Nuclear Events in PTHR1 Action on Bone

甲状旁腺激素（PTH）是钙稳态的重要调节剂， 一种治疗骨质疏松症的骨合成代谢激素。另一方面，甲状旁腺功能亢进， 骨分解本身和慢性肾衰竭中是一个主要问题。激素通过其 成骨细胞上的G蛋白偶联受体，以引起破骨细胞增强的骨吸收。要执行此操作， 成骨细胞响应PTH产生核因子κ-β配体（RANKL）的受体激活剂。 RANKL被广泛认为是PTH诱导的骨松解的主要介质， 甲状旁腺机能亢进导致骨质流失我们在上一轮资助中的工作显示，PTH激活了 cAMP/蛋白激酶A（PKA）抑制盐诱导激酶（SIKs），并需要蛋白磷酸酶（PP） 刺激成骨细胞中Rankl表达。敲低盐诱导激酶（SIKs）2和3以及cAMP- 调节转录辅激活因子2和3（CRTC 2和3）表明所有四个都是该途径的一部分。 此外，丝氨酸-苏氨酸蛋白磷酸酶的抑制降低了PTH诱导的Rankl表达， PTH（1-34）刺激CRTC 2和CRTC 3向细胞核转位。一旦进入细胞核， 这些CRTC与未知的碱性亮氨酸拉链结构域（bZip）转录因子结合，激活 Rankl转录通过结合其远端PTH-应答cAMP-应答元件（克雷斯）。从这些 根据培养细胞的数据和体内的初步数据，我们提出了一个中心假设， CRTC 2/3的调节对于它们在骨骼中的功能是必不可少的，并且PTH控制这种调节。很长的- 这项工作的长期目标是描绘信号和转录调控机制， PTH在骨中的作用。因此，具体的目的是测试我们的假设，这重新提交的竞争 更新提案的重点是SIK和PP对CRTC 2/3功能和Rankl行动的监管，并将，1） 通过a.确定PTH调节成骨细胞中Rankl转录的机制。阐明 成骨细胞中涉及CRTC 2/3核转位的调节机制，B.识别bZip 负责CRTC 2/3诱导的Rankl转录的转录因子，2）决定CRTC 2/3的作用 在骨骼发育和PTH的合成代谢和分解代谢的影响。确定成骨细胞的位置和作用 骨发育中的CRTC 2/3，B.确定CRTC 2/3缺失对PTH合成代谢的影响， 成年老鼠骨头中的分解代谢作用。这项工作的成果将对我们的 了解PTH如何通过SIK/PP/CRTC/bZip通路对骨骼功能发挥其核效应。 在这样做的过程中，这些数据也将为钙代谢紊乱的治疗提供新的视角， 通过PTHR 1途径发挥功能的其他严重疾病，如詹森干骺端 软骨发育不良和麦-奥二氏综合征。

Parathyroid hormone (PTH) is an essential regulator of calcium homeostasis and became the prototypic osteoanabolic hormone for treating osteoporosis. On the other hand, hyperparathyroidism, with its consequent bone breakdown, is a major problem in and of itself and in chronic renal failure. The hormone acts through its G-protein-coupled receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. To do this, the osteoblast produces receptor activator of nuclear factor kappa-β ligand (RANKL) in response to PTH. RANKL is widely considered a principal mediator of PTH-induced bone catabolism and has been associated with bone loss in hyperparathyroidism. Our work in the last cycle of this grant revealed that PTH activates cAMP/protein kinase A (PKA) to inhibit salt-inducible kinases (SIKs) and requires protein phosphatases (PPs) to stimulate Rankl expression in osteoblasts. Knockdown of salt-inducible kinases (SIKs) 2 and 3 and cAMP- regulated transcription coactivators 2 and 3 (CRTC2 and 3) indicate that all four are part of this pathway. Moreover, inhibition of serine-threonine protein phosphatases decreased both PTH-induced Rankl expression and the stimulation by PTH(1-34) of CRTC2 and 3 translocation into the nucleus. Upon entry into the nucleus, these CRTCs associate with unknown basic leucine zipper domain (bZip) transcription factor(s), activating Rankl transcription through binding to its distal PTH-responsive cAMP-response elements (CREs). From these data of cells in culture and preliminary data in vivo, we have developed the central hypothesis that SIK/PP regulation of CRTC2/3 is essential to their function in the skeleton, and PTH controls this regulation. The long- term goals of this work are to delineate the signaling and transcriptional regulatory mechanisms conveying PTH action in bone. Consequently, the specific aims to test our hypothesis of this resubmitted competing renewal proposal focus on the SIK and PP regulation of CRTC2/3 function and action on Rankl, and will, 1) determine the mechanism of PTH regulation of Rankl transcription in osteoblasts by a. elucidating the regulatory mechanisms involved in CRTC2/3 nuclear translocation in osteoblasts, b. identifying the bZip transcription factor(s) responsible for CRTC2/3-induced Rankl transcription, 2) determine the role of CRTC2/3 in bone development and PTH’s anabolic and catabolic effects by a. determining the site and role of osteoblast CRTC2/3 in bone development, b. ascertaining the effects of deletion of CRTC2/3 on PTH’s anabolic and catabolic actions in bones of adult mice. The results of this work will make major contributions to our knowledge of how PTH exerts its nuclear effects on skeletal function through the SIK/PP/CRTC/bZip pathway. In so doing, the data will also provide new perspectives into treatment of disorders of calcium metabolism and other severe diseases functioning through the PTHR1 pathway such as Jansen’s metaphyseal chondrodysplasia and McCune-Albright syndrome.