plays an important role in the occurrence of EMT in TGF-β1-stimulated NRK-52E cells. The underlying mechanisms are mediated, at least in part, through the upregulation of CIP4, which occurrs due to stimulation with TGF-β1; subsequently, CIP4 increases the phosphorylation of Par6, which accelerates the process of EMT.
转化生长因子-β（TGF-β）主要通过 Smad 依赖性机制诱导上皮-间充质转化（EMT）。然而，关于 TGF-β 通过非典型途径诱导 EMT 的研究甚少。在本研究中，研究了 TGF-β1 刺激的 NRK-52E 细胞中的 Cdc42 相互作用蛋白 4（CIP4）/分裂缺陷蛋白 6（Par6）途径。获得大鼠 NRK-52E 细胞并用 TGF-β1 刺激。然后通过 Western blot 分析检查 E-钙粘蛋白、α-平滑肌肌动蛋白（α-SMA）和 CIP4 的表达水平。用 Par6 或 CIP4 小干扰 RNA（siRNA）转染大鼠 NRK-52E 细胞，并以对照的 scrambled siRNA 转染作为对照。将细胞在 20ng/ml 的 TGF-β1 中孵育 72h，以观察 Par6 和 CIP4 沉默的影响。还应用共聚焦荧光显微镜揭示 E-钙粘蛋白、α-SMA、Par6 和 CIP4 的表达和分布。结果表明，TGF-β1 刺激的细胞中 E-钙粘蛋白表达降低，α-SMA 表达增加。同时，Western blot 分析证实 CIP4 和 p-Par6 的表达增加。共聚焦荧光显微镜的结果表明，大鼠 CIP4 表现出位于细胞外周附近的簇状形成；然而，对于 Par6 的蛋白表达和分布，对照细胞与暴露于 TGF-β1 的细胞之间没有明显差异。使用能够敲低 CIP4 和 Par6 的 siRNA 分子证明了 TGF-β1 诱导的 EMT 的逆转。此外，CIP4 功能丧失逆转了 TGF-β1 刺激的 NRK-52E 细胞中 p-Par6 蛋白表达的增加。由于 Par6 功能丧失导致 CIP4 蛋白表达减少，观察到类似的结果。因此，我们的数据表明 CIP4/Par6 复合物在 TGF-β1 刺激的 NRK-52E 细胞 EMT 发生中起重要作用。潜在机制至少部分是通过 TGF-β1 刺激引起的 CIP4 上调介导的；随后，CIP4 增加 Par6 的磷酸化，从而加速 EMT 过程。
