Second Messengers in PTH Action

PTH 行动中的第二使者

• 批准号: 7062730
• 负责人: F RICHARD BRINGHURST
• 金额: $ 31.28万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7062730
• 关键词: adenylate cyclase; biological signal transduction; bone development; bone metabolism; calcium metabolism; cell differentiation; cell growth regulation; cell line; gene mutation; hormone receptor; hormone regulation /control mechanism; molecular cloning; osteoblasts; osteoclasts; parathyroid hormones; peptide hormone analog; phospholipase C; photon absorptiometry; protein kinase A; protein kinase C; second messengers; transfection

Parathyroid hormone (PTH), acting via PTH/PTHrP receptors (PTHRs), regulates bone cell activity in a complex manner. Intermittent (once daily) administration increases bone mass, whereas continuous treatment causes net bone resorption. PTHRs simultaneously activate several different intracellular effector pathways, including adenylyl cyclase (AC), phospholipase C (PLC) and protein kinase C (PKC). This project addresses the hypothesis that the anabolic and catabolic effects on bone of intermittent vs. continuous PTH reflect different patterns of activation of these effector pathways. We have developed unique analogs of PTH that preferentially activate or suppress generation of AC-independent signals via the PTHR, as well as mutant (DSEL) PTHRs selectively defective in PLC/PKC signaling. These tools enable us to dissect contributions of these pathways, relative to that of AC/cAMP signaling, to PTH regulation of bone formation and resorption. At doses that comparably activate AC, these "signal-selective" analogs will be administered once daily (4 weeks) or continuously (2 weeks) to intact male or ovariectomized female mice. Skeletal responses at different sites will be assessed by DEXA, microCT and dynamic histomorphometry, and changes in serum biochemistry, bone markers and skeletal gene expression will be measured. Alterations in numbers of marrow osteoprogenitors induced in vivo will be sought using ex vivo marrow cultures, and mechanisms of observed differences further analyzed by treatment of normal marrow cells with PTH or analogs in vitro. Signal-specific differences in regulation of osteoblast proliferation, differentiation and apoptosis will be assessed using established cell lines, including PTHR-null cells reconstituted with either wild type or DSEL mutant PTHRs, to study contributions of PLC/PKC signals in defined in vitro systems. Roles of ERK1/2, Ras/Raf and Rap-1/B-Raf signaling in proliferative responses, and of p38 MAPK in osteoblastic differentiation, will be specifically addressed, and the possibility that different PKC isoforms may be activated by PLC-dependent vs. PLC-independent signaling will be examined. Preliminary evidence in DSEL knock-in mice that PLC/PKC signaling is important for PTH regulation of osteoclastogenesis will be further addressed using different PTH analogs in the continuous-treatment protocols in vivo and in cultures of intact calvarial bone, normal femoral marrow or wild-type vs. DSEL PTHR-expressing marrow stromal cells in vitro. These studies will provide important new information concerning the manner and extent to which specific PTHR-generated messenger signals regulate cellular activity in bone and may thereby open new avenues to the design of PTH analogs with improved net anabolic activity.

甲状旁腺激素 (PTH) 通过 PTH/PTHrP 受体 (PTHR) 发挥作用，以复杂的方式调节骨细胞活性。间歇（每天一次）给药会增加骨量，而连续治疗会导致净骨吸收。 PTHR 同时激活几种不同的细胞内效应通路，包括腺苷酸环化酶 (AC)、磷脂酶 C (PLC) 和蛋白激酶 C (PKC)。该项目提出了这样的假设：间歇性 PTH 与连续性 PTH 对骨骼的合成代谢和分解代谢影响反映了这些效应通路的不同激活模式。我们开发了独特的 PTH 类似物，可通过 PTHR 优先激活或抑制 AC 独立信号的产生，以及在 PLC/PKC 信号传导中选择性缺陷的突变体 (DSEL) PTHR。这些工具使我们能够剖析这些途径（相对于 AC/cAMP 信号传导）对 PTH 骨形成和骨吸收调节的贡献。在相当激活 AC 的剂量下，这些“信号选择性”类似物将每天一次（4 周）或连续（2 周）给予完整的雄性或卵巢切除的雌性小鼠。不同部位的骨骼反应将通过以下方式评估 将测量 DEXA、显微 CT 和动态组织形态计量学，以及血清生化、骨标志物和骨骼基因表达的变化。将使用离体骨髓培养物来寻找体内诱导的骨髓骨祖细胞数量的变化，并通过在体外用PTH或类似物处理正常骨髓细胞来进一步分析观察到的差异的机制。将使用已建立的细胞系（包括用野生型或 DSEL 突变体 PTHR 重建的 PTHR-null 细胞）评估成骨细胞增殖、分化和凋亡调节中的信号特异性差异，以研究 PLC/PKC 信号在特定体外系统中的贡献。将专门讨论 ERK1/2、Ras/Raf 和 Rap-1/B-Raf 信号在增殖反应中的作用，以及 p38 MAPK 在成骨细胞分化中的作用，并将检查不同 PKC 亚型可能被 PLC 依赖性与 PLC 独立信号激活的可能性。 DSEL 敲入小鼠的初步证据表明 PLC/PKC 信号对于破骨细胞生成的 PTH 调节很重要，将在体内和体外连续治疗方案中使用不同的 PTH 类似物进一步解决这一问题。 完整颅骨、正常股骨髓或野生型培养物与 DSEL PTHR 表达的培养物 体外骨髓基质细胞。这些研究将提供关于特定 PTHR 产生的信使信号调节骨中细胞活性的方式和程度的重要新信息，并可能因此为设计具有改善的净合成代谢活性的 PTH 类似物开辟新途径。