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 TGFβ1 is the crucial inducer of EMT. Phosphorylation of Smad proteins is required for TGFβ1-induced EMT. It was reported that C-Jun N-terminal kinase (JNK) was involved in the TGFβ1/Smad signaling pathway and might regulate the activation of Smad proteins. However, whether JNK is activated by TGFβ1 in rat peritoneal mesothelial cells (RPMCs) and the role taken by JNK signaling in EMT induced by TGFβ1 remains undetermined. In the present study, we investigated the role of JNK-Smad pathway in EMT induced by TGFβ1 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 TGFβ1. The protein expression levels of phosphorylated JNK, Smad2, and Smad3 were detected by Western blotting. The messenger RNA levels and protein expression of α-smooth muscle actin (α-SMA), E-cadherin, and collagen I were determined with reverse transcriptase polymerase chain reaction and Western blotting respectively.
⋄ Results
Expression of α-SMA and collagen I were significantly increased and expression of E-cadherin decreased with TGFβ1 in RPMCs. Transforming growth factor β1 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 TGFβ1-induced phosphorylation of Smad3, but not Smad2. Also, our results showed that SP600125 effectively suppressed TGFβ1-induced high expression of α-SMA and collagen I, and prevented TGFβ1-induced downregulation of E-cadherin expression in RPMCs.
⋄ Conclusions
This study demonstrated that JNK signaling may play an important role in EMT induced by TGFβ1 in RPMCs through activation of Smad3, suggesting that JNK inhibitor may prove to be a novel therapeutic agent for peritoneal fibrosis.
GokalR.Peritoneal dialysis in the 21st century: an analysis of current problems and future developments. J Am Soc Nephrol2002; 13(Suppl 1):S104–16.
2.
WilliamsJDCraigKJTopleyNVon RuhlandCFallonMNewmanGR. Morphologic changes in the peritoneal membrane of patients with renal disease. J Am Soc Nephrol2002; 13:470–9.
3.
YangAHChenJYLinJK. Myofibroblastic conversion of mesothelial cells. Kidney Int2003; 63:1530–9.
4.
Yanez–MoMLara–PezziESelgasRRamirez–HuescaMDominguez–JimenezCJimenez–HeffernanJA. Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells. N Engl J Med2003; 348:403–13.
5.
ZavadilJBöttingerEP. TGF-β and epithelial-to-mesenchymal transitions. Oncogene2005; 24:5764–74.
6.
OhKHMargettsPJ. Cytokines and growth factors involved in peritoneal fibrosis of peritoneal dialysis patients. Int J Artif Organs2005; 28:129–34.
7.
MiyazonoK.TGF-beta signaling by Smad proteins. Cytokine Growth Factor Rev2000; 11:15–22.
8.
TahashiYMatsuzakiKDateMYoshidaKFurukawaFSuganoY. Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury. Hepatology2002; 35:49–61.
9.
LanHYMuWTomitaNHuangXRLiJHZhuHJ. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J Am Soc Nephrol2003; 14:1535–48.
10.
ShiYMassagueJ.Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell2003; 113:685–700.
11.
HocevarBABrownTLHowePH. TGF-β induces fibronectin synthesis through a c-Jun N-terminal kinase dependent, Smad4-independent pathway. EMBO J1999; 18:1345–56.
12.
BogoyevitchMABoehmIOakleyAKettermanAJBarrRK. Targeting the JNK MAPK cascade for inhibition: basic science and therapeutic potential. Biochim Biophys Acta2004; 1697:89–101.
13.
DavisRJ. Signal transduction by the JNK group of MAP kinases. Cell2000; 103:239–52.
14.
KyriakisJM. The stress-activated protein kinase subfamily of c-Jun kinase. Nature1994; 369:156–60.
15.
EngelMEMcDonnellMALawBKMosesHL. Interdependent SMAD and JNK signaling in transforming growth factor-beta mediated transcription. J Biol Chem1999; 274:37413–20.
16.
MoriSMatsuzakiKYoshidaKFurukawaFTahashiYYamagataH. TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions. Oncogene2004; 23:7416–29.
17.
YoshidaKMatsuzakiKMoriSTahashiYYamagataHFurukawaF. Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury. Am J Pathol2005; 166:1029–39.
18.
ZhangYFYangXZhangYJSunYLZouXLKongQY. Peroxisome proliferator-activated receptor–gamma is expressed by rat peritoneal mesothelial cells: its potential role in peritoneal cavity local defense. Am J Nephrol2006; 26:602–11.
19.
BottingerEPBitzerM.TGF-β signaling in renal disease. J Am Soc Nephrol2002; 13:2600–10.
20.
JavelaudDMauvielA.Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-β: implications for carcinogenesis. Oncogene2005; 24:5742–50.
21.
FreyRSMulderKM. Involvement of extracellular signal-regulated kinase 2 and stress-activated protein kinase/Jun N-terminal kinase activation by transforming growth factor beta in the negative growth control of breast cancer cells. Cancer Res1997; 57:628–33.
22.
SlussHKBarrettTDerijardBDavisRJ. Signal transduction by tumor necrosis factor mediated by JNK protein kinases. Mol Cell Biol1994; 14:8376–84.
23.
GuptaSBarrettTWhitmarshAJCavanaghJSlussHKDerijardB. Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J1996; 15:2760–70.
24.
AtfiADjelloulSChastreEDavisRGespachC.Evidence for a role of Rho-like GTPases and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in transforming growth factor β–mediated signaling. J Biol Chem1997; 272:1429–32.
25.
DerynckRZhangYE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature2003; 425:577–84.
26.
YamagataHMatsuzakiKMoriSYoshidaKTahashiYFurukawaF. Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH2-terminal kinase during human colorectal carcinogenesis. Cancer Res2005; 65:157–65.
27.
MoustakasASouchelnytskyiSHeldinCH. Smad regulation in TGF-β signal transduction. J Cell Sci2001; 114:4359–69.
28.
DengYRenXYangLLinYWuX.A JNK-dependent pathway is required for TNF alpha-induced apoptosis. Cell2003; 115:61–70.
29.
LiuJLinA.Role of JNK activation in apoptosis: a double-edged sword. Cell Res2005; 15:36–42.
30.
MaFYFlancRSTeschGHHanYAtkinsRCBennettBL. A pathogenic role for c-Jun amino-terminal kinase signaling in renal fibrosis and tubular cell apoptosis. J Am Soc Nephrol2007; 18:472–84.
31.
UtsugiMDobashiKIshizukaTMasubuchiKShimizuYNakazawaT. C-Jun-NH2-terminal kinase mediates expression of connective tissue growth factor induced by transforming growth factor–beta1 in human lung fibroblasts. Am J Respir Cell Mol Biol2003; 28:754–61.
32.
HayashidaTde CasesteckerMSchnaperHW. Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta–dependent responses in human mesangial cells. FASEB J2003; 17:1576–8.
33.
RhyuDYYangYHaHLeeGTSongJSUhST. Role of reactive oxygen species in TGF-β1–induced mitogenactivated protein kinase activation and epithelial–mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol2005; 16:667–75.
34.
FurukawaFMatsuzakiKMoriSTahashiYYoshidaKSuganoY. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology2003; 38:879–89.
