Heat Shock Protein 70 and CHIP Promote Nox4 Ubiquitination and Degradation within the Losartan Antioxidative Effect in Proximal Tubule Cells

• Main Authors: Andrea F. Gil Lorenzo, Valeria V. Costantino, Martin López Appiolaza, Valeria Cacciamani, Maria E. Benardon, Victoria Bocanegra, Patricia G. Vallés
• Format: Article
• Language: English
• Published: Cell Physiol Biochem Press GmbH & Co KG 2015-07-01
• Series: Cellular Physiology and Biochemistry
• Subjects: Proximal tubule cells; Angiotensin II; Losartan; Nox4/Hsp70/CHIP; Ubiquitination
• Online Access: http://www.karger.com/Article/FullText/430184

Background: Angiotensin II/Angiotensin II type 1 receptor (AT1R) effects are dependent on ROS production stimulated by NADPH oxidase activation. Hsp70 regulates a diverse set of signaling pathways through their interactions with proteins. CHIP is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation. Aim: We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT1R blockage. Methods/Results: Primary culture of proximal tubule epithelial cells (PTCs) from SHR and WKY were stimulated with Angiotensin II (AII) or treated with Losartan (L) or Losartan plus Angiotensin II (L+AII). Losartan decreased AT1R and Nox4 while enhancing caveolin-1 and Hsp70 protein expression in SHR PTCs. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP with decreased Nox4 in SHR PTCs (L) vs (All). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate AII effects on Nox4 expression and oxidative activity. Moreover, MG132 exposed PTCs (L) demostrated blocked ubiquitinated Nox4 degradation and increased colocalization of Nox4/Ubiquitin by inmunofluorescence. Conversely, Hsp72 depletion reduced Nox4/Ubiquitin colocalization causing Nox4 upregulation due to proteosomal degradation inhibition, although Losartan treatment. Conclusion: Our study demonstrates that Hsp70 and CHIP mediates the ubiquitination and proteasomal degradation of Nox4 as part of the antioxidative effect of Losartan in SHR.