MOLECULAR MECHANISMS OF THE SKELETAL EFFECTS OF ESTROGEN IN OLD AGE

雌激素对老年骨骼影响的分子机制

• 批准号: 7094994
• 负责人: STAVROS C. MANOLAGAS
• 金额: $ 21.49万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7094994
• 关键词: aging; animal old age; antioxidants; apoptosis; biological signal transduction; bone density; bone imaging /visualization /scanning; cellular pathology; computed axial tomography; cytokine; developmental genetics; estrogen receptors; estrogens; female; free radical oxygen; genetically modified animals; glutathione; laboratory mouse; mitogen activated protein kinase; molecular pathology; nuclear factor kappa beta; osteoblasts; osteoclasts; osteoporosis; oxidative stress; tissue /cell culture

Estrogen loss increases osteoclastogenesis, decreases osteoclast and increases osteoblast apoptosis, and causes loss of bone by decreasing anti-oxidant defense in these cell types. In addition, an age-dependent decrease in bone strength and mass in ovary-intact C57BL/6 mice is temporally associated with an agedependent increase in the prevalence of osteoblast apoptosis, decreased glutathione reductase (GSR) activity and corresponding increases in the production of reactive oxygen species (ROS) in the bone marrow and the phosphorylation of p66shc in vertebrae. Based on this evidence, it is hypothesized that increased ROS levels with advancing age is a fundamental mechanism of the age-dependent decline of bone strength and mass, and loss of estrogens exaggerates the adverse effects of aging on bone by decreasing defense against ROS, thereby, contributing perpetually to the loss of bone mass and strength that persists for decades after menopause, and is associated with old age. Estrogens enhance defense against ROS by modulating the activity of anti-oxidant enzymes, attenuating ROS-mediated induction of cytokines, and modulating signaling cascades that have been implicated in the defense against oxidative stress, apoptosis, and aging. All these effects are mediated by extranuclear (nongenotropic) actions of the ER on cytoplasmic kinases and result in changes in the birth and lifespan of osteoblasts, osteoclasts, and osteocytes. To test these interrelated hypotheses, in vitro studies are proposed to investigate whether the effects of estrogens on osteoblast and osteoclast apoptosis, as well as on osteoclastogenesis, are, at least in part, a direct consequence of the ability of these hormones to: a) antagonize the actions of ROS by increasing the levels of glutathione; and/or b) counteract ROS-activated signals on cytoplasmic kinases, and/or downstream transcription factors and the resulting upregulation of cytokines. Further, the role of oxidative stress in the molecular and cellular mechanism responsible for the loss of bone strength and mass associated with aging and the extent to which estrogen deficiency and the resulting attenuation of defense against oxidative stress in osteoblasts or osteoclasts contributes to these changes will be studied in mice over-expressing GSR in osteoblasts or osteoclasts. Finally, mice in which ERa will be deleted from osteoblasts or osteoclasts by means of CreXLoxP recombination; mice heterozygotes for an ERa knock-in mutant which cannot bind to ERE; and mice over-expressing an ERa mutant, which is incapable of membrane localization and prevents kinase-mediated signaling of the endogenous receptor, will be used to examine whether the antioxidant actions of estrogens on osteoblasts and osteoclasts in vivo are ER-dependent and nongenotropic. This work should help us understand how women become increasingly more susceptible to fractures as they grow old. Moreover, it mav lead to the discovery of more effective treatments or even a cure for osteoporosis.

雌激素损失增加破骨细胞生成，减少破骨细胞，增加成骨细胞凋亡， 通过降低这些细胞类型中的抗氧化剂防御而导致骨质流失。此外，年龄依赖性 卵巢完整的C57 BL/6小鼠骨强度和骨质量的降低与年龄依赖性 成骨细胞凋亡发生率增加，谷胱甘肽还原酶（GSR）降低 活性和骨髓中活性氧（ROS）产生的相应增加 和椎骨中p66 shc的磷酸化。根据这一证据，可以推测， 随着年龄的增长，活性氧水平是骨强度随年龄增长而下降的基本机制 而雌激素的丢失会通过降低防御能力来夸大衰老对骨骼的不利影响 对抗ROS，因此，永久地导致骨量和强度的损失，持续 几十年后，更年期，并与老年有关。雌激素通过以下方式增强对ROS的防御： 调节抗氧化酶的活性，减弱ROS介导的细胞因子诱导，和 调节与抗氧化应激，凋亡， 和衰老。所有这些作用都是由ER对细胞质的亲核（非遗传性）作用介导的。 激酶，并导致成骨细胞、破骨细胞和骨细胞的出生和寿命的变化。测试 这些相互关联的假设，在体外研究提出，以调查是否影响雌激素 对成骨细胞和破骨细胞凋亡以及破骨细胞生成的影响，至少部分是直接的， 这些激素的能力的结果：a）通过增加ROS的水平来拮抗ROS的作用， 和/或B）抵消细胞质激酶上的ROS激活信号，和/或下游 转录因子和由此产生的细胞因子的上调。此外，氧化应激在 与衰老相关的骨强度和质量损失的分子和细胞机制 以及雌激素缺乏和由此导致的对氧化应激的防御减弱的程度 在成骨细胞或破骨细胞中导致这些变化的基因将在小鼠中研究， 成骨细胞或破骨细胞。最后，在小鼠中，ER α将通过以下方法从成骨细胞或破骨细胞中删除： CreXLoxP重组的方法; ER α敲入突变体的小鼠杂合子，其不能结合 ERE;和过表达ER α突变体的小鼠，其不能膜定位并阻止ER α的表达。 激酶介导的内源性受体的信号传导，将被用来检查是否抗氧化剂 雌激素在体内对成骨细胞和破骨细胞的作用是ER依赖性的和非遗传性的。这项工作 应该可以帮助我们理解女性随着年龄的增长是如何变得越来越容易骨折的。 此外，它可能会导致发现更有效的治疗方法，甚至治愈骨质疏松症。