Study of the level of ERM proteins in pulmonary microvascular endothelial cells induced by tumor necrosis factor-α

Abstract

Objective To investigate the effect of tumor necrosis factor -α (TNF -α) on the levels of ezrin -radixin -moesin (ERM) proteins and the phosphorylated ERM proteins (p -ERM) in rat pulmonary microvascular endothelial cells (PMVEC) , and to explore Rho kinase (ROCK) influencing on modulation of the ERM proteins phosphorylation. Methods Cultured rat pulmonary microvascular endothelial cells were randomly divided into dose -dependent and time -dependent groups. In dose - dependent group, cells were cultured with different doses of TNF -α (0, 0.1 , 1 , 10 μg/LTNF -α) for 60 min. In time -dependent group, cells were cultured with TNF -α (10 μg/L) for 0, 15, 30, 60, 90, 120, 180 min. In ROCK inhibitor (Y27632) intervention group, cells were cultured with TNF -α(10 μg/L) or Y27632 (30 μmol/L) +TNF -α (10 μg/L) for 60 min respectively. The levels of ERMproteins and p -ERM were determined by western blot. One way analysis of variance (ANOVA) was employed for statistical analysis by using SPSS version 16.0 software to compare values among all groups. A significant difference was presumed as aP value <0.05. Results Western blot revealed that ERM and p-ERM proteins were present in rat PMVEC. Stimulation withTNF-α gradually up -regulated the level of p-ERM proteins in a dose -dependent manner [0 μg/LTNF-α group: (0.648 ± 0.102) , 0.1 μg/LTNF-α group: (0.728 ±0.082) , 1 μg/LTNF-α group: (0.926 ±0.121) , 10 μg/LTNF-α group: (1.245 ± 0.134) , allP =0.000 ]. In time -dependent group, the level of p -ERM proteins rose at 15 min (0.777 ± 0.151) , peaked at 90 min (1.295 ± 0.176) , then decreased gradually at 120 min (0.802 ± 0.139) , but stayed higher level at 180 min (0.669 ± 0.128) than that in un -stimulated 0 min group (0.631 ±0.123 , P =0.004 , 0.000 , 0.001, 0.016 , respectively). When PMVEC pre -incubated with ROCK inhibitor and TNF -α, the level of p -ERM proteins caused a marked attenuation of TNF-α stimulation [(0.634 ±0.112) vs. (0.875 ±0.164) , P =0.002 ] , however, there are no significant differences compared to ROCK inhibitor alone group (0.661 ±0.108) and no intervention group (0.654 ± 0.125) , P =0.973 , P =0.900 , respectively). Conclusions TNF-α could induce up -regulation of the level of the phosphorylated ERM proteins in rat PMVEC, and ROCK signal molecules might involve in modulation of the ERM proteins phosphorylation. Key words: TNF-α; Pulmonary microvascular endothelial cell; Ezrin-Radixin-Moesin; Inflammation; Acute respiratory distress syndrome; Rho kinase