PTH and Mineralization: Role of Matrix Gla Protein

描述(申请人提供)：骨和牙齿的矿化是一个复杂的过程，由羟基磷灰石晶体形成的各种成核剂和抑制剂之间的相互作用控制。在这些蛋白质中，基质GLA蛋白(MGP)是最重要的，有体内证据表明它在调节矿化中起到了作用。最近，我们发现甲状旁腺激素(PTH)诱导MGP，这种反应可能在PTH介导的成骨细胞矿化抑制中起关键作用。然而，参与MGP甲状旁腺激素调控的转录机制尚不清楚。对这一规则的详细了解将有助于理解可用作矿化缺陷患者预防和治疗干预目标的因素。本项目的总体目标是使用遗传学和分子方法来确定MGP在甲状旁腺素调节成骨细胞矿化中的重要性，并确定参与这一调节的转录机制。本项目将验证两个假说：(A)甲状旁腺激素治疗后MGP的诱导与成骨细胞矿化的抑制有关，(B)MGP的刺激是通过第二信使(S)的激活而介导的，第二信使调节MGP基因启动子中特定的顺式作用反应元件。我们将追求以下两个目标：1.确定MGP缺陷或抑制的成骨细胞对甲状旁腺激素治疗的反应；2.确定成骨细胞中MGP对甲状旁腺激素反应所必需的DNA反应元件。目的1将利用MGP缺陷(MGP-/-)小鼠和使用RNAi抑制MGP合成的成骨细胞来确定甲状旁腺素对矿化的调节。我们预计甲状旁腺素对MGP缺失/抑制的成骨细胞的治疗不会抑制矿化。目的2研究MGP启动子的特性，以了解甲状旁腺激素诱导的调控机制。根据我们的初步数据，我们预计Cre和AP-1位点是PTH诱导MGP转录的关键。我们的长期目标将在未来的R01应用中进行测试，我们的长期目标是表征参与PTH介导的MGP调控的特定转录因子，并对MGP进行详细的结构功能分析，因为它与PTH介导的矿化抑制有关。

DESCRIPTION (provided by applicant): Mineralization of bone and teeth is a complex process controlled by the interplay between various nucleators and inhibitors of hydroxyapatite crystal formation. Among these proteins, matrix Gla protein (MGP) is the most important with in vivo evidence for a role in regulating mineralization. Recently, we showed that MGP is induced by parathyroid hormone (PTH), and this response might play a crucial role in PTH-mediated inhibition of mineralization in osteoblasts. However, the transcriptional mechanisms involved in PTH regulation of MGP are not known. Detailed knowledge of this regulation will help in understanding a factor that can be used as a target for preventive and therapeutic interventions for patients with mineralization defects. The overall goal of this project is to use genetic and molecular approaches to determine the importance of MGP in the regulation of osteoblast mineralization by PTH; and to determine the transcriptional mechanisms involved in this regulation. This project will test two hypotheses: (a) induction of MGP is responsible for the inhibition of mineralization seen in osteoblasts following parathyroid hormone treatment, and (b) stimulation of MGP is mediated through activation of second messenger(s) which regulate specific cis-acting response elements in the promoter of the MGP gene. The following two specific aims will be pursued: 1. Determine the response of Mgp-deficient or -suppressed osteoblasts to parathyroid hormone treatment, and 2. Identify the DNA response elements necessary for MGP responsiveness to parathyroid hormone in osteoblasts. Aim 1 will use both Mgp-deficient (Mgp -/-) mice and osteoblasts in which MGP synthesis is suppressed using RNAi to determine the regulation of mineralization by PTH. We expect that PTH treatment of MGP-deficient/suppressed osteoblasts will not inhibit mineralization. Aim 2 will characterize the MGP promoter to understand the regulatory mechanisms involved in PTH induction. We expect, based on our preliminary data that CRE and AP-1 sites are key to PTH induced transcription of MGP. Our long-range goal, which will be tested in a future R01 application, is to characterize specific transcriptional factors involved in PTH-mediated regulation of MGP, and perform detailed structure-function analyses of MGP as it relates to PTH-mediated inhibition of mineralization.