Chronic effects of angiotensin II on NHE3 promoter involve several intracellular signaling pathways and Sp1/Egr‐1 binding site

Abstract

Previous studies have shown that 24h exposure to 10−11 M angiotensin II (AII) stimulated NHE3 activity and increased NHE3 protein content and mRNA levels in OKP cells. The purpose of this study was to verify the effects of AII on the NHE3 gene promoter activity and its regulatory mechanisms. OKP cells were used in transient transfection assays. Four fragments of NHE3 promoter cloned into a vector containing the reporter gene Firefly luciferase were studied (−2095/+55, −152/+55, −85/+31 and −65/+31). The promoter activity of the fragment −65/+31 was significantly increased after 24h‐treatment with 10−11M AII. This increase was abolished by pretreatment with losartan (AT1R inhibitor). Two mutations were performed in this fragment. The mutation at the Sp1/Egr‐1 binding site completely abolishes the AII stimulatory effect, whereas the mutation at the AP2 binding site partially revert this effect. In order to verify the mechanisms involved in this stimulatory response, we used intracellular signaling pathways inhibitors. Our results showed that cytochrome P450, PI3K, PKA and MAPK pathways are involved in this process, while PLC and JAK/STAT pathways do not participate. These data reveal that AII chronic stimulatory effects on NHE3 proximal promoter occur through AT1R and are dependent on the Sp1/Egr‐1 binding site integrity. This response involves activation of different intracellular signaling pathways.Financial Support: FAPESP and CNPq