Regulation of estrogen receptors and MMP-2 expression by estrogens in human retinal pigment epithelium
Regulation of estrogen receptors and MMP-2 expression by estrogens in human retinal pigment epithelium
复制标题
DOI：
10.1167/iovs.01-1276
复制
发表时间：
2003-01-01
影响因子：
4.4
通讯作者：
Cousins, SW
中科院分区：
文献类型：
--
作者：
Marin-Castaño, ME;Elliot, SJ;Cousins, SW
目的.视网膜相关性黄斑变性（ARMD）的特征在于视网膜色素上皮（RPE）下进行性增厚和各种富含脂质的细胞外基质（ECM）沉积物的积累。ECM的失调可能参与了ARMD的病理过程。通过激活雌激素受体（Elks），雌激素调节ECM周转相关基因的表达，其中包括基质金属蛋白酶（MMP）-2。雌激素缺乏可能使ECM的合成和降解失调，导致胶原和其他蛋白质在RPE及其基底膜之间积聚。本研究的目的是证实ER在人RPE中的表达，阐明ER是否具有功能，以及17 β-雌二醇（E-2）是否调节ER和MMP-2的表达。通过使用用于RT-PCR的总RNA和用于Western印迹分析的蛋白提取物，在新鲜分离的人RPE单层和培养的人RPE细胞中检测ER的表达。从新鲜分离的人RPE和培养的人RPE收集上清液，通过酶谱法和蛋白质印迹法评估MMP-2活性和蛋白质表达。ER的转录活性进行了研究，在转染实验中与雌激素反应的报告构建。所有这些研究均在存在或不存在E-2（10（-11）和10（-7）M）的情况下进行。从女性和男性个体分离的人RPE在mRNA和蛋白质水平表达ER亚型α和β。用10（-10）M E-2处理培养的RPE细胞增加两种受体亚型的mRNA和蛋白表达。E-2（10（-10）M）也增加MMP-2活性（接近2.2倍）和蛋白表达（接近2.5倍）。与此相反，在较高的E-2浓度（10（-8）M）下，ER水平和MMP-2活性与基线相比没有变化。用10（-7）M的吡咯烷二硫代氨基甲酸酯（PDTC）（一种核因子（NF）-κ B的抑制剂）预孵育细胞，可消除E-2在10（-10）M时诱导的MMP-2活性和蛋白表达的增加。两种ER亚型均在RPE中表达，并以剂量依赖性方式受E-2调节。雌激素同样调节MMP-2。这种雌激素诱导的作用至少部分是通过NF-κ B介导的。这些数据支持这样的假设，即雌激素可能通过ER在RPE中发挥生物学功能，并且雌激素缺乏或过量可能导致影响与ARMD相关的Bruch膜中ECM周转的分子失调。
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 (Elks), 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 (similar to2.2-fold) and protein expression (similar to2.5-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.