Nuclear Events in PTHR1 Action on Bone

甲状旁腺激素(PTH)是钙稳态的重要调节器，是钙稳态的原型 治疗骨质疏松症的骨合成激素。另一方面，甲状旁腺机能亢进症及其后果 骨质破坏本身和慢性肾功能衰竭都是一个主要问题。这种荷尔蒙通过它的 成骨细胞上G蛋白偶联受体诱导破骨细胞增强骨吸收。要做到这点， 成骨细胞在甲状旁腺激素刺激下产生核因子-β受体激活剂配体(RANKL)。 RANKL被广泛认为是甲状旁腺素诱导的骨分解代谢的主要介质，并已被认为与 甲状旁腺功能亢进症的骨质丢失。我们在这笔赠款的最后一个周期中的工作表明，PTH激活了 CAMP/蛋白激酶A(PKA)抑制盐诱导的蛋白激酶(SIKs)，需要蛋白磷酸酶(PPS)来 刺激成骨细胞RANKL的表达。盐诱导蛋白激酶(SIKs)2和3的基因敲除和cAMP- 受调控的转录共激活因子2和3(CRTC2和3)表明所有四个都是这一途径的一部分。 此外，抑制丝氨酸-苏氨酸蛋白磷酸酶可降低PTH诱导的RANKL表达 PTH(1-34)刺激CRTC2和3易位入核。一旦进入原子核， 这些CRTC与未知的碱性亮氨酸拉链结构域(BZip)转录因子(S)结合，激活 RANKL通过与其远端PTH反应cAMP反应元件(Cres)结合而转录。从这些 细胞培养的数据和体内的初步数据，我们发展了SIK/PP的中心假设 CRTC2/3的调节对于它们在骨骼中的功能是必不可少的，而PTH控制着这一调节。长的- 这项工作的学期目标是描绘信号和转录调控机制，传递 甲状旁腺素在骨骼中的作用。因此，具体目的是检验我们关于这一重新提交的竞争的假设 更新建议侧重于CRTC2/3的SIK和PP调节功能和对RANKL的作用，并将，1) 甲状旁腺激素调节成骨细胞RANKL转录的机制 成骨细胞中CRTC2/3核转位的调控机制，B.鉴定bZip 转录因子(S)负责CRTC2/3诱导的RANKL转录，2)决定CRTC2/3的作用 在骨发育和甲状旁腺激素的合成代谢和分解代谢中的作用 CRTC2/3在骨发育中的作用，B.确定CRTC2/3缺失对PTH合成代谢和 成年小鼠骨骼的分解代谢作用。这项工作的成果将为我们的 了解甲状旁腺激素如何通过Sik/PP/CRTC/bZip途径对骨骼功能产生核效应。 通过这样做，这些数据还将为治疗钙代谢紊乱和 其他通过PTHR1途径发挥作用的严重疾病，如Jansen干骺端 软骨发育不良和麦肯-奥尔布赖特综合征。

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.