Oursler M J, Pederson L, Pyfferoen J, Osdoby P, Fitzpatrick L, Spelsberg T C
Department of Biochemistry and Molecular Biology, Mayo Clinic, Mayo Foundation, Rochester, Minnesota 55905.
Endocrinology. 1993 Mar;132(3):1373-80. doi: 10.1210/endo.132.3.8440193.
We have previously demonstrated that avian osteoclasts contain high levels of 17 beta-estradiol (17 beta E2) receptors and respond to 17 beta E2 treatment with a dose-dependent decrease in in vitro resorption of [3H] proline-labeled bone particles. To more accurately assess the influence of 17 beta E2 on osteoclastic activity, the specificity of estrogen modulation of resorption levels was determined using a quantitative pit resorption assay. Treatment with 17 beta E2 significantly decreased the number of osteoclast resorption pits formed compared with that after either vehicle or 17 alpha E2 treatment. Cotreatment with 17 beta E2 and hydroxytamoxifen (a complete 17 beta E2 antagonist in birds) abrogated the influence of 17 beta E2 on resorption activity. To elucidate the mechanism by which 17 beta E2 inhibits osteoclast activity, the effects of 17 beta E2 on the steady state mRNA levels of two avian osteoclast lysosomal proteins, lysozyme and a lysosomal membrane protein (LEP-100), were examined. Using highly purified avian osteoclasts, 17 beta E2 was shown to decrease lysosomal protein mRNA levels in a dose-dependent manner within 8 h of treatment in a process that required de novo protein synthesis. This response was specific for 17 beta E2, since the inactive stereoisomer 17 alpha E2 had no effect. Furthermore, coincubation of 17 beta E2 with hydroxytamoxifen eliminated the 17 beta E2 influence. After removal of 10(-8) M 17 beta E2, lysosomal gene mRNA levels returned to near-normal levels within 24 h. This is consistent with the previously reported ability of avian osteoclast-mediated resorption activity to recover from 17 beta E2 treatment. Lysozyme protein levels similarly decreased after 17 beta E2 treatment. These data suggest that avian osteoclasts are target cells for 17 beta E2 in vitro, that osteoclast activity in vivo is likely to be modulated by circulating levels of 17 beta E2, and that the 17 beta E2 inhibition of osteoclast resorption activity may be mediated at least in part via regulation of osteoclast lysosomal gene expression.
我们之前已经证明，鸟类破骨细胞含有高水平的17β-雌二醇（17β-E2）受体，并且对17β-E2处理有反应，[3H]脯氨酸标记的骨颗粒的体外吸收呈剂量依赖性降低。为了更准确地评估17β-E2对破骨细胞活性的影响，使用定量凹坑吸收试验确定雌激素对吸收水平调节的特异性。与载体或17α-E2处理后相比，17β-E2处理显著减少了形成的破骨细胞吸收凹坑的数量。17β-E2与羟基他莫昔芬（鸟类中的一种完全17β-E2拮抗剂）共同处理消除了17β-E2对吸收活性的影响。为了阐明17β-E2抑制破骨细胞活性的机制，研究了17β-E2对两种鸟类破骨细胞溶酶体蛋白（溶菌酶和一种溶酶体膜蛋白（LEP-100））稳态mRNA水平的影响。使用高度纯化的鸟类破骨细胞，显示17β-E2在处理8小时内以剂量依赖性方式降低溶酶体蛋白mRNA水平，这一过程需要从头合成蛋白质。这种反应对17β-E2具有特异性，因为无活性的立体异构体17α-E2没有影响。此外，17β-E2与羟基他莫昔芬共同孵育消除了17β-E2的影响。去除10(-8)M 17β-E2后，溶酶体基因mRNA水平在24小时内恢复到接近正常水平。这与先前报道的鸟类破骨细胞介导的吸收活性从17β-E2处理中恢复的能力一致。17β-E2处理后溶菌酶蛋白水平同样降低。这些数据表明，鸟类破骨细胞在体外是17β-E2的靶细胞，体内破骨细胞活性可能受到循环中17β-E2水平的调节，并且17β-E2对破骨细胞吸收活性的抑制可能至少部分通过调节破骨细胞溶酶体基因表达来介导。
