Beloti Márcio Mateus, Bellesini Larissa Sverzut, de Oliveira Paulo Tambasco, Rosa Adalberto Luiz
Department of Oral and Maxillofacial Surgery, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Av do Cafe, s/n, 14040-904, Ribeirao Preto, SP, Brazil.
Mol Cell Biochem. 2006 Apr;285(1-2):101-9. doi: 10.1007/s11010-005-9063-9. Epub 2006 Feb 14.
TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERalpha, ERbeta, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERalpha and ERbeta antagonist) or MPP (ERalpha antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERalpha and ERbeta was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERalpha and ERbeta was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10(-5) M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERalpha-dependent pathway by binding to this receptor and not by increasing the expression of ER.
TAK - 778已被证明在体外和体内均能刺激骨生成。然而，TAK - 778发挥其作用的机制仍不清楚。有证据表明TAK - 778通过雌激素受体（ER）介导的信号传导发挥作用；因此，本研究旨在探讨ERα、ERβ和膜ER在TAK - 778的成骨作用中所起的作用。为此，将人骨髓间充质细胞与TAK - 778一起培养，同时加入ICI182,780（ERα和ERβ拮抗剂）或MPP（ERα拮抗剂）或PD98059（作用于膜ER途径的细胞外调节激酶抑制剂）。评估了以下参数：细胞增殖、胶原蛋白含量、碱性磷酸酶（ALP）活性和类骨形成。使用方差分析比较数据。通过免疫标记研究TAK - 778对ERα和ERβ表达的影响。为了研究TAK - 778是否与ER结合，进行了ER结合试验。免疫标记和结合试验均使用来自人牙槽骨的细胞进行。TAK - 778的成骨作用被ICI182,780和MPP抑制；然而，它不受PD98059的影响。TAK - 778对ERα和ERβ的表达均无影响。使用TAK - 778进行结合试验得到的竞争曲线显示，当使用10(-5) M TAK - 778时出现最大置换。本研究结果表明，TAK - 778通过与该受体结合，通过ERα依赖性途径增强成骨细胞分化，而不是通过增加ER的表达。
