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


OBJECTIVE
We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT1R blockage.


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.


CONCLUSIONS
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.
IMBECU-CONICET (National Council of Scientific and Technical Research of Argentina), Universidad Nacional de Cuyo, Mendoza, Argentina.
Andrea F
AF
Gil Lorenzo
Valeria V
VV
Costantino
Martin López
ML
Appiolaza
Valeria
V
Cacciamani
Maria E
ME
Benardon
Victoria
V
Bocanegra
Patricia G
PG
Vallés
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001794:Blood Pressure / Q000187:drug effects; D001835:Body Weight / Q000187:drug effects; D051242:Caveolin 1 / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019808:Losartan / Q000494:pharmacology*; D008297:Male; D008954:Models, Biological; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects*; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects*
D000975:Antioxidants; D051242:Caveolin 1; D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D044140:Receptor, Angiotensin, Type 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; C540442:Stub1 protein, rat; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D019808:Losartan