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

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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.