Losartan induces Hsp70/CHIP interaction and Nox4 ubiquitination in proximal tubule cells (ptcs) from spontaneously hypertensive rats

COSTANTINO VALERIA; GIL LORENZO ANDREA; BOCANEGRA VICTORIA; CACCIAMANI VALERIA; BENARDON MARÍA EUGENIA; GARRAMUÑO VALLÉS, PATRICIA

San Luis

Congreso; Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2014

The chaperone Hsp70 regulates a diverse set of signaling pathways through its interaction with proteins. CHIP (carboxy terminus of Hsp70 interacting protein), is a cytoplasmic protein that U-box domain contains its E3 ubiquitin ligase activity. CHIP regulates the chaperone function in part by regulating the molecular triage decision and determining whether proteins enter the productive folding pathway or results in client substrate ubiquitination and proteosomal degradation. We investigated Hsp70/CHIP contribution to Nox4 regulation after AT1R receptor blockade with Losartan, in primary culture of PTCs. PTCs from 8-week SHR and WKY rats were stimulated with Angiotensin II (100 nmol/L, 15min) (AII), pretreated with Losartan (100 μmol/L, 90min) (L) and with Losartan 75min plus Angiotensin II 15min (L+AII). Losartan, increased Hsp70 and decreased Nox4 protein levels in SHR (L) membranes fraction. Decreased Hsp70 in SHR (L) vs SHR (AII) in cytosolic fraction confirm Hsp70 translocation to membranes. Immunoprecipitation and immunofluorescence confocal microscopy proved interaction and colocalization of increased Hsp70/CHIP that contrasts with decreased Nox4 in membranes from SHR PTCs (L) vs PTCs (AII). PTCs (L) exposed to MG132 blocked the degradation of ubiquitinated Nox4. Immunofluorescence analysis demonstrated Nox4 ubiquitination by increased colocalization of Nox4/ Ubiquitin in PTCs (L) exposed to MG132. Conversely, Hsp72 knockdown PTCs (L) reduced Nox4/Ubiquitin colocalization, resulting in Nox4 upregulation due to the proteosomal degradation inhibition, even Losartan treatment. In conclusion, our data suggest that Hsp70 and CHIP interaction mediates the ubiquitination and proteasomal degradation of Nox4 as part of the antioxidative effect of Losartan in PTCs from SHR.