Role Of Ezrin In The Injury Of Rat Pulmonary Microvascular Endothelial Cells Induced By TNF-a And The Impact Of Rac1

Background and ObjectiveEzrin, radixin, and moesin are widely expressed proteins known as ERMs that link the actin cytoskeleton to membrane and membrane-associated proteins.As a member of the family, Ezrin are localized at the cell cortex and have the ability to cross-link proteins of the plasma membrane with the sub-cortical cytoskeleton via the characteristic FERM domain and were shown to be important for the regulation of cell morphology, growth, survival, adhesion, proliferation and migration through several signal transduction pathways. Studies showed that the expression of p-Ezrin was significantly reduced and the increased permeability of HPMVEC was significantly inhibited by small interfering RNA. But in the process of pneumonia injury, which signaling pathways has Ezrin taken part in is unclear. The aim of this study to investigate the role of Ezrin in the modulation of rat pulmonary microvascular endothelial cells(RPMVEC) injury induced by TNF-α and the impact of Rac1.Methods1.RPMVECs were successfully isolated and cultured in vitro.2.Western-blotting technique was used to determine the Ezrin and p-Ezrin expression in RPMVECs induced by TNF-α at different time points.3.Western-blotting technique was used to determine the Ezrin and p-Ezrin expression in RPMVECs stimulated by Rac1 inhibitor NSC23766 at different time points.Results1. RPMVECs were isolated and cultured successfully in vitro,and were confirmed by morphology and fluorescein isothiocyanate-banderiraea simplicifolia I isolectin B4(FITC-BSI)integration experiment.2. Few Ezrin expression was found in RPMVECs. Treatment with 10 ug/l TNF-α induced a significant increase in Ezrin phosphorylation in a time-dependent manner:At 0h, 0.25 h, 0.5h, 1h, 3h, 6h, 12 h, 24 h, the relative expression levels of p-Ezrin were(0.21 ± 0.33),(0.53 ± 0.19),(0.95 ±0.24),(1.07±0.30),(1.68±0.30),(1.32±0.37),(0.93±0.20),(0.87±0.18),elevating at 0.25h(0.53 ± 0.19), peaking at 3 hours(1.68±0.30), then it lowered gradually, but it was still higher at 24 hours(0.87±0.18) than 0 hour group(0.21 ± 0.33). This occurred without detectable changes in the protein expression of Ezrin(P<0.001).3. Compared with blank control group(0.68 ± 0.16), in single NSC 23766(1.33 ± 0.24) or TNF-α(0.92 ± 0.12) simulation group,p-Ezrin expression was induced. When NSC 23766 was pre-treated with PMVECs, the expression of p-Ezrin(2.14 ± 0.18)was significantly increased compared with that in single TNF-α simulation group(P<0.001).Conclusion1. Few Ezrin expression was found in PMVECs, and TNF-α could not affect Ezrin expression.2. TNF-α could increase Ezrin phosphorylation in a time-dependent manner.3. Rac 1 signaling pathway inhibition plays an important role in TNF-α-induced injury by up-regulation of p-Ezrin in PMVECs.