5599

MAPK8

Transmembrane 4L six family member 5 (TM4SF5) can regulate cell-cell adhesion and cellular morphology via cytoplasmic p27(Kip1)-mediated changes in RhoA activity. However, how TM4SF5 causes cytosolic p27(Kip1) stabilization remains unknown. In this study we found that TM4SF5-mediated Ser10 phosphorylation of p27(Kip1) required for cytosolic localization was not always correlated with Akt activity. Inhibition or suppression of c-Jun N-terminal kinase (JNK) in TM4SF5-expressing cells decreased Ser10 phosphorylation of p27(Kip1) and rescued expression levels and localization of adherence junction molecules to cell-cell contacts. These observations suggest involvement of JNKs in TM4SF5-mediated p27(Kip1) Ser10 phosphorylation and localization during epithelial-mesenchymal transition.CI - Copyright (c) 2011 Elsevier Ireland Ltd. All rights reserved.

Transforming growth factor (TGF)-beta1 is a key mediator of lung remodeling and fibrosis. epithelial cells are both a source of and can respond to TGF-beta1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-beta1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-beta1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-beta1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-beta1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-beta1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-beta1-induced EMT.

BACKGROUND: Peritoneal fibrosis is a serious complication in long-term peritoneal dialysis (PD) patients. epithelial-mesenchymal transition (EMT) plays an important role in peritoneal fibrosis, and TGFbeta1 is the crucial inducer of EMT. Phosphorylation of Smad proteins is required for TGFbeta1-induced EMT. It was reported that C-Jun N-terminal kinase (JNK) was involved in the TGFbeta1/Smad signaling pathway and might regulate the activation of Smad proteins. However, whether JNK is activated by TGFbeta1 in rat peritoneal mesothelial cells (RPMCs) and the role taken by JNK signaling in EMT induced by TGFbeta1 remains undetermined. In the present study, we investigated the role of JNK-Smad pathway in EMT induced by TGFbeta1 in RPMCs. METHODS: We harvested RPMCs from the peritoneum of male Sprague-Dawley rats and then cultured the cells in Dulbecco modified Eagle medium/F12 medium with 15% (volume:volume) fetal bovine serum. The cells were pretreated with SP600125, a specific inhibitor of JNK, for 4 hours before incubation with TGFbeta1. The protein expression levels of phosphorylated JNK, Smad2, and Smad3 were detected by Western blotting. The messenger RNA levels and protein expression of alpha-smooth muscle actin (alpha-SMA), E-cadherin, and collagen I were determined with reverse transcriptase polymerase chain reaction and Western blotting respectively. RESULTS: expression of alpha-SMA and collagen I were significantly increased and expression of E-cadherin decreased with TGFbeta1 in RPMCs. Transforming growth factor beta1 can stimulate phosphorylated JNK expression from 5 minutes, with the peak at 10 minutes, and phosphorylated Smad2 and Smad3 expression from 10 minutes, with the peak at 30 minutes. The addition of SP600125, which blocked activation of JNK, effectively inhibited TGFbeta1-induced phosphorylation of Smad3, but not Smad2. Also, our results showed that SP600125 effectively suppressed TGFbeta1-induced high expression of alpha-SMA and collagen I, and prevented TGFbeta1-induced downregulation of E-cadherin expression in RPMCs. CONCLUSIONS: This study demonstrated that JNK signaling may play an important role in EMT induced by TGFbeta1 in RPMCs through activation of Smad3, suggesting that JNK inhibitor may prove to be a novel therapeutic agent for peritoneal fibrosis.