Regulation of estrogen receptors and MMP-2 expression by estrogens in human retinal pigment epithelium.
PURPOSE: Age-related macular degeneration (ARMD) is characterized by progressive thickening and accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE). ECM dysregulation probably contributes to the pathologic course of ARMD. By activating estrogen receptors (ERs), estrogens regulate the expression of genes relevant in the turnover of ECM, among them matrix metalloproteinase (MMP)-2. Estrogen deficiency may predispose to dysregulated synthesis and degradation of ECM, leading to accumulation of collagens and other proteins between the RPE and its basement membrane. The purposes in the current study were to confirm the expression of ERs in human RPE, to elucidate whether these ERs are functional, and to test whether 17beta-estradiol (E(2)) regulates expression of ERs and MMP-2. METHODS: Expression of ERs was examined in freshly isolated human RPE monolayer and in cultured human RPE cells, by using total RNA for RT-PCR and protein extracts for Western blot analysis. Supernatants were collected from freshly isolated human RPE and from cultured human RPE to assess MMP-2 activity by zymography and protein expression by Western blot. The transcriptional activity of ERs was studied in transfection experiments with an estrogen-responsive reporter construct. All these studies were preformed in the presence or absence of E(2) (10(-11) and 10(-7) M). RESULTS: Human RPE isolated from female and male individuals expressed both ER subtypes alpha and beta at the mRNA and protein levels. Treatment of cultured RPE cells with 10(-10) M E(2) increased expression of mRNA and protein of both receptor subtypes. E(2) (10(-10) M) also increased MMP-2 activity (approximately 2.2-fold) and protein expression (approximately 2.5-fold). In contrast, there was no change in ER levels and MMP-2 activity at higher E(2) concentrations (10(-8) M), compared with baseline. Preincubation of cells with 10(-7) M pyrrolidinedithiocarbamate (PDTC), an inhibitor of nuclear factor (NF)-kappaB, abolished the increase in MMP-2 activity and protein expression induced by E(2) at 10(-10) M. CONCLUSIONS: Both ER subtypes are expressed in RPE and regulated in a dose-dependent fashion by E(2). Estrogens similarly regulate MMP-2. This estrogen-induced effect is, at least in part, mediated through NF-kappaB. These data support the hypothesis that estrogens may exert biological function in RPE through ERs and that estrogen deficiency or excess may cause dysregulation of molecules that influence the turnover of ECM in Bruch's membrane associated with ARMD.
Marin-Castaño, ME; Elliot, SJ; Potier, M; Karl, M; Striker, LJ; Striker, GE; Csaky, KG; Cousins, SW
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