Purchase this article with an account.
Eri Takahashi, Akira Haga, Hidenobu Tanihara; Merlin regulates hyaluronan endocytosis and epithelial mesenchymal transition in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4239.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To study the function of merlin, neurofibromatosis 2 coded protein, in the process of epithelial mesenchymal transition (EMT) in human RPE (ARPE-19) cells.
ARPE-19 cells were stimulated with tumor necrosis factor-α (TNF-α) and dynamin inhibitor, dynasore. Cells were transfected with specific two merlin siRNAs (siMerlin #1, 2) to knockdown of endogenous merlin expression and used control siRNA (siCTL). Expression levels of proteins were assessed by western blot analysis. We used Fluorescein-conjugated hyaluronan (Fl-HA) to assess hyaluronan endocytosis and internalized Fl-HA was evaluated by a fluorescence microscopy and a BZ-X Analyzer software. All experiments were performed in serum-free medium.
Western blot analysis showed that TNF-α induced merlin downexpression and immunoprecipitation revealed that merlin/CD44 interaction decreased in the presence of TNF-α. We also found that phosphorylated form of ERM proteins (other CD44 binding partners) was increased in siMerlin-transfected cells. Fl-HA endocytosis was increased in siMerlin-transfected cells (siMerlin #1: p=0.02, #2: p=0.006 vs. siCTL) and internalized Fl-HA positive vesicles were co-localized with clathrin heavy chain. Clathrin-dynamin endocytosis inhibitor, dynasore treatment blocked Fl-HA endocytosis in siMerlin-transfected cells (p<0.001 vs. siMerlin). In addition, treatment with dynasore inhibited TNF-α-induced EMT such as morphological change (spindle shape), upexpression of mesenchymal proteins (fibronectin and α-smooth muscle actin) and merlin downregulation. Further, dynasore treatment inhibited TNF-α-promoted phosphorylation of p38 mitogen-activated protein kinase.
Our findings demonstrated that merlin had inhibitory effects on TNF-α-induced EMT through regulation of hyaluronan endocytosis. Proliferative mesenchymal phenotypes in RPE cells play important roles in formation of intraocular fibrosis such as proliferative vitreoretinopathy (PVR) and our findings provide new therapeutic strategies to prevent PVR.
This PDF is available to Subscribers Only