Second Messengers in PTH Action

甲状旁腺激素(PTH)通过PTH/PTHrP受体(PTHR)作用，以一种复杂的方式调节骨细胞的活动。间歇(每天一次)给药会增加骨量，而持续给药会导致净骨吸收。PTHR同时激活几种不同的细胞内效应通路，包括腺酰环化酶(AC)、磷脂酶C(PLC)和蛋白激酶C(PKC)。这个项目提出的假设是，间歇性和持续性甲状旁腺激素对骨骼的合成代谢和分解代谢效应反映了这些效应通路的不同激活模式。我们已经开发了独特的PTH类似物，它们优先通过PTHR激活或抑制AC非依赖信号的产生，以及选择性地在PLC/PKC信号转导中存在缺陷的突变型(Dsel)PTHR。这些工具使我们能够剖析这些通路相对于AC/cAMP信号在甲状旁腺素调节骨形成和吸收中的作用。在类似激活AC的剂量下，这些“信号选择性”类似物将每天给药一次(4周)或连续给药(2周)给完整的雄性或去卵巢的雌性小鼠。不同部位的骨骼反应将通过以下方式进行评估 DXA、MicroCT和动态组织形态计量学，以及血清生化、骨标记物和骨骼基因表达的变化。我们将通过体外骨髓培养来寻找体内诱导的骨祖细胞数量的变化，并通过甲状旁腺素或类似物体外处理正常骨髓细胞来进一步分析观察到的差异的机制。为了研究PLC/PKC信号在特定的体外系统中的作用，将使用已建立的细胞系，包括用野生型或Dsel突变型PTHR重组的PTHR-空细胞，来评估信号特异性差异对成骨细胞增殖、分化和凋亡的调控。ERK1/2，Ras/Raf和Rap-1/B-Raf信号在增殖反应中的作用，以及p38MAPK在成骨细胞分化中的作用，以及不同的PKC亚型可能被PLC依赖和非PLC依赖的信号激活的可能性将被具体讨论。在dsel基因敲入小鼠中的初步证据表明，PLC/PKC信号在PTH调节破骨细胞生成中是重要的，在体内和体内连续治疗方案中，将使用不同的PTH类似物进一步解决 培养完整的颅骨、正常的股骨骨髓或表达PTHR的野生型或dsel 骨髓基质细胞体外培养。这些研究将提供关于特定的甲状旁腺素受体产生的信使信号调节骨骼中细胞活动的方式和程度的重要新信息，从而可能为设计具有改善净合成代谢活性的甲状旁腺素类似物开辟新的途径。

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.