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O. Alcazar, M. Pons, M. E. Marin-Castano; MAPK Signaling in the Angiotensin II-Mediated MMP-2 Activity in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6158.
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Accumulation of extracellular molecules under the retinal pigment epithelium has been previously observed in eyes with age-related macular degeneration (AMD) and may play a role in the pathogenesis of AMD. Clinical studies have revealed hypertension (HTN) as a systemic risk factor and Angiotensin II (Ang II) as the most important hormone associated with HTN. Our laboratory has previously shown that Ang II increases MMP-2 activity in vivo and in vitro. In the present study, we aim to explore the regulation of MAPK signaling by AngII in ARPE-19 cells, and the potential signaling connection between AngII stimulation and its well characterized effect of increasing MMP-2 activity.
ARPE-19 cells were cultured to semiconfluent or confluence and the medium was first deprived of phenol red and then serum reduced. Cells were stimulated with Ang II for either 5 min (phosphorylation analyses) or 24 h, and further collected for protein determination. Western blot, cytoimmunostaining and zymography analyses were performed. Gene expression silencing experiments were evaluated 48 h after transient transfection with siRNA.
Immunocytochemistry and Western blot analyses showed that stimulation with AngII increased both Erk(1/2) and p38 MAPK phosphorylation while JNK phosphorylation remained unaffected. Regulation of Erk(1/2) and p38 MAPK phosphorylation by Ang II was dose-dependent and abolished in the presence of candesartan, which is an AngII Type 1 (AT1) receptor blocker. Interestingly, incubation of RPE cells with the Erk(1/2) upstream kinase MEK inhibitor PD98059 and/or the p38MAPK inhibitor SB203580 blunted the increase of MMP-2 activity induced by AngII. Moreover, using specific siRNA to decrease the expression level of Erk(1/2) and/or p38MAPK showed a concomitant decrease in the Ang II ability to stimulate the MMP-2 activity.
Our study suggests that intracellular activation of Erk(1/2) and p38 MAPK signaling may be necessary for Ang II to induce an increase in MMP-2 activity in ARPE-19 cells. This effect seems to be mediated by the AT1 receptor. Further investigation is necessary to evaluate potential crosstalk between both signaling pathways.
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