Role of ATF-4 in the Anabolic Actions of PTH on Bone

描述（由申请人提供）：骨质疏松症是一个主要的公共卫生问题，在美国影响超过1000万人，每年导致150万例骨折。间歇性给予甲状旁腺激素（PTH）是目前治疗骨质疏松症最有效的方法。甲状旁腺激素改善骨量和骨微结构，增加骨强度，降低骨折风险。然而，甲状旁腺激素对骨形成的合成代谢作用的分子机制仍然知之甚少。基因缺失研究表明，激活转录因子4 （ATF4）是骨形成的关键因素。我们的初步研究结果强烈表明，PTH至少在一定程度上通过ATF4控制成骨细胞特异性基因表达和骨形成。该项目的长期目标是了解PTH信号如何调节骨形成过程中的成骨细胞活性。我们的假设是：i) PTH通过促进ATF4的磷酸化和与Runx2的蛋白相互作用来激活ATF4， ii) ATF4介导PTH对骨的合成代谢作用。为了检验这些假设，我们将追求以下具体目标：目标1。确定PTH调节ATF4和Runx2转录活性的机制。为了实现这一目标：i)我们将确定PTH在ATF4中的应答磷酸化位点，并评估PTH诱导的ATF4磷酸化在培养细胞中的功能意义；ii)我们将定义ATF4和Runx2中介导ATF4-Runx2相互作用的区域/氨基酸残基；iii)我们将确定PTH信号是否会增加ATF4-Runx2结合，以及这些相互作用是否对体外成骨细胞中PTH的作用是必要的。目标2。确定甲状旁腺的合成代谢作用是否需要ATF4。我们将使用ATF4缺陷小鼠和在成骨细胞中过表达ATF4的转基因小鼠来确定体内PTH的合成代谢作用是否需要ATF4。目标3。确定体内正常骨形成和甲状旁腺激素诱导的骨形成是否需要ATF4-Runx2相互作用。本项目将探索甲状旁腺激素合成代谢作用的新机制，并为改善骨质疏松症和其他代谢性骨病的治疗确定新的潜在治疗靶点。

DESCRIPTION (provided by applicant): Osteoporosis is a major public health problem that affects more than 10 million people in the US and results in 1.5 million fractures annually. Intermittent administration of parathyroid hormone (PTH) is the most effective current treatment for osteoporosis. PTH improves bone mass and bone microarchitecture, increases bone strength, and reduces the risk for fractures. However, the molecular mechanism underlying the anabolic actions of PTH on bone formation remains poorly understood. Gene deletion studies showed that activating transcription factor 4 (ATF4) is a critical factor for bone formation. The findings from our preliminary studies strongly suggest that PTH controls osteoblast-specific gene expression and bone formation, at least in part, through ATF4. The long-term goal of this project is to understand how PTH signals regulate osteoblast activity in bone formation. Our hypotheses are: i) PTH activates ATF4 by promoting its phosphorylation and protein-protein interactions with Runx2, and ii) ATF4 mediates the anabolic actions of PTH on bone. To test these hypotheses, we will pursue the following Specific Aims: Aim 1. Determine the mechanism used by PTH to regulate ATF4 and Runx2 transcriptional activity. To accomplish this aim: i) we will identify the PTH responsive phosphorylation sites in ATF4 and assess the functional significance of PTH-induced phosphorylation in ATF4 in cultured cells; ii) We will define the regions/amino acid residues within ATF4 and Runx2 that mediate the ATF4-Runx2 interactions; and iii) we will determine whether PTH signals increase ATF4-Runx2 binding and if these interactions are necessary for PTH actions in osteoblasts in vitro. Aim 2. Establish whether the anabolic actions of PTH require ATF4. We will determine if ATF4 is required for the anabolic actions of PTH in vivo using the ATF4-deficient mice and transgenic mice overexpressing ATF4 in osteoblasts. Aim 3. Determine whether ATF4-Runx2 interactions are required for normal and PTH-induced bone formation in vivo. This project will explore a novel mechanism to explain the anabolic actions of PTH and define new potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases.