Estrogens, androgens, aging, and bone loss in males

描述（由申请人提供）： 性腺切除术增加了小鼠骨骼中活性氧（ROS）的产生，抗氧化剂可以预防卵巢或睾丸功能丧失对骨骼的不良影响。相反，雌激素或非芳香化雄激素降低氧化应激。雌激素的作用来自ER 1的DNA结合独立作用。与这种机制完全一致，不能刺激ER 1的核引发作用的雌二醇树枝状聚合物缀合物（EDC）再现雌二醇对破骨细胞和成骨细胞的作用;并且在预防氧化应激和卵巢切除术诱导的骨丢失方面与雌二醇一样有效，而不影响子宫重量。在雌性小鼠中，破骨细胞谱系（表达Lys-M）细胞中的ER 1缺失导致松质骨而不是皮质骨的丢失;而在雌性和雄性小鼠中，间充质祖细胞（表达Prx 1的细胞）中的ER 1缺失降低了皮质厚度。与上述一致，ROS减弱成骨细胞生成并缩短成骨细胞和骨细胞的寿命。另一方面，ROS是破骨细胞生成、功能和存活所必需的。此外，FoxOs -转录因子是对抗氧化应激（OS）的重要防御机制-功能的丧失或获得显著改变了骨骼的稳态。基于这些发现，我们推测，类似于雌激素的ER 1介导的作用，雄激素的AR介导的作用通过细胞自主机制降低了成骨细胞和破骨细胞中ROS的产生。相反，雄激素缺乏会增加任一细胞类型中ROS的产生，并且在成年男性骨骼中，这会导致松质骨和皮质骨的损失。雄激素对松质骨的保护作用主要通过破骨细胞AR介导，并由破骨细胞生成减少和破骨细胞寿命缩短继发于细胞凋亡增加所致。雄激素对皮质骨的保护作用是通过AR和ER 1介导的，并且是由ROS诱导的FoxO激活的减弱以及由此产生的Wnt信号传导和成骨细胞生成的增强引起的。EDC将保护性腺切除雄性的皮质骨，与雌性相同，不会影响雄性生殖器官，如精囊。为了推进这些相互关联的假设，雄激素对成骨细胞的细胞自主作用对骨骼稳态的贡献将通过确定雄性小鼠骨骼中Prx 1，Osx或DMP 1表达细胞中AR缺失的影响进行研究;以及AR介导的雄激素信号传导对体内和从这些小鼠分离的成骨细胞中ROS和FoxO激活途径的影响。此外，雄激素对破骨细胞的细胞自主作用对骨骼稳态的贡献将通过确定雄性小鼠骨骼中表达LysM和组织蛋白酶K的细胞中AR缺失的影响，以及AR介导的雄激素信号传导对体内和从这些小鼠分离的骨细胞中ROS活化途径的影响来研究。最后，将确定成骨细胞中ER 1缺失对雄性动物骨骼稳态的贡献以及EDC在雄激素缺乏野生型成年雄性小鼠中的骨保护功效。 公共卫生相关性： 女性缺乏雌激素或男性缺乏雄激素和雌激素都会导致任何性别的骨质疏松症。然而，缺乏任何一类性类固醇对男性或女性骨骼产生不良影响的机制尚未完全了解;雌激素有助于维持男性骨骼的分子机制也未完全了解。本申请中提出的工作将研究雄激素的损失如何影响骨细胞的氧化应激，以及它如何有助于这种疾病的发展。

DESCRIPTION (provided by applicant): Gonadectomy increases the generation of reactive oxygen species (ROS) in the murine skeleton and the adverse effects of the loss of ovarian or testicular function on bone can be prevented by anti-oxidants. Conversely, estrogens or non-aromatizable androgens decrease oxidative stress. The effects of estrogens result from DNA-binding independent actions of the ER1. In full agreement with such a mechanism, an estradiol dendrimer conjugate (EDC) that is not capable of stimulating the nuclear- initiated actions of ER1 reproduces the effects of estradiol on osteoclasts and osteoblasts ; and is as potent as estradiol in preventing oxidative stress and ovariectomy-induced bone loss, without affecting uterine weight. ER1 deletion in cells of the osteoclast lineage (expressing Lys-M) causes loss of cancellous, but not cortical, bone in female mice; while ER1 deletion from mesenchymal progenitors (Prx1 expressing cells) decreases cortical thickness in both females and males. Consistent with the above, ROS attenuate osteoblastogenesis and shorten the lifespan of osteoblasts and osteocytes. On the other hand ROS are required for osteoclast generation, function, and survival. Moreover, loss or gain of function of FoxOs - transcription factors that are an important defense mechanism against oxidative stress (OS) -dramatically alters skeletal homeostasis. Based on these findings it is hypothesized that similar to ER1-mediated effects of estrogens, AR-mediated effects of androgens decrease ROS generation in both osteoblasts and osteoclasts via cell autonomous mechanisms. Conversely, androgen deficiency increases ROS production in either cell type, and in the adult male skeleton this leads to loss of both cancellous and cortical bone. The protective effect of androgens on cancellous bone is mediated primarily via the osteoclast AR and results from decreased osteoclastogenesis and shortened osteoclast lifespan secondary to increased apoptosis. The protective effect of androgens on cortical bone is mediated via both the AR and the ER1 and is caused by attenuation of ROS-induced FoxO activation and the resulting potentiation of Wnt signaling and osteoblastogenesis. The EDC will be protective of cortical bone in gonadectomised males, as it is in females, without affecting male reproductive organs such as seminal vesicles. To advance these interrelated hypotheses, the contribution of cell autonomous effects of androgens on osteoblasts to skeletal homeostasis will be investigated by determining the effects of AR deletion from Prx1, Osx, or DMP1 expressing cells in the skeleton of male mice; and the effects of AR-mediated androgen signaling on ROS and FoxO-activated pathways in vivo and in osteoblastic cells isolated from these mice. Further, the contribution of cell autonomous effects of androgens on osteoclasts to skeletal homeostasis will be studied by determining the effects of AR deletion from LysM and Cathepsin K expressing cells in the skeleton of male mice; and the effects of AR-mediated androgen signaling on ROS-activated pathways in vivo and in osteoclastic cells isolated from these mice. Lastly, the contribution of the ER1 deletion from osteoblastic cells to skeletal homeostasis in the male and the bone sparing efficacy of the EDC in androgen deficient wild type adult male mice will be determined. PUBLIC HEALTH RELEVANCE: A deficiency of estrogens in females or both androgens and estrogens in males contributes to the development of osteoporosis in either sex. However, the mechanism(s) responsible for the adverse effects of the deficiency of either class of sex steroid on the male or female skeleton are not well understood; nor is the molecular mechanism(s) by which estrogens contribute to the maintenance of the male skeleton. The work proposed in this application will investigate how loss of androgens influences oxidative stress on bone cells and how it contributes to the development of this disease.