Second Messengers in PTH Action

甲状旁腺激素（PTH）通过PTH/PTHrP受体（PTHR）起作用，以复杂的方式调节骨细胞活性。间歇性（每日一次）给药可增加骨量，而连续给药可导致净骨吸收。PTHR同时激活几种不同的细胞内效应物途径，包括腺苷酸环化酶（AC）、磷脂酶C（PLC）和蛋白激酶C（PKC）。本项目阐述了间歇性与连续性PTH对骨的合成代谢和分解代谢作用反映了这些效应途径激活的不同模式的假设。我们已经开发了独特的PTH类似物，其通过PTHR优先激活或抑制AC非依赖性信号的产生，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的贡献，相对于AC/cAMP信号传导，PTH调节骨形成和吸收。在不激活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）施用给完整的雄性或卵巢切除的雌性小鼠。将通过以下方式评估不同研究中心的Skeleton反应： 将测量DEXA、microCT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寻找体内诱导的骨髓骨祖细胞数量的改变，并通过用PTH或类似物体外处理正常骨髓细胞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PTHR重建的PTHR-无效细胞）评估成骨细胞增殖、分化和凋亡调控中的信号特异性差异，以研究PLC/PKC信号在定义的体外系统中的贡献。ERK 1/2，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，将被专门讨论，不同的PKC亚型可能被PLC依赖与PLC独立信号激活的可能性将被检查。在DSEL基因敲入小鼠中的初步证据表明，PLC/PKC信号传导对于PTH调节破骨细胞生成是重要的，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。 完整颅骨、正常股骨骨髓或野生型与DSEL PTHR表达的培养物 体外培养骨髓基质细胞。这些研究将提供重要的新信息的方式和程度，具体的PTH产生的信使信号调节骨细胞活性，从而可能开辟新的途径PTH类似物的设计与改善净合成代谢活性。

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.