NHERF-Mediated PTH Signaling in Mineral Ion Homeostasis

在对骨骼、肾脏和整体矿物质离子动态平衡的生物学作用中，PTH刺激PTH/PTHrP受体，并激活靶细胞内独特的信号通路(包括腺苷环化酶、磷脂酶C、磷脂酶-C非依赖性蛋白激酶C和钙通道)。越来越多的证据表明，这些不同的通路中有一些在骨和肾脏细胞功能上有明显的差异，这导致了对信号选择性及其细胞作用的更多研究。我们的研究小组最近发现，Na+/H+交换调节因子(NHERF)(细胞内信号调节支架蛋白的组成部分)可以作为“分子开关”，促进甲状旁腺素激活PLC途径，抑制腺苷环化酶途径。由合作者进行的NHERF-1基因敲除具有骨量减少的骨骼表型。现在已经在骨细胞系中发现了NHERF。缺乏NHERF的负鼠肾细胞系(OK)缺乏通常甲状旁腺素刺激的磷酸盐吸收阻断。NHERF基因可逆转上述缺陷，恢复野生型OK细胞的功能。该细胞系可作为甲状旁腺素介导的肾小管上皮细胞抑制的模型。 磷酸盐的吸收。因此，我们的具体目标是：目的I.在负鼠肾细胞模型中，分析NHERF在PTH信号转导和细胞形态方面的功能作用，比较NHERF完整、缺陷和复制的细胞系。目的II.研究NHERF/PTH1R复合体在体外培养骨细胞系甲状旁腺激素信号转导中的作用。目的III.研究NHERF-1在体外和体内调节甲状旁腺激素在骨中的作用，与我们的同事提供的NHERF-1-/-小鼠合作，以评估导致骨骼表型的细胞机制。

In its biological actions on bone and kidney and overall mineral ion homeostasis, PTH stimulates the PTH/PTHrP receptor and activates distinctive signalling pathways within target cells (including adenylyl cyclase, phospholipase C, phospholipase-C independent protein kinase C and calcium channels). Growing evidence that some of these distinctive pathways have discernable differences in bone and renal cell function has led to increased study of signal selectivity and its cellular role. Na+/H+exchange regulatory factors (NHERFs) (components of signal modulating intracellular scaffold proteins) have recently been shown by our group to act as "molecular switches" that increase activation by PTH of the PLC pathway and suppress the adenylyl cyclase pathway. A gene knockout of NHERF-1, performed by collaborators, has an osteopenic skeletal phenotype. NHERFs have now been identified in bone cell lines. An opossum kidney cell line (OK) which is deficient in NHERF lacks the usual PTH stimulated blockade of phosphate uptake. The deficiency is reversed and wild type OK cell line function restored by NHERF transfection. This cell line serves as a model of PTH-mediated inhibition of tubular re- absorption of phosphate. Our specific aims are therefore: AIM I. Analyze the functional role of NHERFs with respect to PTH signaling and cell morphology in an opossum kidney cell model, comparing cell lines that are NHERF intact, defective and repleted. AIM II. Study the role of the NHERF/PTH1R complex in PTH signaling in bone cell lines in vitro. AIM III. Study the role of NHERF-1 in mediating the actions of PTH in bone, both in vitro and in vivo, working with the NHERF- 1 -/- mice provided by our colleagues for assessment of cellular mechanisms responsible for the skeletal phenotype.