Carboxyl-Terminal PTH Receptors in Bone Cell

描述（由申请人提供）：甲状旁腺激素（PTH）强烈调节骨和软骨中的细胞分化和功能。完整PTH（1-84）的氨基末端激活1型PTH/PTHrP受体（PTH 1 R），产生cAMP和其他第二信使，控制骨骼组织中的基因表达和细胞功能。现在清楚的是，骨细胞也表达不同类别的PTH受体（“CPTHR”）。这些仅识别完整PTH（1-84）的羧基（C）末端部分，该结构域在整个进化过程中广泛保守，但不能结合或激活PTH 1 R。甲状旁腺以钙调节的方式分泌PTH C片段，并且也通过完整PTH的外周切割产生。它们构成血液中的大部分PTH，并在肾衰竭时积累到高水平，这与生长迟缓和骨营养不良有关。CPTH片段hPTH（7-84）是血液中存在的扩展CPTH片段的模型，其在体内发挥抗钙化作用，在体外发挥抗吸收作用，似乎不涉及PTH 1 R活化。克隆、PTH 1 R-无效鼠骨细胞、成骨细胞、骨髓基质细胞和软骨细胞特异性结合CPTHR放射性配体125 I [Tyr 34]hPTH（19-84），并对一系列截短的CPTH肽显示出相同的配体选择性模式。在体外培养的骨骼肌细胞中，CPTH片段可引起细胞内Ca++、PKC和MAP激酶的增加，调控基因表达，调节破骨细胞的形成，促进成骨细胞和骨细胞凋亡。本项目将阐明骨细胞正常表达CPTHR，并通过完整的PTH和循环CPTH片段介导骨骼功能调节的假设。将从建立的骨细胞cDNA文库中克隆CPTHR cDNA，通过在无CPTHR的宿主细胞中表达这些cDNA来重建CPTHR功能，并通过原位杂交和北方分析（Aim I）来分析小鼠中的组织特异性CPTHR表达。将定义体外骨吸收和破骨细胞形成的CPTHR调节中涉及的细胞靶点和作用，以及负责这些作用的CPTH配体结构域（目的II）。将鉴定PTH 1 R缺失克隆骨细胞中受CPTHR强烈调控的基因，并将寻求通过CPTH肽在体内控制此类基因的表达（Aim III）。这项研究将提供新的信息和试剂，进一步确定CPTHR在正常骨生理学中的作用，也许，在与慢性肾功能衰竭等疾病相关的骨骼疾病的病因学中。

DESCRIPTION (provided by applicant): Parathyroid hormone (PTH) strongly regulates cellular differentiation and function in bone and cartilage. The amino-terminus of intact PTH(1-84) activates type-1 PTH/PTHrP receptors (PTH1Rs) to generate cAMP and other second messengers that control gene expression and cellular function in skeletal tissue. It now is clear that bone cells also express a distinct class of PTH receptors ("CPTHRs"). These recognize only the carboxyl(C)-terminal portion of intact PTH(1-84), a domain extensively conserved throughout evolution yet unable to bind or activate PTH1Rs. PTH C-fragments are secreted in a calcium-regulated manner by the parathyroid glands and generated also by peripheral cleavage of intact PTH. They comprise most of the PTH in blood and accumulate to high levels in renal failure, which is associated with growth retardation and osteodystrophy. The CPTH fragment hPTH(7-84), a model of extended CPTH fragments present in blood, exerts anticalcemic effects in vivo and antiresorptive effects in vitro that appear not to involve PTH1R activation. Clonal, PTH1R-null murine osteocytes, osteoblasts, marrow stromal cells and chondrocytes specifically bind the CPTHR radioligand 125I [Tyr34]hPTH(19-84) and show identical patterns of ligand selectivity for a series of truncated CPTH peptides. In skeletal cells in vitro, CPTH fragments can elicit increases in cytosolic Ca++, PKC and MAP kinase; control gene expression; regulate osteoclast formation and promote osteoblast and osteocyte apoptosis. This project will address the hypothesis that CPTHRs are expressed normally by bone cells and can mediate regulation of skeletal function by intact PTH and circulating CPTH fragments. CPTHR cDNA(s) will be cloned from an established osteocyte cDNA library, CPTHR function will be reconstituted by expressing these cDNAs in CPTHR-null host cells and tissue-specific CPTHR expression will be analyzed in mice by in situ hybridization and Northern analysis (Aim I). Cellular targets and actions involved in CPTHR regulation of bone resorption and osteoclast formation in vitro will be defined, as will CPTH ligand domains responsible for these actions (Aim II). Genes strongly regulated by CPTHRs in PTH1R-null clonal bone cells will be identified, and control of the expression of such genes by CPTH peptides in vivo will be sought (Aim III). This research will provide new information and reagents required to further define the roles of CPTHRs in normal bone physiology and, perhaps, in the etiology of skeletal diseases associated with disorders such as chronic renal failure.