REGULATION OF CELL DIFFERENTIATION BY 1,25(OH)2D3

本项目旨在研究三个主要假设：1) 1，25(OH)2-D3诱导细胞外钙快速进入靶点 细胞；2)钙离子的进入依赖于“未知”的存在。 质膜中的因子，推测为蛋白质；和3)1，25(OH)2- D3介导的钙内流是细胞内钙离子表达的触发因素 靶细胞的分化表型。调查人员计划 以解决当前提案中的三个具体目标。在第一个 目的他们建议描述维生素D和其他类固醇的作用 巨噬细胞系细胞内游离钙及钙内流的研究 钙代谢系统的细胞和靶细胞。在第二个 目的研究1，25(OH)2-D3的调节机制。 钙离子进入靶细胞；包括近端肾小管 细胞、成骨细胞、成骨细胞样成骨肉瘤细胞、 巨噬细胞和巨噬细胞样HL-60细胞。在最后一个目标中，他们 将分析1，25(OH)2-D3作为分化因子的作用 近端肾小管细胞和成骨细胞。在…的过程中 在具体目标1中概述的实验，研究人员将应用 大多数用于研究肾上皮细胞的方法都是从 1，25(OH)2-D3介导的钙离子通量和磷脂的研究 交通人骨髓来源的巨噬细胞样细胞。他们 假设三种不同程度的不同表型 分化是存在的，这样的原生文化应该做出回应 不同于1，25(OH)2-D3。非粘附性CPU前体细胞(其 显然缺乏1，25(OH)2-D3的受体)将与更多 成熟贴壁细胞和更多分化的单核巨噬细胞样细胞 细胞。据报道，后两种人群对 1，25(OH)2-D3。目标2建议对 1，25(OH)2-D3介导的大鼠近端肾小管钙离子内流 上皮细胞使用基底外侧膜小泡(BLMV)，钙 通量技术，Fura II在分离细胞中的记录，荧光 视频成像分析和膜片钳记录。AIM中的实验 3将探讨1，25(OH)2-D3在促进分化中的作用 人UMR-106细胞、负鼠肾(OK)细胞的原代培养 成骨细胞和犬近端肾小管细胞。标记物： 将被监测的成熟表型都与 甲状旁腺素受体的表达及其转导能力 生物可探测信号。

This project is designed to investigate three major hypotheses: 1) 1,25(OH)2-D3 induces rapid entry of extracellular calcium into target cells; 2) Calcium entry is dependent upon the presence of an "unknown" factor in the plasma membrane, presumably a protein; and 3) 1,25(OH)2- D3-mediated calcium entry is a trigger for the expression of the differentiated phenotype of the target cell. The investigators plan to address three specific aims in the current proposal. In the first aim they propose to describe the effects of vitamin D and other sterols on cytosolic free calcium and calcium influx of macrophage lineage cells and target cells of the calcium metabolism system. In the second aim they will investigate the mechanisms of 1,25(OH)2-D3 -regulated calcium entry into target cells; including proximal renal tubular cells, osteoblasts, osteoblast-like osteogenic sarcoma cells, macrophages and the macrophage-like HL-60 cells. In the last aim they will analyze the role of 1,25(OH)2-D3 as a factor for differentiation of proximal renal tubular cells and osteoblasts. In the course of experiments outlined in specific aim 1, the investigators will apply much of the methodology used to study renal epithelium to an investigation of 1,25(OH)2-D3-mediated calcium flux and phospholipid traffic in human bone marrow derived, macrophage-like cells. They postulate that three distinct phenotypes of varying degrees of differentiation are present and such primary cultures should respond differently to 1,25(OH)2-D3. Non-adherent CPU precursor cells (which apparently lack receptors for 1,25(OH)2-D3) will be compared to more mature adherent cells and more differentiated monocyte macrophage-like cells. The latter two populations are reported to respond to 1,25(OH)2-D3. Aim 2 proposes investigations into the mechanism of 1,25(OH)2-D3-mediated calcium entry into rat proximal tubular epithelial cells using basolateral membrane vesicles (BLMV), calcium flux technology, Fura II recording in isolated cells, fluorescence video imaging analysis and patch-clamp recording. Experiments in Aim 3 will prob e the role of 1,25(OH)2-D3 in promoting the differentiation of UMR-106 cells, Opossum Kidney (OK) cells, primary cultures of human osteoblasts and canine proximal renal tubular cells. Markers of the mature phenotype that will be monitored are all linked to the expression of the PTH receptor and its ability to transduce biodetectable signals.