Estrogens, androgens, aging, and bone loss in males

描述（由申请人提供）： 性腺切除术会增加小鼠骨骼中活性氧（ROS）的产生，并且抗氧化剂可以预防卵巢或睾丸功能丧失对骨骼的不利影响。相反，雌激素或不可芳香化的雄激素可减少氧化应激。雌激素的作用是由 ER1 的 DNA 结合独立作用引起的。与这种机制完全一致，雌二醇树枝状大分子缀合物（EDC）不能刺激 ER1 的核启动作用，但会重现雌二醇对破骨细胞和成骨细胞的作用；与雌二醇一样有效预防氧化应激和卵巢切除引起的骨质流失，而不影响子宫重量。破骨细胞谱系细胞（表达 Lys-M）中的 ER1 缺失会导致雌性小鼠松质骨而非皮质骨的损失；而从间充质祖细胞（表达 Prx1 的细胞）中删除 ER1 会降低女性和男性的皮质厚度。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）功能的丧失或增强会显着改变骨骼稳态。基于这些发现，推测与雌激素 ER1 介导的作用类似，雄激素 AR 介导的作用通过细胞自主机制减少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，是由于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 ER1 介导的，是由 ROS 诱导的 FoxO 激活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 将像女性一样保护性腺切除男性的皮质骨，而不影响精囊等男性生殖器官。为了推进这些相互关联的假设，将通过确定雄性小鼠骨骼中 Prx1、Osx 或 DMP1 表达细胞 AR 缺失的影响来研究雄激素对成骨细胞的细胞自主效应对骨骼稳态的贡献。以及 AR 介导的雄激素信号对体内 ROS 和 FoxO 激活途径以及从这些小鼠中分离的成骨细胞的影响。此外，通过确定雄性小鼠骨骼中 LysM 和组织蛋白酶 K 表达细胞 AR 缺失的影响，研究雄激素对破骨细胞的细胞自主效应对骨骼稳态的贡献；以及 AR 介导的雄激素信号对体内 ROS 激活途径以及从这些小鼠中分离的破骨细胞的影响。最后，将确定成骨细胞 ER1 缺失对雄性骨骼稳态的贡献以及 EDC 在雄激素缺乏的野生型成年雄性小鼠中的骨保留功效。 公共卫生相关性： 女性雌激素缺乏或男性雄激素和雌激素两者缺乏都会导致男女骨质疏松症的发生。然而，缺乏任一类性类固醇对男性或女性骨骼产生不利影响的机制尚不清楚；雌激素有助于维持男性骨骼的分子机制也不是。本申请中提出的工作将研究雄激素的丧失如何影响骨细胞的氧化应激以及它如何促进这种疾病的发展。

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.