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

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Abstract

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 NFκB, 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 α (TNFα) in endothelial cells (EC).

Methods and results: Our data demonstrate that RIP140 is up-regulated at the transcriptional and translational level by TNFα in human umbilical vein EC (HUVEC) (p<0.01). In silico analysis revealed two NFκB binding sites within the RIP140 promoter. NFκB inhibition with BAY11-7085, or by over-expression of the IκBα super-repressor protein, abolished RIP140 induction by TNFα (p<0.01). Functional experiments demonstrated that TNFα-induced expression of the NFκB target gene E-selectin is prolonged in RIP140-deficient HUVEC, while TNFα 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 TNFα treatment, RIP140 associates with the p65 subunit of NFκB, 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 NFκB 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 TNFα 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 TNFα 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 TNFα and plays an important role in the control of inflammation, acting as a negative regulator of NFκB, 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.

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