Suppression of CIP4/Par6 attenuates TGF-beta 1-induced epithelial-mesenchymal transition in NRK-52E cells

Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) primarily via a Smad-dependent mechanism. However, there are few studies available on TGF-beta-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-beta 1-stimulated NRK-52E cells. Rat NRK-52E cells were obtained and stimulated with TGF-beta 1. The expression levels of E-cadherin, alpha-smooth muscle actin (alpha-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-beta 1 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, alpha-SMA, Par6 and CIP4. The results demonstrated that E-cadherin expression was decreased, and alpha-SMA expression was increased in the TGF-beta 1-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-beta 1. siRNA molecules capable of CIP4 and Par6 knockdown were used to demonstrate reversed TGF-beta 1-induced EMT. Moreover, CIP4 loss of function reversed the increase in p-Par6 protein expression in the TGF-beta 1-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-beta 1-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-beta 1; subsequently, CIP4 increases the phosphorylation of Par6, which accelerates the process of EMT.