Retinoid X Receptor´s activation modulates a crosstalk between NrF2 and NFkB pathway in retinal pigment epithelium cells upon H2O2 treatment

Abstract

Age-related macular degeneration (AMD) is the main pathology leading to blindness in adults and has no cure or effective treatment. Retinal pigment epithelial (RPE) cells have immunomodulatory properties and their degeneration contributes in AMD development. Oxidative stress is also involved in the pathogenesis of this disease. We have demonstrated that RXR activation with HX630 protects RPE (D407) cells from H2O2-induced apoptosis, and prevents NFkB nuclear translocation. Also, our previous results suggested RXR- PPARgamma as the main heterodimer involved. In this work we investigate the RXR-PPARgamma involvement in the mentioned protective effect and its mechanism of action. For that, D407 were treated or not with H2O2, HX630 and/or a PPARgamma specific agonist (Pioglitazone: PG). We analyzed cell viability by MTT assay and DAPI stained; and studied NFkB pathway (which modulates inflammation and apoptosis) and Nrf2 (which activates cytoprotective genes and regulates NFkB pathway) by qRT-PCR, florescence microscopy and Western-blot. PG reproduced the inhibition of NFkB translocation and the protective effect of RXR activation against oxidative damage. PG inhibited the IKBalpha phosphorylation more than HX630, while it promoted IKBalpha synthesis. When HX630 and PG were together, the inhibition of NFkB translocation was higher than the agonists alone, although, there was no synergism in apoptosis prevention. HX630 increases Nrf2 synthesis, PG does not, and both agonists together decrease NrF2 levels suggesting proteosomal degradation. As a whole, our results show that RXR and PPARgamma agonists together potentiate the anti- inflammatory response but not the antiapoptotic effects of each agonist alone on RPE cells upon oxidative stress, and suggest that both agonists are necessary together to alter the crosstalk between NrF2 and NFkB pathway.