Caveolin-1 Regulates Rac1 Activation and Rat Pulmonary Microvascular Endothelial Hyperpermeability Induced by TNF-α
Figure 7
Down-regulation of caveolin-1 expression enhanced Rac1 activity, which was abolished by NSC-23766.
A.Primary PMVECs transfected with control or caveolin-1 shRNA were challenged with TNF-α(100 ng/ml) for 2 hours. Rac1 activity in the cell lysates was evaluated using GTPase pull-down assays and normalized to the total GTPase content in cell lysates. Caveolin-1 deficient cells showed a significant increase in GTP-bound Rac1 compared with control shRNA cells in the resting state (2.5±0.2-fold increase, P>0.001) and after TNF-α stimulation (2.7±0.4-fold increase, P>0.01).B. Measurement of Rac1 activity by GTPase pulldown assays. Down-regulation of caveolin-1 expression resulted in increased activity of Rac1 to 250±20% of controls shRNA. Incubation with NSC-23766(200 mM, 30 min) abolished this effect. Co-treated with NSC-23766 and TNF-α, the activity of Rac1 decreased more than that treated with TNF-α alone. Each data point represents the mean±SD. derived from six independent experiments, *P<0.05, **P<0.01, ***P<0.001.