Quaedackers M E, Van Den Brink C E, Wissink S, Schreurs R H, Gustafsson J A, Van Der Saag P T, Van Der Burg B B
Hubrecht Laboratory , Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.
Endocrinology. 2001 Mar;142(3):1156-66. doi: 10.1210/endo.142.3.8003.
Estrogens are important mediators of bone homeostasis, and postmenopausal estrogen replacement therapy is extensively used to prevent osteoporosis. The biological effects of estrogen are mediated by receptors belonging to the superfamily of steroid/thyroid nuclear receptors, estrogen receptor (ER)alpha and ER beta. ER alpha, not only trans-activates target genes in a hormone-specific fashion, but it can also neutralize other transcriptional activators, such as nuclear factor (NF)-kappa B, causing repression of their target genes. A major mechanism by which estrogens prevent osteoporosis seems to be repression of transcription of NF-kappa B target genes, such as the osteoclast-activating cytokines interleukin-6 and interleukin-1. To study the capacity of both ERs in repression of NF-kappa B signaling in bone cells, we first carried out transient transfections with ER alpha or ER beta of the human osteoblastic U2-OS cell line, in which endogenous NF-kappa B was stimulated by tumor necrosis factor alpha. Repression by ER alpha was already observed without 17 beta-estradiol, whereas addition of the ligand increased repression to 90%. ER beta, however, was able to repress NF-kappa B activity only in the presence of ligand. Because it is known that some antiestrogens can also display tissue-specific agonistic properties, 4-hydroxytamoxifen was tested for its capacity in repressing NF-kappa B activity and was found to be active (albeit less efficient than 17 beta-estradiol) and, interestingly, only with ER alpha. The pure antagonist ICI 164,384 was incapable of repressing through any ER subtypes. Deletion analysis and the use of receptor ER alpha/ER beta-chimeras showed that the A/B domain, containing activation function-1, is essential for this suppressive action. Next, we developed stable transfectants of the human osteoblastic U2-OS cell line containing ER alpha or ER beta in combination with an NF-kappa B luciferase reporter construct. In these cell lines, repression of NF-kappa B activity was only mediated through ER alpha and not through ER beta. These findings offer new insights into the specific role of both ER subtypes in bone homeostasis and could eventually help in developing more specific medical intervention strategies for osteoporosis.
雌激素是骨稳态的重要调节因子，绝经后雌激素替代疗法被广泛用于预防骨质疏松症。雌激素的生物学效应是由属于类固醇/甲状腺核受体超家族的受体介导的，即雌激素受体（ER）α和ERβ。ERα不仅以激素特异性方式反式激活靶基因，还能中和其他转录激活因子，如核因子（NF）-κB，从而抑制其靶基因。雌激素预防骨质疏松症的主要机制似乎是抑制NF-κB靶基因的转录，如破骨细胞激活细胞因子白细胞介素-6和白细胞介素-1。为了研究两种ER在骨细胞中抑制NF-κB信号传导的能力，我们首先用人成骨U2-OS细胞系的ERα或ERβ进行瞬时转染，其中内源性NF-κB由肿瘤坏死因子α刺激。在没有17β-雌二醇的情况下已经观察到ERα的抑制作用，而添加配体可将抑制作用提高到90%。然而，ERβ仅在存在配体的情况下才能抑制NF-κB活性。因为已知一些抗雌激素也可表现出组织特异性激动特性，所以测试了4-羟基他莫昔芬抑制NF-κB活性的能力，发现其具有活性（尽管效率低于17β-雌二醇），有趣的是，仅对ERα有活性。纯拮抗剂ICI 164,384不能通过任何ER亚型进行抑制。缺失分析和受体ERα/ERβ嵌合体的使用表明，包含激活功能-1的A/B结构域对于这种抑制作用至关重要。接下来，我们构建了人成骨U2-OS细胞系的稳定转染体，其包含ERα或ERβ以及NF-κB荧光素酶报告基因构建体。在这些细胞系中，NF-κB活性的抑制仅通过ERα介导，而不是通过ERβ。这些发现为两种ER亚型在骨稳态中的特定作用提供了新的见解，并最终可能有助于开发更具特异性的骨质疏松症医学干预策略。
