Calcium requirement for the lymphokine and vitamin D mediated differentiation of monoblastic U937 cells

The monocyte-osteoclast hypothesis states that osteoclasts are derived from the differentiation of monocytes. Recently we reported that the U937 cell line, a monoblastic cell of human origin, differentiated to an osteoclast precursor when cultured in the presence of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and lymphokines. Using this model of monocytic differentiation we have tested the role of calcium (Ca) in the culture medium by the addition of CaCl2 in the presence or absence of EGTA, a chelating agent, on the growth and differentiation of the U937 cells. Increasing the Ca concentration ([Ca]) to 2.0 mM inhibited the differentiation of resting or 1,25(OH)2D3 exposed cells, but had no effect on the cells exposed to lymphokines or lymphokines and 1,25 (OH)2D3. Reducing the [Ca] to 10(-7) M inhibited differentiation but also reduced cell viability. Addition of CaCl2 to medium containing EGTA to produce [Ca] in the range of 10(-5) M to 10(-3) M restored the cellular growth and viability. Inhibition of differentiation associated with the reduced calcium concentration over the range of 10(-5) M to 10(-3) M was not due to growth inhibition. These findings show the importance of the extracellular [Ca] in an in vitro model of monocytic differentiation induced by vitamin D and lymphokines.