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.
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.
Authors: Torry A Tucker; LaTerrica Williams; Kathleen Koenig; Hema Kothari; Andrey A Komissarov; Galina Florova; Andrew P Mazar; Timothy C Allen; Khalil Bdeir; L Vijaya Mohan Rao; Steven Idell Journal: Am J Respir Cell Mol Biol Date: 2012-02 Impact factor: 6.914