PROMOTION OF HL-60 CELL-DIFFERENTIATION BY 1,25-DIHYDROXYVITAMIN D-3 REGULATION OF PROTEIN-KINASE-C LEVELS AND ACTIVITY

The hormone 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] promotes diffe rentiation of a number of cell types including HL-60 promyelocytic leu kemia cells. It is now established that protein kinase C beta (PKC bet a) plays a critical role in HL-60 cell maturation to a monocyte/macrop hage phenotype. In the present study, we investigated the importance o f PKC beta levels and activation in 1,25-(OH)(2)D-3-mediated different iation of HL-60 cells. Cell differentiation promoted by 1,25-(OH)(2)D- 3 at 48 hr was 39 +/- 3% (mean +/- SEM) nitroblue tetrazolium (NET) po sitive and at. 72 hr it was 35 +/- 2% NET positive and 70% CD14 positi ve. Thus, promotion of cell differentiation by 20 nM 1,25-(OH)(2)D-3 t reatment was maximal at 48-72 hr. When PKC beta levels and cell differ entiation were assayed at 72 hr, treatment with 20 nM 1,25-(OH)(2)D-3 for the initial 6 hr increased PKC beta levels by 175% but had little effect on cell differentiation (7 +/- 2% NET positive; 11% CD14 positi ve). The effect of ionomycin, a calcium ionophore, on PKC beta levels and cell differentiation also was examined. Alone, 5 mu M ionomycin pr omoted few cells (3% CD14 positive) to differentiate. In contrast, cel ls treated with 5 mu M ionomycin for 66 hr after a 6-hr pretreatment w ith 20 nM 1,25-(OH)(2)D-3 resulted in 34 +/- 5% NET positive cells and 73% CD14 positive cells. Quantitatively, this induction of differenti ation was identical to that observed in cultures continuously treated with 1,25-(OH)(2)D-3 (35 +/- 2% NBT positive; 70% CD14 positive). Ther efore, ionomycin seemed to replace the requirement for the continuous presence of 1,25-(OH)(2)D-3. Chelerythrine chloride (3 mu M), a specif ic PKC inhibitor, blocked differentiation promoted by 1,25-(OH)(2)D-3 alone (82 +/- 2% inhibition) or in sequence with ionomycin (86 +/- 3% inhibition). Taken together, our data show that the capacity of 1,25-( OH)(2)D-3 to both increase PKC beta levels and activate PKC is utilize d to promote HL-60 cell differentiation. These data further suggest th at 1,25-(OH)(2)D-3 has a genomic action to increase PKC beta levels an d also a nongenomic action requiring its continuous presence to promot e HL-60 cell differentiation. (C) 1997 Elsevier Science Inc.