MOLECULAR MECHANISMS OF ESTROGEN-REGULATED BONE RESORPTION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这个项目的主要目的是加深我们对伴随雌激素缺乏而导致的骨吸收增加的机制的理解。比较了6个月龄对照组、去卵巢(OVX)组、激素替代疗法(HRT)组、钙通道拮抗剂(地尔硫卓、硝苯地平、维拉帕米)组和联合用药组的骨吸收参数。正如预期的那样，OVX大鼠显示出来自两个骨骼间隔室的骨吸收速度增加，而HRT降低了这一影响。不同拮抗剂对无定形脑室的作用与雌激素的作用呈拮抗作用，而对钙化脑室的作用与雌激素的作用呈协同作用，说明作用机制不同。钙通道拮抗剂对平均尾动脉压的影响已按假设进行，表明三类钙通道拮抗剂与对照组相比，即使与HRT联合使用，也能显著降低血压。用来检测尿液中各种骨降解标志物(脱氧吡啶啉(DPD)、吡啶啉(PYD)和螺旋肽)的酶联免疫吸附试验为监测骨骼代谢提供了另一种途径。OVX女性的DPD、PYD和螺旋肽的排泄量显著增加，这表明骨转换增加，而接受HRT的女性与对照组没有显著差异。钙拮抗剂的使用减少了DPD、PYD和螺旋肽的排泄，表明骨转换减少，提示其作用模式与雌激素相似。为了确定雌激素诱导成骨样细胞(7F2和UMR-106)这一作用的机制，在不含雌激素化合物(FBS和酚红)的强化培养液(OPTI-MEM)中进行了实验。与用胎牛血清培养的对照组相比，该方案没有改变细胞的活性或形态。实验结果表明，在一定浓度范围内(0.1 nM到1.9 mM)，雌激素在培养24小时后刺激细胞增殖，表明雌激素具有成骨活性。我们最初的基因组研究表明，雌激素上调了转化生长因子β诱导的早期生长反应(TIEG)和骨保护素(OPG)的转录。这些实验结果支持雌激素在骨骼转换中具有抗吸收作用的假说。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The primary objective of this project is to enhance our understanding of the mechanisms of increased bone resorption, which accompanies estrogen deficiency. Bone resorption parameters have been compared between control females six months of age, ovariectomized females (OVX), females receiving hormone replacement therapy (HRT), and females receiving a calcium channel antagonists (Diltiazem, Nifedipine, Verapamil), or females receiving a combination of the two agents. As expected OVX rats displayed an increased rate of bone resorption from both skeletal compartments and HRT reduced that effect. The actions of the various antagonists were antagonistic to that of estrogen on the amorphous compartment and synergistic on the calcified compartment indicating differing mechanisms of action. The effects of the calcium channel antagonists on mean tail arterial pressure have proceeded as hypothesized demonstrating that all three classes of calcium channel antagonists significantly lowered blood pressure as compared to controls even when given in combination with HRT. ELISA assays designed to detect various bone degradation makers (deoxypyridinoline (DPD), pyridinoline (PYD), and helical peptide) in urine have provided an alternate avenue by which to monitor skeletal metabolism. OVX females display a significant increase in the excretion of DPD, PYD, and helical peptide, indicating an increase in bone turnover, while those receiving HRT were not significantly different from controls. The utilization of calcium antagonists decreases the excretion of DPD, PYD, and helical peptide, indicating a decrease in bone turnover and suggesting a similar mode of action as estrogen. In order to determine the mechanism by which estrogen elicits this action in osteoblast-like cells (7F2 & UMR-106), experiments were carried out in an enriched media (OPTI-MEM) in the absence of estrogenic compounds (FBS & phenol red). This protocol did not alter the viability or morphology of the cells as compared to controls grown with FBS. Experimental results indicate that estrogen over a range of concentrations (0.1nM to 1.9mM) stimulates cell proliferation following 24 hr incubation, demonstrating estrogen's osteoblastic activity. Our initial genomic studies suggest that estrogen up regulates the transcription of Transforming Growth Factor Beta Inducible Early Growth Response (TIEG) and osteoprotegerin (OPG). These experimental findings support the hypothesis of an antiresorptive role for estrogen in skeletal turnover.