Somjen Dalia, Kohen Fortune, Gayer Batya, Knoll Esther, Limor Rona, Baz Merav, Sharon Orly, Posner Gary H, Stern Naftali
Institute of Endocrinology, Metabolism and Hypertension, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel.
J Steroid Biochem Mol Biol. 2004 May;89-90(1-5):397-9. doi: 10.1016/j.jsbmb.2004.03.006.
In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.
在培养的人血管平滑肌细胞（VSMC）中，17β-雌二醇（E2）对DNA合成产生双相效应，即在低浓度时刺激，高浓度时抑制。此外，E2增加了这些细胞中肌酸激酶（CK）的比活性。有观察表明，新型蛋白结合的膜不透性雌激素复合物可抑制DNA合成，提示通过膜结合位点相互作用。然而，其他效应，如增加CK活性，仅在天然E2存在时可见，而在E2-牛血清白蛋白（E2-BSA）存在时未见，因此表明涉及经典的核受体途径。在本报告中，我们证实人VSMC同时表达雌激素受体α（ERα）和雌激素受体β（ERβ）。此外，用维生素D非钙类似物JK 1624 F2-2（JKF）预处理培养的VSMC后，通过实时聚合酶链反应（PCR）检测发现，ERα信使核糖核酸（mRNA）表达增加（100 - 200%），而ERβ mRNA表达下降（30 - 40%）。通过蛋白质免疫印迹分析评估的ERα蛋白表达平行增加（25 - 50%），而ERβ蛋白表达下降（25 - 55%）。使用与铕（Eu）连接的与E2结合的卵清蛋白（Ov-E2，即Eu-Ov-E2）来评估特异性膜结合位点，我们观察到JKF使膜结合下调70 - 80%。相反，3[H] E2的总细胞结合（几乎完全代表细胞内E2结合）在相同的维生素D类似物作用下增加了60 - 100%。结果提供了证据，表明JKF对ERα/ERβ以及雌激素的膜结合与核结合的影响是不同的，且显示出差异调节。
