Sex Steroids and Runx Signaling in Bone

描述（由申请人提供）：Runx 2是一种主要的成骨细胞转录因子，在骨骼发育和体内平衡中发挥关键作用。在人类中，Runx 2单倍型有助于骨量的变化。Runx 1在成骨细胞中表达，与Runx 2具有相似的DNA结合特性，也与骨代谢有关。性类固醇激素及其受体（SHR）在骨骼健康和疾病中也起着关键作用，并且是积极或消极影响骨量和脆性的药物的目标。性类固醇的促骨作用是通过成骨细胞的合成代谢作用介导的，但更重要的是通过减弱骨吸收。性类固醇的抗吸收作用可归因于破骨细胞凋亡增加和通过成骨细胞和其他间充质细胞中的机制知之甚少而间接抑制骨转换。我们发现激活的雌激素受体a（ER a）和雄激素受体（AR）各自抑制Runx 2，并且AR而不是ER a抑制Runx 1。根据转基因小鼠的最新数据，这些抑制活性是重要的，转基因小鼠的成骨细胞过度表达Runx 2或Runx 2的显性阴性形式。这两种小鼠模型都表明，抑制Runx 2的活性有助于控制骨转换并预防骨质疏松症。因此，我们建议深入研究Runx蛋白和SHR之间的物理相互作用，介导Runx 2和/或Runx 1抑制的机制，以及生理意义。这将通过分析重组和瞬时表达的蛋白质，以及成骨细胞中的内源性SHR和Runx蛋白质，包括它们彼此之间、与共调节因子以及与基因组Runx靶点之间的关联来完成。具体目标1是剖析ER α和Runx 2之间的功能和分子相互作用。具体目标2是剖析AR和Runx 2之间以及AR和Runx 1之间的功能和分子相互作用。根据我们的初步数据，我们假设这些相互作用都存在相似和独特的特征。具体目标3是在体内建立成骨细胞ER α信号传导的要求，以及成骨细胞分化期间的时机，其中它赋予对骨的保护，并测试和表征成骨细胞SHR信号传导在共培养环境中的抗Runx 2抗破骨细胞生成特性。目的1-3中包含了解决选择性雌激素受体调节剂（SERM）的新作用机制的实验。与雌二醇一样，SERMs促进ER a和Runx 2之间的物理相互作用。然而，SERM引起不同的功能结果，可能解释这些药物的可变骨骼效应。我们的研究将为性激素调节骨骼代谢提供新的见解，并将在分子水平上揭示性别之间的共性和差异。他们将破译现有SERM的神秘作用机制，并基于其对Runx蛋白的影响支持新SERM的合理开发。 公共卫生相关性：该项目将揭示性激素下降时发生的骨质疏松症的基本机制。它是基于这样的观察，即控制骨代谢的Runx蛋白与雌激素和雄激素的受体相互作用。这项工作最终可能导致开发出治疗绝经后骨质疏松症的改进药物。

DESCRIPTION (provided by applicant): Runx2 is a master osteoblast transcription factor playing pivotal roles in skeletal development and homeostasis. In humans, Runx2 haplotypes contribute to variations in bone mass. Runx1, which is expressed in osteoblasts and shares similar DNA-binding properties with Runx2, has been implicated in bone metabolism as well. Sex steroid hormones and their receptors (SHRs) also play critical roles in bone health and disease, and are targets for drugs that affect bone mass and fragility either positively or negatively. The proskeletal effects of sex steroids are mediated by anabolic effects in osteoblasts, but more importantly by attenuating bone resorption. The anti-resorptive effects of sex steroids are attributable to both increasing osteoclast apoptosis and indirect inhibition of bone turnover via poorly understood mechanisms in osteoblasts and other mesenchymal cells. We found that the activated estrogen receptor a (ERa) and the androgen receptor (AR) each inhibits Runx2, and that AR, but not ERa, inhibits Runx1. These inhibitory activities are important in light of recent data from transgenic mice whose osteoblasts over-express either Runx2 or a dominant negative form of Runx2. Both mouse models indicate that restraining the activity of Runx2 helps keep bone turnover in check and prevent osteoporosis. We therefore propose to investigate in depth the physical interactions between Runx proteins and SHRs, the mechanisms mediating the resulting inhibition of Runx2 and/or Runx1, and the physiological implications. This will be done by analyses of recombinant and transiently expressed proteins, as well as the endogenous SHR and Runx proteins in osteoblasts, including their associations with each other, with co-regulators, and with genomic Runx targets. Specific Aim 1 is to dissect the functional and molecular interactions between ERa and Runx2. Specific Aim 2 is to dissect the functional and molecular interactions between AR and Runx2, as well as between AR and Runx1. Based on our preliminary data, we hypothesize the existence of both similar and unique features for each of these interactions. Specific Aim 3 is to establish in vivo the requirement for osteoblastic ERa signaling, and the timing during osteoblast differentiation, in which it confers protection on bone, and to test and characterize the anti-Runx2 anti-osteoclastogenic properties of osteoblastic SHR signaling in a co-culture setting. Incorporated into Aims 1-3 are experiments addressing novel mechanisms of action of selective estrogen receptor modulators (SERMs). Like estradiol, SERMs promote a physical interaction between ERa and Runx2. However, SERMs elicit different functional outcomes, possibly explaining the variable skeletal effects of these drugs. Our studies will provide novel insights into the regulation of skeletal metabolism by sex hormones, and will reveal commonalities and differences between the genders at the molecular level. They will decipher cryptic mechanisms of action of existing SERMs, and support the rationale development of novel ones, based on their influence on Runx proteins. PUBLIC HEALTH RELEVANCE: This project will unravel fundamental mechanisms of osteoporosis that occurs as sex hormones decline. It is based on the observation that Runx proteins, which control bone metabolism, interact with receptors for both estrogens and androgens. The proposed work can ultimately lead to the development of improved drugs to treat postmenopausal osteoporosis.