Promotion of HL-60 cell differentiation by 1,25-dihydroxyvitamin D₃ regulation of protein kinase C levels and activity

The hormone 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] promotes differentiation of a number of cell types including HL-60 promyelocytic leukemia cells. It is now established that protein kinase Cβ (PKCβ) plays a critical role in HL-60 cell maturation to a monocyte/macrophage phenotype. In the present study, we investigated the importance of PKCβ levels and activation in 1,25-(OH)₂D₃-mediated differentiation of HL-60 cells. Cell differentiation promoted by 1,25-(OH)₂D₃ at 48 hr was 39 ± 3% (mean ± SEM) nitroblue tetrazolium (NBT) positive and at 72 hr it was 35 ± 2% NBT positive and 70% CD14 positive. Thus, promotion of cell differentiation by 20 nM 1,25-(OH)₂D₃ treatment was maximal at 48-72 hr. When PKCβ levels and cell differentiation were assayed at 72 hr, treatment with 20 nM 1,25-(OH)₂D₃ for the initial 6 hr increased PKCβ levels by 175% but had little effect on cell differentiation (7 ± 2% NBT positive; 11% CD14 positive). The effect of ionomycin, a calcium ionophore, on PKCβ levels and cell differentiation also was examined. Alone, 5 μM ionomycin promoted few cells (3% CD14 positive) to differentiate. In contrast, cells treated with 5 μM ionomycin for 66 hr after a 6-hr pretreatment with 20 nM 1,25-(OH)₂D₃ resulted in 34 ± 5% NBT positive cells and 73% CD14 positive cells. Quantitatively, this induction of differentiation was identical to that observed in cultures continuously treated with 1,25-(OH)₂D₃ (35 ± 2% NBT positive; 70% CD14 positive). Therefore, ionomycin seemed re, replace the requirement for the continuous presence of 1,25-(OH)₂D₃. Chelerythrine chloride (3 μM), a specific PKC inhibitor, blocked differentiation promoted by 1,25-(OH)₂D₃ alone (82 ± 2% inhibition) or in sequence with ionomycin (86 ± 3% inhibition). Taken together, our data show that the capacity of 1,25-(OH)₂D₃ to both increase PKCβ levels and activate PKC is utilized to promote HL-60 cell differentiation. These data further suggest that 1,25-(OH)₂D₃ has a genomic action to increase PKCβ levels and also a nongenomic action requiring its continuous presence to promote HL-60 cell differentiation.