Second Messengers in PTH Action

PTH 行动中的第二使者

• 批准号: 7627068
• 负责人: F RICHARD BRINGHURST
• 金额: $ 17.64万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7627068
• 关键词: Address; Adenylate Cyclase; Apoptosis; Back; Biochemical Pathway; Biological Markers; Calcium; Calvaria; Cell Line; Cell physiology; Cells; Continuous Infusion; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DEXA; DNA Binding; Densitometry; Diet; Dose; EP300 gene; Exhibits; Exposure to; Fracture; Genetic; Histology; Homeostasis; Hormonal; In Vitro; Ions; Knock-out; Knockout Mice; Ligands; Link; Mechanics; Mediating; Mediator of activation protein; Minerals; Molecular; Mus; Mutation; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Parathyroid Hormone Receptor; Parathyroid Hormones; Pathological fracture; Pathway interactions; Pattern; Phospholipase C; Play; Property; Protein Kinase C; Protein Kinase C Activation Pathway; Proteins; RNA Interference; Relative (related person); Role; Second Messenger Systems; Serum; Signal Pathway; Signal Transduction; Structure; Testing; Therapeutic Effect; Therapeutic Uses; Vertebral Bone; Wild Type Mouse; Work; analog; bone; bone cell; bone mass; calcium metabolism; calcium phosphate; citrate carrier; defined contribution; design; feeding; hormone analog; human PTH protein; in vitro Assay; in vivo; inhibitor/antagonist; insight; mouse model; mutant; novel; osteoclastogenesis; osteoprogenitor cell; response; second messenger; skeletal; substantia spongiosa; tomography

Parathyroid hormone (PTH) is a principal regulator of bone and mineral-ion homeostasis. When given exogenously, PTH can increase or decrease bone mass, depending upon the dose and temporal pattern of administration. Once-daily injected PTH uniquely augments osteoblastic bone formation and is the only anabolic therapy currently approved for the treatment of osteoporosis. Osteoblasts (Obs), the main targets of PTH action in bone, express PTH/PTHrP receptors (PTHRIs) which can activate, in parallel, multiple effector pathways, including cyclic AMP/protein kinase A, phospholipase C (PLC)-dependent protein kinase C(s) (PKCs) and PLC-independent PKC(s). Activation of these pathways via normal PTHRIs can be separated by use of novel mutant "signal-selective" PTH analogs. Using such analogs, we have shown that PLC-independent signaling mechanism(s) play a positive modulatory role in the anabolic action of PTH in normal mice and that PKC6 is activated by PTHRIs via a PLC-independent mechanism that contributes to PTH stimulation of Ob differentiation in vitro. Further, mice lacking PKC6 have reduced bone mass and develop fractures on a low-calcium diet. In other preliminary studies we also found that the transcriptional co-regulator protein CITED1 is up-regulated in Obs following intermittent but not continuous PTH exposure. CITEDI-knockout (KO) Obs in vitro show increased basal differentiation and strikingly augmented responsiveness to cAMP-dependent cyclic PTH stimulation of Ob differentiation. Using PKC6 and CITED1 KO mice, we will address the following hypotheses: (I) that PKC6 mediates positive modulatory effects of cAMP- and PLC-independent PTHR1 signaling on the anabolic PTH response and may constrain the catabolic action of continuous PTH; and (II) that CITED1 induction by intermittent PTH may feed back to preferentially suppress cAMP-dependent responses and limit the anabolic effect of PTH. Responses of bone in KO mice to intermittent vs. continuous PTH treatment will be assessed by densitometric, microstructural, mechanical, histological, histomorphometric and molecular (mRNA) analyses, supplemented by ex vivo and in vitro assays of Ob commitment, differentiation, proliferation and apoptosis as well as of osteoclast formation and activity. Molecular mechanisms of PKC6 activation and action and of CITED1 action will be defined using established Ob cell lines and calvarial Obs from KO mice. This work will contribute important new insight into how the multiple signals triggered by PTH in target cells of bone are integrated to increase or reduce bone mass. Information gained could guide design of new forms of PTH with selective signaling properties and/or of adjunctive agents directed at PKC5 or CITED1 to augment the therapeutic effect of PTH in osteoporosis.

甲状旁腺激素(PTH)是骨和矿物质离子动态平衡的主要调节因子。当给定时 外源性，甲状旁腺激素可以增加或减少骨量，取决于剂量和时间模式 行政管理。每天注射一次PTH独特地促进成骨细胞骨形成，是唯一 合成代谢疗法目前被批准用于治疗骨质疏松症。成骨细胞(Obs)，主要靶点 甲状旁腺素在骨骼中的作用，表达甲状旁腺素/甲状旁腺素受体(PTHrI)，它可以并行地激活多个 效应通路，包括环磷酸腺苷/蛋白激酶A、磷脂酶C(PLC)依赖的蛋白激酶 C(S)(PKCS)和不依赖PLC的PKC(S)。通过正常的PTHRI激活这些通路可以 通过使用新的突变型“信号选择性”PTH类似物来分离。使用这样的类比，我们已经证明了 非PLC信号转导机制(S)在甲状旁腺素合成代谢中的正向调节作用 而PKC6是由PTHRIs通过一种不依赖PLC的机制激活的，这种机制有助于 PTH对体外培养的Ob分化的刺激作用此外，缺乏PKC6的小鼠骨量减少， 低钙饮食会导致骨折。在其他初步研究中，我们还发现转录 协同调节蛋白CITED1在间歇性但不是持续的甲状旁腺素暴露后在Obs中上调。 CITEDI基因敲除(KO)Obs在体外表现为基本分化增加，并显著增强 对cAMP依赖的周期性甲状旁腺素刺激Ob分化的反应性。使用PKC6和CITED1 KO小鼠，我们将解决以下假设：(I)PKC6介导的正向调节作用 CAMP和PLC不依赖的PTHR1信号在合成代谢的PTH反应中的作用，并可能限制 连续甲状旁腺素的分解代谢作用；以及(Ii)间歇性甲状旁腺激素诱导的CITED1可能反馈到 优先抑制cAMP依赖的反应，限制PTH的合成代谢作用。的回应 KO小鼠间歇性甲状旁腺激素治疗与持续甲状旁腺素治疗的骨密度将通过密度计量学进行评估， 显微结构、力学、组织学、组织形态计量学和分子(信使核糖核酸)分析 通过体外和体外检测Ob的迁移、分化、增殖和凋亡以及 破骨细胞的形成和活性。PKC6激活和作用的分子机制及CITED1 将使用已建立的Ob细胞系和来自KO小鼠的头盖骨Ob来定义作用。 这项工作将为了解靶区PTH触发的多种信号提供重要的新见解 骨细胞被整合以增加或减少骨量。获得的信息可以指导新的 具有选择性信号特性的PTH形式和/或针对PKC5或CITED1的辅助剂 增强甲状旁腺素对骨质疏松症的治疗作用。