Nuclear Events in PTH Action on Bone

描述（由申请人提供）：甲状旁腺激素（PTH）在钙代谢调节中起核心作用。该激素通过其g蛋白偶联受体作用于成骨细胞，引起破骨细胞增强骨吸收。成骨细胞对甲状旁腺激素产生蛋白酶。其中之一是基质金属蛋白酶-13 （MMP-13，胶原酶-3）。该酶已被证明在甲状旁腺激素刺激的骨吸收和钙血症反应以及软骨内骨形成中起关键作用。我们已经证明，PTH通过蛋白激酶a （PKA）依赖性途径诱导成骨细胞中MMP-13基因转录，该途径调节许多与MMP-13启动子相关的蛋白质。我们在最后一个周期的工作使我们得出结论，在基础条件下，组蛋白去乙酰化酶- 4 （HDAC4）与Runx2结合在runt结构域结合位点（RD位于MMP-13启动子的-132/-126）的存在抑制了MMP-13基因。PTH引起Runx2和HDAC4的pga依赖性磷酸化，导致HDAC4从MMP-13启动子释放并转运到细胞质中。在细胞核中，Runx2随后募集组蛋白乙酰转移酶（HATs）、p300和p300/CBP相关因子（PCAF）。新转录和合成的Fos/Jun随后结合到MMP-13启动子-48/-42位点的激活蛋白1（AP-1）上，并与p300、CBP和RD位点的Runx2结合蛋白相互作用；然后是最大转录。抑制通过HDAC4重新结合Runx2和III类HDAC SIRT1在AP-1位点与Fos/Jun结合而重新启动。根据这些培养细胞的数据和我们在体内的初步数据，我们已经提出了一个中心假设，即hdac是骨架中runx2依赖基因的重要调节剂，维持这些基因处于基本抑制状态，PTH通过HDAC4的解离引起短暂的转录诱导；抑制通过hdac的重新结合而重新启动。这项工作的长期目标是描述在成骨细胞和骨中传递甲状旁腺激素作用的转录调控机制。因此，我们的具体目标是验证我们的假设，这一修订的竞争延续建议的重点是共同抑制蛋白，HDAC4和SIRT1，并将，1)研究HDAC4在调节Runx2依赖基因表达中的作用，a.与Runx2相互作用，b.在HDAC4缺失小鼠中表达Runx2依赖基因的细胞，c.在HDAC4条件缺失小鼠中MMP-13和骨代谢，2)研究SIRT1在调节MMP-13表达中的作用，通过，a.它与AP-1蛋白的相互作用，b. Sirt1条件缺失小鼠的MMP-13和骨代谢。这项工作的结果将对我们了解甲状旁腺激素如何对骨骼功能施加其核影响做出重大贡献。此外，它将定义hdac如何对此做出贡献。在此过程中，这些数据也将为钙代谢障碍、癌症相关骨病和其他骨疾病的治疗提供新的视角。

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.