As shown in Figures 3A and B, pretreatment with the inhibitor of c Src reduced LPS induced 17-AAG 75747-14-7 VCAM 1 protein and mRNA e pression and promoter activity. In addition, transfection with c Src siRNA also inhibited LPS induced VCAM 1 e pression. LPS could stimulate c Src phos phorylation, which was inhibited by pretreatment with PP1. c Src has been shown to regulate ROS generation in human tracheal smooth muscle cells. Moreover, we also found that LPS induced p47pho trans location, NADPH o idase activation, and ROS generation were inhibited by transfection with c Src siRNA. We further investigated the physical association of TLR4, c Src, and p47pho in LPS induced ROS generation and VCAM 1 e pression. As shown in Figure 3G, the protein levels of TLR4 and p47pho were time dependently increased in c Src immunoprecipitated comple in LPS treated HRMCs.

Thus, these data sug gested that LPS induced VCAM 1 e pression is mediated through c Src dependent NADPH o idase ROS generation in HRMCs. LPS induces VCAM 1 e pression via NADPH o idase ROS dependent p38 MAPK activation in HRMCs MAPKs, including p38 MAPK, JNK1 2, and p42 p44 MAPK have been shown to regulate VCAM 1 induction in various cell types. Here, we determined whether these three MAPKs were involved in LPS induced VCAM 1 e pression in HRMCs. As shown in Figures 4A and B, pretreatment with the inhibitor of p38 MAPK, JNK1 2, or MEK1 2 markedly inhib ited LPS induced VCAM 1 protein and mRNA e pression and promoter activity in HRMCs. It has been shown that ROS dependent activation of MAPKs is required for in flammatory responses.

In HRMCs, LPS stimulated p38 MAPK phosphorylation was inhibited by transfection with either c Src siRNA or p47pho siRNA. However, pretreatment with PP1, but not edaravone inhib ited LPS induced p42 p44 MAPK and JNK1 2 phosphoryl ation. Finally, the involvement of p38 MAPK in LPS induced VCAM 1 e pression was further confirmed by transfection with p38 MAPK siRNA. As shown in Figure 4F, transfection with p38 siRNA reduced the e pression of Batimastat total p38 MAPK protein and subsequently attenuated VCAM 1 e pression induced by LPS. These results indicated that p38 MAPK phosphorylation involved in VCAM 1 induction by LPS was mediated through a c Src NADPH o idase ROS dependent cascade in HRMCs. LPS induces VCAM 1 e pression via p38 MAPK dependent ATF2 activation ATF2 is activated by inflammatory signals transduced by the p38 MAPK pathway.

In addition, LPS has also inhibitor order us been shown to regulate VCAM 1 e pression via an ATF2 signaling. In this study, we investigated whether ATF2 activation was involved in LPS induced VCAM 1 e pression in HRMCs. As shown in Figures 5A, B and C, transfection with ATF2 siRNA inhibited LPS induced VCAM 1 protein and mRNA e pression and promoter activity in HRMCs.