Merlin Regulates Epithelial-To-Mesenchymal Transition Of Arpe-19 Cells Via Tak1-P38mapk-Mediated Activation

PURPOSE. The purpose of this study was to investigate the function of merlin, which is a binding partner of the hyaluronan receptor CD44, during the epithelial-to-mesenchymal transition (EMT) of human retinal pigment epithelium (ARPE-19) cells.METHODS. Human retinal pigment epithelium cells were stimulated with TNF-alpha and treated using epithelial-to-mesenchymal-transition inhibitors (a dynamin inhibitor or transforming growth factor-beta-activated kinase 1 [TAK1] inhibitor). Levels of protein expression were assessed by immunoblot analysis, and localization of the relevant proteins was determined by immunofluorescence microscopy. Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assays. All experiments were performed in serum-free medium.RESULTS. Tumor necrosis factor-alpha treatments downregulated the expression of merlin and led to the dissociation of CD44 and merlin. The ezrin-radixin-moesin (ERM) proteins were phosphorylated, and hyaluronan endocytosis was accelerated in merlin small interfering RNA (siMerlin)-transfected cells. Treatment with the endocytosis inhibitor dynasore blocked hyaluronan endocytosis, whereas treatment with TNF-alpha induced mesenchymal phenotypes and downregulation of merlin. Additionally, siMerlin transfection promoted p38MAPK phosphorylation, which was inhibited not only by TAK1 inhibitor treatment but also by TAK1 small interfering RNA (siRNA, siTAK1) transfection. The increased level of BrdU incorporation in siMerlin cells was reduced by additional siTAK1 transfection. Furthermore, TNF-alpha-induced mesenchymal differentiation and high motility were also inhibited by TAK1 inhibitor treatment and by siTAK1 transfection.CONCLUSIONS. Our findings demonstrated that merlin exerts inhibitory effects on TNF-alpha-induced EMT by regulating hyaluronan endocytosis and the TAK1-p38MAPK signaling pathway. The proliferative and mesenchymal characteristics of RPE cells play important roles in the development of intraocular fibrotic disorders, such as proliferative vitreoretinopathy (PVR), and our findings provide new therapeutic strategies to prevent the development of PVR.