P622The transcriptional co-regulator RIP140 regulates endothelial inflammation by controlling the expression of A20 and repressing NFkB activation.

Cardiovascular Research

PubMedID: 25020348

Calay D, Dufton N, Christian M, Parker M, Haskard DO, Mason JC. P622The transcriptional co-regulator RIP140 regulates endothelial inflammation by controlling the expression of A20 and repressing NFkB activation. Cardiovasc Res. 2014;103 Suppl 1S112-3.
PURPOSE
Chronic inflammation is a central feature of many human diseases, including atherosclerosis. The transcriptional co-regulator RIP140 (Receptor-interacting protein of 140 kDa) is a co-repressor for nuclear receptors and regulates lipid and glucose metabolism in adipocytes and skeletal muscle. RIP140 may also act as a co-activator for NF?B, driving pro-inflammatory responses in murine macrophages. The aim of this study is to elucidate the regulation and the role of RIP140 in the inflammatory response triggered by tumour necrosis factor a (TNFa) in endothelial cells (EC).

METHODS AND RESULTS
Our data demonstrate that RIP140 is up-regulated at the transcriptional and translational level by TNFa in human umbilical vein EC (HUVEC) (p<0.01). In silico analysis revealed two NF?B binding sites within the RIP140 promoter. NF?B inhibition with BAY11-7085, or by over-expression of the I?Ba super-repressor protein, abolished RIP140 induction by TNFa (p<0.01). Functional experiments demonstrated that TNFa-induced expression of the NF?B target gene E-selectin is prolonged in RIP140-deficient HUVEC, while TNFa upregulation of E-selectin is reduced in RIP140-overexpressing cells (p<0.05). Transfection of an E-selectin luciferase reporter confirmed these observations. Co-immunoprecipitation revealed that, upon TNFa treatment, RIP140 associates with the p65 subunit of NF?B, while interaction with the transcriptional repressors histone de-acetylases is reduced, suggesting a co-activator function for RIP140. Further siRNA experiments showed that RIP140 positively regulates expression of A20, a negative regulator of the NF?B pathway. Chromatin immunoprecipitation studies will now investigate the binding of RIP140 and p65 on the A20 promoter. Consistent with the data obtained under static conditions, the expression of E-selectin was higher in RIP140-depleted HUVEC exposed to TNFa under laminar or oscillatory flow, compared to control transfected cells (p<0.05). Finally, using an air pouch model of inflammation, we showed that the inflammatory response to TNFa was significantly attenuated in RIP140 transgenic mice, compared to wild-type littermates (p<0.01).

CONCLUSION
This study demonstrates for the first time a role for RIP140 in vascular EC, where it is induced by TNFa and plays an important role in the control of inflammation, acting as a negative regulator of NF?B, through its effect on the A20 deubiquitinase. Our data broaden the understanding of the mechanisms involved in inflammation and may pave the way for new therapeutic approaches in the treatment of chronic inflammatory vascular diseases.