P717HuR mediate the sinergistic effects of Angiotensin II and interleukin 1beta on vascular COX-2 expression and cell migration

Abstract

Purpose. Angiotensin II (Ang II) and interleukin 1b (IL-1β) are involved in cardiovascular diseases inducing pro-inflammatory enzymes such as cyclooxygenase 2 (COX-2). COX-2 is an early response gene regulated by its transcription and by mRNA stability. Our purpose was to evaluate whether Ang II potentiates the effect of IL-1β on COX-2 expression and activity, the mechanisms involved in this effect and the functional consequence of this interaction. Methods. Rat aortic smooth muscle cells were stimulated with IL-1b or Ang II+IL-1b (4 or 24 h). mRNA and protein levels were measured by qPCR and western blot, respectively. The COX-2 promoter and 3'UTR activity were measured by luciferase assay. Immunofluorescence and cellular fractionation were used to analyze HuR cytoplasmic translocation. The binding of HuR to COX-2 mRNA was assessed by immunoprecipitation. Cell proliferation was analyzed by a commercial kit and cell migration by wound healing and transwell. In a mouse carotid ligation model, COX-2, mPGES-1 and TXAS were measured by immunohistochemistry. Results. 1) IL-1b (10 ng/ml) increased COX-2, mPGES-1 and TXAS expression. 2) Ang II (0.1 mM) increased PGIS and TXAS expression and potentiated and decreased, respectively, COX-2 and mPGES-1 expression induced by IL-1b. 3) The potentiation of COX-2 expression was accompanied by increased mRNA stability and 3'UTR luciferase activity. These effects were blocked by ERK1/2 and HuR inhibitors. 4) Ang II+IL-1b promoted HuR translocation to the cytoplasm and its binding to COX-2 mRNA. 5) Ang II did not affect cell proliferation but potentiated cellular migration induced by IL-1b; this effect was abolished by inhibitors of COX-2, TXAS, ERK1/2 and HuR and by antagonists of TP, EP-1 and EP-3 receptor. 6) PGE2 and a TXA2 analogue increased the cellular migration. 7) After the carotid ligation, COX-2, mPGES-1 and TXAS expression were increased. Conclusions. 1) Ang II differentially modulates COX-2, mPGES-1, PGIS and TXAS vascular expression induced by IL-1b. 2) The potentiation of IL-1β-induced COX-2 expression evoked by Ang II is mediated by ERK1/2 and it is due to an increase in the COX-2 mRNA stability mediated by HuR. 3) TXA2 and PGE2 derived from COX-2 participate in the effects of Ang II plus IL-1β on cell proliferation. 4) These effects could contribute to the vascular pro-inflammatory actions of Ang II and IL-1b.