Estrogen receptor regulation of bone metabolism in adult mice

描述(申请人提供)：尽管雌激素(E)在调节骨代谢和与年龄相关的骨丢失方面很重要，但关于E对骨的作用仍然存在基本的、未回答的问题。更全面地了解E对骨转换的调节是重要的，因为即使绝经后妇女的E治疗由于众所周知的非骨骼风险而下降，了解E调节骨代谢的机制可能会发现新的治疗靶点。现有的雌激素受体(ER)α细胞特异性缺失的小鼠模型提供了重要的机制见解，但所有现有模型的一个重要局限性是从受孕开始就涉及ER缺失，使得无法区分E对骨骼发育的影响和对成人骨骼的影响。为了解决这个问题，我们开发了一个实验模型，在这个模型中，我们可以选择性地删除成年小鼠的ERα。在初步研究中，成年小鼠在保持循环E水平不变的情况下，ERα被全局删除，我们令人惊讶地发现，尽管子宫重量显著减少到卵巢切除后的水平，但没有骨丢失或骨吸收增加。这些数据建立了一种独特的方法来定义ERα在调节成人骨骼骨转换中的作用，并指出了骨中潜在的代偿机制，但在很大程度上被忽视了。我们假设这种机制涉及ER？，在目标1中，我们将在骨骼成熟后全局删除ERα和ER？，并确定我们观察到的成年小鼠单独删除ER？α后没有骨丢失是否归因于ER？补偿。第二个重要的悬而未决的问题是，当E在成年鼠(或人)体内被提取时，哪种细胞类型对引发骨丢失至关重要？在目标2中，我们将测试这一重要细胞是骨细胞的假设，并且骨细胞中ERα和ERç的缺失将需要触发成年小鼠的骨丢失。支持这一假说的现有证据是：1)骨细胞被越来越多地认为是骨重建的“主调节器”；2)阻止骨细胞凋亡也可以防止去卵巢后骨吸收的增加；3)ERα和ER？都激活了细胞外信号相关激酶，介导了E对骨细胞的抗凋亡作用。关于骨细胞是引发E缺乏后骨重建的关键细胞的一个相互竞争的假说是，关键细胞实际上是破骨细胞。因此，从受孕开始，破骨细胞中ERα缺失的小鼠就增加了骨转换，减少了骨量。为了最终解决这个问题，在目标3中，我们将删除成年小鼠破骨细胞中的ERα和/或ERç，并将其与目标2中这些受体的骨细胞特异性缺失进行比较。此外，在目标2和3中嵌入了对高度丰富的骨细胞、破骨细胞前体和成骨细胞群体的分析，这些细胞不需要体外培养，从而为E作用于骨骼提供了重要的机制见解。

DESCRIPTION (provided by applicant): Despite the importance of estrogen (E) in regulating bone metabolism and age-related bone loss, there remain fundamental, unanswered questions regarding E action on bone. A more complete understanding of E regulation of bone turnover is important because even though E treatment of postmenopausal women is declining due to well publicized non-skeletal risks, understanding the mechanisms by which E regulates bone metabolism is likely to identify novel therapeutic targets. Existing mouse models with cell-specific deletion of estrogen receptor (ER)α have provided significant mechanistic insights, but an important limitation of all current models is that they have involved ER deletion from conception onwards, making it impossible to distinguish the effects of E on skeletal development from those on the adult skeleton. To address this issue, we have developed an experimental model in which we can selectively delete ERα in the adult mouse. In preliminary studies in which ERα was globally deleted in adult mice while holding circulating E levels constant, we surprisingly found no bone loss or increase in bone resorption despite marked decreases in uterine weight to levels observed following ovariectomy. These data establish a unique approach to define the role of ERα in regulating bone turnover in the adult skeleton, and also point to a potential compensatory mechanism in bone that has been largely ignored. We hypothesize that this mechanism involves ERß, and in Aim 1, we will globally delete both ERα and ERß following skeletal maturity and determine whether the absence of bone loss we observed following ERα deletion alone in the adult mouse was due to ERß compensation. A second important, unresolved question is that when E is withdrawn in the adult mouse (or human), which cell type is crucial for triggering bone loss? In Aim 2 we will test the hypothesis that this crucial cell is the osteocyte, and that deletion of both ERα and ERß in the osteocyte will be required to trigger bone loss in the adult mouse. The existing evidence in support of this hypothesis is that 1) the osteocyte is increasingly recognized as the "master regulator" of bone remodeling; 2) prevention of osteocyte apoptosis also prevents the increase in bone resorption following ovariectomy; and 3) both ERα and ERß activate extracellular signal- related kinases that mediate the anti-apoptotic effects of E on osteocytes. A competing hypothesis to the osteocyte being the crucial cell triggering bone remodeling following E deficiency is that the key cell is, in fact, the osteoclast. Thus, mice with ERα deletion in osteoclasts from conception onwards have increased bone turnover and reduced bone mass. In order to definitively resolve this issue, in Aim 3 we will delete ERα and/or ERß in osteoclasts in the adult mouse and compare this to osteocyte-specific deletion of these receptors in Aim 2. Moreover, embedded within Aims 2 and 3 will be analyses of highly enriched populations of osteocytes, osteoclast progenitors, and osteoblasts that are performed without in vitro culture, thereby providing important mechanistic insights into E action on bone.