ABSTRACT
Objective To investigate the role of C-Jun N-terminal kinase (JNK) in epithelial mesenchymal transition (EMT) induced by transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells(RPMCs). Methods RPMCs were harvested from the peritoneum of male Sprague-Dawley rats, then cultured in DMEM/F12 medium with 15% (V/V) FBS. After stimulation with TGF-β1, the expression of a-smooth muscle actin (α-SMA), E-cadherin and collagen I were detected in RPMCs. In some groups, the ceils were pretreated with SP600125, a specific inhibitor of JNK, for 4 hours before incubation with TGF-β1. The protein expression of phosphorylated JNK was detected by Western blotting. The mRNA and protein expression ofα-SMA, E-cadherin and collagen I were examined with RT-PCR and Western blotting, respectively.The intracellular distribution and expression of α-SMA was determined by indirect immunofluorescence. Results TGF-β1 could significantly increase the expression of α-SMA and collagen I, and decrease the expression of E-cadherin in RPMCs. TGF-α1 could stimulate the expression of phosphorylated JNK at 5 minutes with the peak at 10 minutes (P<0.01). The addition of SP600125 effectively inhibited TGF-β1-induced high expression of α-SMA and collagen I (P<0.05), and prevented TGF-β1-induced down-regulation of E-cadherin expression in RPMCs (P<0.05). The indirect immunofluorescence showed that the expression of intracellular α-SMA in RPMCs stimulated by TGF-β1 for 48 h increased significantly, which could be inhibited by SP600125. Conclusions JNK regulates epithelial mesenchymal transition induced by TGF-β1 in rat peritoneal mesothelial cells. JNK inhibitor may be used as a novel therapeutic agent for peritoneal fibrosis.
ABSTRACT
AIM: To investigate the expression of osteopontin (OPN) and CD44 in human renal proximal tubular epithelial cells stimulated by human serum albumin (HSA). METHODS: Proximal tubular epithelial HK-2 cells were stimulated by HSA at different concentrations for different time, then OPN mRNA production was detected by RT-PCR, and OPN protein was detected by Western blotting. The expression of OPN and CD44 in HK-2 cells after stimulation for 24 h or 48 h were detected by immunofluorescence with confocal laser scanning microscope. RESULTS: Osteopontin mRNA in HK-2 cells showed a highest expression at 3 h and 48 h after HSA stimulation. However, the expression of OPN protein in HK-2 cells reached the maximum at 24 h after HSA stimulation. OPN mRNA and protein showed a strong dose-dependence relation with the concentration of HSA. HSA also stimulated HK-2 cells to express CD44 protein, the fluorescence of CD44 was most prominent at 48 h after HSA stimulation. CONCLUSION: HSA stimulates human renal proximal tubular epithelial cells to express OPN and CD44.