The proinflammatory actions of angiotensin II are mediated through the phosphorylation of p65/Rel A on Ser 536

Objectif The proinflammatory actions of Angiotensin II (Ang II), through the activation of NF‐κB transcription factor, are though to be involved in the development of atherosclerosis. However the signaling pathways leading to the activation of NF‐κB by Ang II needs further investigations. Methods Vascular smooth muscle cells (VSMC) in culture were left untreated or preexposed to pharmacological inhibitors or siRNA duplexes both targeting the IκB kinase (IKK) complex as well as the MEK‐ERK‐RSK cascade. Cells were then stimulated with Ang II. Different biochemical assays were performed to determine the signaling pathways involved in activation of NF‐κB. These assays included: Electromobility shift assays (EMSA), in vitro kinase assays, Western blot and RT‐PCR analysis, and chromatine immunoprecipitation (ChIP) assays. Results The phosphotransferase activity of the IKK complex, the DNA binding activity of NF‐κB and the inductions of the NF‐κB‐related gene IL‐6 were all increased in Ang II‐treated VSMC. Intriguinly no serine 32 phosphorylation nor degradation of IκBα were observed. Instead, we observed that Ang II treatment led to the phosphorylation of p65 on Ser‐536 in an RSK‐independent manner. In addition, we observed a significative recruitment of p65 Ser‐536 to the IL‐6 promoter in Ang II‐treated cells. Conclusion Our data demonstrate that part of the proinflammatory activities of AngII are likely dependent on phosphorylation of p65 on Ser‐536. Since RSK is not involved in this process, we suggest that the IKK complex is responsible of the detected phosphop65 signal in Ang II‐treated VSMC. This study is supported by the IRSC.