Nuclear Events in PTH Action on Bone

描述(申请人提供)：甲状旁腺激素(PTH)在调节钙代谢中起核心作用。这种激素通过其在成骨细胞上的G蛋白偶联受体起作用，促使破骨细胞增强骨吸收。成骨细胞在甲状旁腺素的作用下产生蛋白水解酶。其中之一是基质金属蛋白酶-13(MMP13，胶原酶-3)。这种酶已被证明在甲状旁腺素刺激的骨吸收、钙化反应和软骨内骨形成中起关键作用。我们发现，甲状旁腺激素通过蛋白激酶A(PKA)依赖的途径诱导成骨细胞中的基质金属蛋白酶-13基因转录，该途径调节与基质金属蛋白酶-13启动子相关的许多蛋白质。我们在这项资助的最后一个周期的工作使我们得出结论，在基础条件下，组蛋白脱乙酰酶-4(HDAC4)的存在被组蛋白脱乙酰酶-4(HDAC4)所抑制，组蛋白脱乙酰酶-4(HDAC4)与Runx2结合在Runt结构域的位置(MMP13启动子的-132/-126处)。PTH导致依赖于PKA的Runx2和HDAC4的磷酸化，导致HDAC4从MMP-13启动子释放并转运到细胞质。在细胞核中，Runx2然后招募组蛋白乙酰转移酶(HATS)、p300和p300/CBP相关因子(PCAF)。新转录和合成的Fos/Jun随后与激活蛋白-1(MMP-13启动子的-48/-42处的AP-1)结合，并在RD位点与p300、CBP和与Runx2结合的蛋白相互作用；然后进行最大转录。通过HDAC4与Runx2重新结合以及通过III类HDAC SIRT1与AP-1位点的Fos/Jun结合来重新启动抑制。从这些培养细胞的数据和我们在体内的初步数据，我们发展了一个中心假设，即HDACs是骨架中依赖Runx2的基因的重要调节因子，维持基因的碱基抑制状态，PTH通过解离HDAC4导致瞬时转录诱导；通过HDACs的重新结合重新启动抑制。这项工作的长期目标是描绘在成骨细胞和骨中传递甲状旁腺素作用的转录调控机制。因此，为了验证我们的假设，我们修订的竞争性延续方案集中在共抑制蛋白HDAC4和SIRT1上，并将：1)研究HDAC4在调节Runx2依赖基因表达中的作用，A)它与Runx2的相互作用，B.HDAC4缺失小鼠中表达Runx2依赖基因的细胞，c.MMP-13与HDAC4条件缺失小鼠的骨代谢，2)研究SIRT1通过A与AP-1蛋白的相互作用，b.Sirt1条件缺失小鼠的骨代谢，调节MMP13的表达。这项工作的结果将对我们了解甲状旁腺激素是如何发挥其对骨骼功能的核作用做出重大贡献。此外，它还将定义HDAC如何为此做出贡献。通过这样做，这些数据还将为钙代谢障碍、癌症相关骨病和其他骨病的治疗提供新的视角。 与公共健康相关：这项研究将研究一种蛋白质激素(甲状旁腺激素，PTH)如何能够与骨骼细胞表面相互作用，并将信号传递到细胞的DNA，以调节与骨骼和软骨更新相关的基因的表达。甲状旁腺素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导致开发新药来取代甲状旁腺素，治疗骨质疏松症和其他骨骼疾病。

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.