This research grant application addresses a novel interaction between vitamin D metabolism and phagocytic cells. The central hypothesis of this application is that the normal growth and differentiation of phagocytes depends on vitamin D and its metabolites and that the differentiated phagocyte alters the metabolism of vitamin D by producing metabolites with unique functions or catabolic metabolites or both. Phagocytic cells can metabolize 25-hydroxyvitamin D3 to at least two other metabolites of which one is novel and as yet unidentified. We propose to produce novel metabolites in sufficient quantity for chemical and spectroscopic identity. The bioactivity of the novel metabolites as either agonists or antagonists of vitamin D will be measured by several assays. The stimuli thought to regulate the phagocyte metabolism of vitamin D including ionic calcium, lymphokines and cyclic AMP-elevating substances will be studied to ascertain their role in this process. Attention will be given to the possibility that oxygen reduction products formed by phagocytes participate in the metabolism of vitamin D by these cells. Phagocytes with defects in oxidative metabolism, e.g., chronic granulomatous disease, will be utilized in these studies in an attempt to separate these concomitant metabolic events. Bisphosphonates are chemicals known to alter mineral metabolism and produce direct effects on phagocytes. Their influence on the phagocytic metabolism of vitamin D and the oxidative metabolism of these cells will be studied. Several laboratories reported that 1Alpha,25-dihydroxyvitamin D3 induced the differentiation of transformed cells of hematogenous origin. We have recently observed that U937 cells cloned from a human histiocytic lymphoma differentiated into macrophages when incubated with 1Alpha,25(OH)2D3. We propose experiments designed to understand the role of ionic calcium and cyclic AMP in this induction process. The physiological relevance of this phenomenon will be tested in macrophage precursors isolated from vitamin D-deficient rats and incubated in vitro with 1Alpha,25(OH)2D3. Membrane proteins such as the C3 receptor can be detected after the induction. Phagocytes incubated with 1Alpha,25(OH)2D3 will be labeled with 35S-methionine. Radiolabeled membrane proteins will be isolated and tentatively identified by gel electrophoresis. A working hypothesis states that these proteins function as calcium transport proteins or hormone receptors, both of which may be involved in the normal growth and differentiation of the cells.
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