Critical Role of Non-Muscle Myosin Light Chain Kinase in Thrombin-Induced Endothelial Cell Inflammation and Lung PMN Infiltration
Figure 2
Role of nmMLCK in thrombin-induced NF-κB activation.
(A) Effect of RNAi knockdown of nmMLCK on thrombin-induced NF-κB transcriptional activity. EC were transfected with control or nmMLCK siRNA using DharmaFect1. Twenty-four hours later, cells were again transfected with NF-κBLUC construct using DEAE-Dextran as described in Materials and Methods. Cells were challenged with thrombin (5 U/ml) for 6 h. Cell extracts were prepared and assayed for Firefly (F) and Renilla (R) luciferase activities. Data are means±S.E. (n = 4 for each condition). ***, p<0.001 compared with untreated controls; ###, p<0.001 compared with thrombin-stimulated controls. (B) Effect of inhibition of nmMLCK on thrombin-induced IκBα degradation. Confluent EC monolayers were pretreated with ML-7 (10 µM) and then challenged with thrombin (5 U/ml) for 1 h. Total cell lysates were immunoblotted with an anti-IκBαantibody. Actin levels were used to monitor loading. The bar graph represents the effect of inhibition of nmMLCK on thrombin-induced IκBα degradation normalized to actin level. Data are means±S.E. (n = 3–4 for each condition). *, p<0.05 compared with untreated control or ML-7-treated control. (C) Effect of nmMLCK knockdown on thrombin-induced IκBα phosphorylation and degradation. EC were transfected with control or nmMLCK siRNA using DharmaFect1. After 24–36 h, cells were challenged for 1 h with thrombin (5 U/ml). Total cell lysates were prepared and immunoblotted with an anti-phospho IκBα (Ser32 and Ser36) or anti-IκBαantibody. RelA/p65 levels were used to monitor loading. The blot is representative of 2 separate experiments. (D) Effect of inhibition of nmMLCK on thrombin-induced DNA binding of RelA/p65. EC were pretreated with ML-7 (10 mM) prior to challenge with thrombin for 1 h. Nuclear extracts were prepared and assayed for RelA/p65 DNA binding activity by ELISA as described in Materials and Methods. Data are means±S.E. (n = 3 for each condition). **, p<0.01 compared with untreated control; #, p<0.05 compared with thrombin-stimulated control. (E) Effect of nmMLCK knockdown on thrombin-induced DNA binding of RelA/p65. EC were transfected with control or nmMLCK siRNA for 24–36 h as described in Materials and Methods. Cells were challenged with thrombin (5 U/ml) for 1 h. Nuclear extracts were prepared and assayed for RelA/p65 DNA binding activity by EMSA as described in Materials and Methods. Representative of two experiments.