The focus of this research proposal is the interaction between phagocytes, vitamin D and mineral metabolism including bone. Phagocytes metabolize 25(OH)D3 in vitro to 19 nor, 10 keto, 250HD3 and 24,25(OH)2D3. We propose to study in detail the formation of either 1,25(OH)2D3 or 19/10 by cultured cells including renal cells from vitamin D deficient mice, peritoneal macrophages from these mice and, when the conditions have been optimized, pulmonary alveolar macrophages. Preliminary evidence indicates that the U937 cell line, a transformed monocytic cell line, lacks cytochrome P450 in the undifferentiated state and expresses this substance when exposed to 250HD3 or 1,25(OH)2D3. The possibility of altered metabolism of 250HD3 in the differentiated phagocyte compared to its precursor will be examined. Next we will study phagocytic functions in vitamin D deficient mice. If any defects are found, the effects of 1,25(OH)2D3 or 19/10 on these defects will be determined. The U937 cell model will be converted to a D- deficient model which will then be induced to differentiate by 1,25(OH)2D3 or 19/10. The concentrations of metabolites active in the D-deficient model will be compared to previous results. When optimal conditions for induced differentiation have been defined, the role of ionic calcium will be studied using calcium ionophores, calcium channel blockers and changes in the calcium concentration of the medium. The role of protein kinase C in the differentiation will be examined in experiments involving stimulation or suppression of this enzyme. Phagocytes also secrete a soluble factor that resorbs devitalized bone particles. The isolation, partial purification and regulation of this resorption factor will be undertaken in an effort to increase our knowledge about the role of phagocytes in bone resorption.
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