Sex Steroids and Runx Signaling in Bone

描述（申请人提供）：Runx2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起关键作用。在人类中，Runx2单倍型导致骨量的变化。Runx1在成骨细胞中表达，与Runx2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（SHRs）在骨骼健康和疾病中也起着至关重要的作用，并且是影响骨量和脆弱性的药物的目标，无论是积极的还是消极的。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。性类固醇的抗骨吸收作用可归因于增加破骨细胞凋亡和间接抑制骨转换，其机制尚不清楚成骨细胞和其他间充质细胞。我们发现激活的雌激素受体a （ERa）和雄激素受体（AR）均抑制Runx2，而AR抑制Runx1，而ERa不抑制Runx1。这些抑制活性是重要的，根据最近的数据，转基因小鼠的成骨细胞过表达Runx2或Runx2的显性阴性形式。两种小鼠模型都表明，抑制Runx2的活性有助于控制骨转换，预防骨质疏松症。因此，我们建议深入研究Runx蛋白与SHRs之间的物理相互作用，介导Runx2和/或Runx1抑制的机制，以及生理意义。这将通过分析重组蛋白和瞬时表达蛋白，以及成骨细胞中的内源性SHR和Runx蛋白，包括它们之间的关联，与共同调节因子的关联，以及与基因组Runx靶点的关联来完成。具体目的1是剖析ERa和Runx2之间的功能和分子相互作用。具体目的2是剖析AR与Runx2之间以及AR与Runx1之间的功能和分子相互作用。根据我们的初步数据，我们假设存在这些相互作用的相似和独特的特征。具体目标3是在体内建立成骨细胞ERa信号的需求，以及成骨细胞分化过程中对骨的保护时间，并在共培养环境下测试和表征成骨细胞SHR信号的抗runx2抗破骨特性。纳入目标1-3的是研究选择性雌激素受体调节剂（SERMs）作用新机制的实验。与雌二醇一样，serm促进ERa和Runx2之间的物理相互作用。然而，serm引起不同的功能结果，可能解释了这些药物对骨骼的不同影响。我们的研究将为性激素对骨骼代谢的调节提供新的见解，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，并基于它们对Runx蛋白的影响，支持新serm的基本原理发展。

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.