Jisa E, Dornstauder E, Ogawa S, Inoue S, Muramatsu M, Jungbauer A
Institute of Applied Microbiology, University of Agricultural Sciences, Vienna, Austria.
Biochem Pharmacol. 2001 Oct 1;62(7):953-61. doi: 10.1016/s0006-2952(01)00731-6.
Raloxifene represents a potent compound for the prevention and treatment of osteoporosis and cardiovascular disease in postmenopausal women. Raloxifene exhibits targeted antiestrogenicity in breast and uterus, but acts as an agonist in bone and liver. This synthetic selective estrogen receptor modulator binds both estrogen receptors alpha and beta. The molecular mechanisms by which raloxifene exerts agonistic or antagonistic activity are still not resolved. Therefore, the binding behavior of raloxifene to estrogen receptors and its effects on DNA binding and transactivation were studied. The equilibrium binding affinity of raloxifene by displacing radiolabeled 17beta-estradiol exhibited a similar affinity behavior to that of its natural ligand. Using BIACORE technology with an immobilized estrogen response element, we showed that 17beta-estradiol and raloxifene increased the binding of estrogen receptor alpha to the DNA, suggesting a ligand-dependent dimerization. The influence of the ligands to the binding of estrogen receptor beta was lower. We may conclude that unliganded estrogen receptor alpha binds as a monomer whereas in the presence of 10(-8) M 17beta-estradiol or higher, homodimers are formed that interact with the estrogen response element. Transactivation studies in a yeast reporter system in a ligand-dependent manner resulted in a similar potency of raloxifene to estrogen receptor beta compared to the control testosterone. Subeffective doses of raloxifene combined with 17beta-estradiol did not shift the efficiency, whereas saturating concentrations of 17beta-estradiol combined with increasing concentrations of raloxifene altered the response induced by 17beta-estradiol. In this pure system, the antagonistic activity of raloxifene could not be detected as was expected by the results from ligand competition analysis.
雷洛昔芬是预防和治疗绝经后女性骨质疏松症及心血管疾病的一种有效化合物。雷洛昔芬在乳腺和子宫中表现出靶向抗雌激素作用，但在骨骼和肝脏中起激动剂作用。这种合成的选择性雌激素受体调节剂能与雌激素受体α和β结合。雷洛昔芬发挥激动或拮抗活性的分子机制仍未明确。因此，研究了雷洛昔芬与雌激素受体的结合行为及其对DNA结合和反式激活的影响。通过置换放射性标记的17β-雌二醇，雷洛昔芬的平衡结合亲和力表现出与其天然配体相似的亲和力行为。使用固定化雌激素反应元件的BIACORE技术，我们发现17β-雌二醇和雷洛昔芬增加了雌激素受体α与DNA的结合，提示存在配体依赖性二聚化。配体对雌激素受体β结合的影响较小。我们可以得出结论，未结合配体的雌激素受体α以单体形式结合，而在存在10(-8)M 17β-雌二醇或更高浓度时，会形成与雌激素反应元件相互作用的同源二聚体。在酵母报告系统中以配体依赖性方式进行的反式激活研究表明，与对照睾酮相比，雷洛昔芬对雌激素受体β的效力相似。雷洛昔芬与17β-雌二醇的亚有效剂量组合并未改变效率，而17β-雌二醇的饱和浓度与不断增加的雷洛昔芬浓度组合则改变了17β-雌二醇诱导的反应。在这个纯系统中，正如配体竞争分析结果所预期的那样，未检测到雷洛昔芬的拮抗活性。
