Carboxyl-Terminal PTH Receptors in Bone Cell

描述(申请人提供)：甲状旁腺激素(PTH)强烈调节骨骼和软骨中的细胞分化和功能。完整的甲状旁腺激素(1-84)的氨基末端激活1型甲状旁腺素/甲状旁腺素受体(PTH1Rs)，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。现在很清楚，骨细胞也表达一类不同的甲状旁腺素受体(“CPTHR”)。它们只识别完整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，但无法结合或激活PTH1Rs。甲状旁腺素C片段由甲状旁腺以钙调节的方式分泌，也由完整甲状旁腺的外周裂解产生。它们构成了血液中甲状旁腺激素的大部分，在肾功能衰竭时积聚到高水平，这与生长迟缓和骨营养不良有关。CPTH片段hPTH(7-84)是存在于血液中的扩展CPTH片段的模型，在体内发挥抗癌作用，在体外发挥似乎不涉及PTH1R激活的抗吸收作用。克隆的PTH1R缺失的小鼠骨细胞、成骨细胞、骨髓基质细胞和软骨细胞特异性地结合CPTHR放射性配体125I[Tyr34]hPTH(19-84)，并对一系列截短的CPTH多肽显示出相同的配体选择性模式。在体外培养的骨骼细胞中，CPTH片段可诱导细胞内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的凋亡。这个项目将解决这样的假设，即CPTHR由骨细胞正常表达，并可以通过完整的PTH和循环中的CPTH片段介导骨骼功能的调节。CPTHR基因(S)将从已建立的骨细胞文库中克隆出来，通过在CPTHR缺失的宿主细胞中表达这些cDNA来重建CPTHR的功能，并将通过原位杂交和Northern分析(Aim I)来分析CPTHR在小鼠中的组织特异性表达。将定义CPTHR在体外调节骨吸收和破骨细胞形成所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(AIM II)。在PTH1R缺失的克隆骨细胞中，CPTHR强烈调控的基因将被确定，并将寻求在体内通过CPTH多肽控制这些基因的表达(目标III)。这项研究将提供新的信息和试剂，以进一步确定CPTHR在正常骨生理学中的作用，并可能在与慢性肾功能衰竭等疾病相关的骨骼疾病的病因学中发挥作用。

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.