Nuclear Events in PTH Action on Bone

性状（由申请人提供）：甲状旁腺激素（PTH）在钙代谢的调节中起核心作用。该激素通过其G蛋白偶联受体作用于成骨细胞，以引起破骨细胞增强的骨吸收。成骨细胞产生蛋白酶以响应PTH。其中之一是基质金属蛋白酶-13（MMP-13，胶原酶-3）。这种酶已被证明在PTH刺激的骨吸收和钙离子反应以及软骨内骨形成中具有关键作用。我们已经表明，甲状旁腺素诱导MMP-13基因转录成骨细胞通过蛋白激酶A（PKA）依赖的途径，调节许多与MMP-13启动子相关的蛋白质。我们在该资助的最后一个周期中的工作使我们得出结论，在基础条件下，MMP-13基因被在runt结构域结合位点（MMP-13启动子的-132/-126处的RD）处与Runx 2结合的组蛋白脱乙酰基酶-4（HDAC 4）的存在所抑制。PTH引起Runx 2和HDAC 4的PKA依赖性磷酸化，导致HDAC 4从MMP-13启动子释放并运输到细胞质。在细胞核中，Runx 2然后募集组蛋白乙酰基转移酶（HAT）、p300和p300/CBP相关因子（PCAF）。新转录和合成的Fos/Jun随后与激活蛋白-1（MMP-13启动子的-48/-42处的AP-1）位点结合，并与p300、CBP和在RD位点与Runx 2结合的蛋白质相互作用;最大转录然后增强。通过HDAC 4重新结合Runx 2和通过III类HDAC SIRT 1在AP-1位点结合Fos/Jun重新启动抑制。从这些培养细胞的数据和我们在体内的初步数据，我们已经开发了中心假设，HDAC是骨架中Runx 2依赖性基因的重要调节因子，将基因维持在基础抑制状态，PTH通过HDAC 4的解离引起转录的瞬时诱导;通过HDAC的重新结合重新启动抑制。这项工作的长期目标是描绘成骨细胞和骨中传递PTH作用的转录调节机制。因此，测试我们的这种修订的竞争性延续提议的假设的具体目的集中在共阻遏蛋白HDAC 4和SIRT 1上，并且将：1）通过a.其与Runx 2的相互作用，B.在Hdac 4缺失小鼠中表达Runx 2依赖性基因的细胞，c. MMP-13与Hdac 4条件性缺失小鼠骨代谢的关系; 2）通过a.其与AP-1蛋白的相互作用，B. MMP-13和Sirt 1条件性缺失小鼠的骨代谢。这项工作的结果将作出重大贡献，我们的知识，PTH如何发挥其对骨骼功能的核影响。此外，它将定义HDAC如何为此做出贡献。在这样做的过程中，这些数据也将为钙代谢紊乱、癌症相关性骨病和其他骨病的治疗提供新的视角。 公共卫生相关性：这项研究将研究蛋白质激素（甲状旁腺激素，PTH）如何能够与骨细胞表面相互作用，并将信号传递到细胞的DNA，以调节参与骨和软骨转换的基因的表达。PTH是维持血清钙水平所必需的，也被用于治疗骨质疏松症。我们的研究结果可能会导致新的药物正在开发，代替PTH，治疗骨质疏松症和其他骨骼和骨骼疾病。

DESCRIPTION (provided by applicant): Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. The hormone acts through its G-protein-coupled receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. The osteoblast produces proteases in response to PTH. One of these is matrix metalloproteinase-13 (MMP-13, collagenase-3). This enzyme has been shown to have a critical role in PTH-stimulated bone resorption and calcemic responses and endochondral bone formation. We have shown that PTH induces MMP-13 gene transcription in osteoblastic cells through a protein kinase A (PKA)-dependent pathway which regulates many of the proteins associated with the MMP-13 promoter. Our work in the last cycle of this grant has led us to conclude that, under basal conditions, the MMP-13 gene is repressed by the presence of histone deacetylase- 4 (HDAC4) bound to Runx2 at the runt domain binding site (RD at -132/-126 of the MMP-13 promoter). PTH causes the PKA-dependent phosphorylation of Runx2 and HDAC4, resulting in the release of HDAC4 from the MMP-13 promoter and its trafficking to the cytoplasm. In the nucleus, Runx2 then recruits the histone acetyl transferases (HATs), p300 and p300/CBP associated factor (PCAF). Newly transcribed and synthesized Fos/Jun subsequently bind to the activator protein-1(AP-1 at -48/-42 of the MMP-13 promoter) site and interact with p300, CBP and the proteins bound to Runx2 at the RD site; maximal transcription then ensues. Repression is re-initiated by HDAC4 re-binding to Runx2 and by the class III HDAC, SIRT1, binding to Fos/Jun at the AP-1 site. From these data of cells in culture and our preliminary data in vivo, we have developed the central hypothesis that HDACs are essential regulators of Runx2-dependent genes in the skeleton, maintaining the genes in a basally repressed state and PTH causes transient induction of transcription through dissociation of HDAC4; repression is reinitiated by re-binding of HDACs. The long-term goals of this work are to delineate the transcriptional regulatory mechanisms conveying PTH action in osteoblasts and bone. Consequently, the specific aims to test our hypothesis of this revised competing continuation proposal focus on the co-repressor proteins, HDAC4 and SIRT1, and will, 1) investigate the role of HDAC4 in regulating the expression of Runx2-dependent genes by, a. its interaction with Runx2, b. the cells expressing Runx2- dependent genes in Hdac4 null mice, c. MMP-13 and bone metabolism in Hdac4 conditional deletion mice, 2) investigate the role of SIRT1 in regulating the expression of MMP-13 by, a. its interaction with AP-1 proteins, b. MMP-13 and bone metabolism in Sirt1 conditional deletion mice. The results of this work will make major contributions to our knowledge of how PTH exerts its nuclear effects on skeletal function. Moreover, it will define how HDACs contribute to this. In so doing, the data will also provide new perspectives into treatment of disorders of calcium metabolism, cancer-associated bone disease and other bone disorders. PUBLIC HEALTH RELEVANCE: This research will investigate how a protein hormone (parathyroid hormone, PTH) is able to interact with the surface of a cell in bone and transmit signals to the cell's DNA to regulate the expression of genes involved in bone and cartilage turnover. PTH is essential for maintaining serum calcium levels, and is also being used to treat osteoporosis. The results of our research could lead to new drugs being developed, in place of PTH, to treat osteoporosis and other bone and skeletal disorders.