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 Cbeta (PKCbeta) plays a critical role in HL-60 cell maturation to a monocyte/macrophage phenotype. In the present study, we investigated the importance of PKCbeta 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 PKCbeta levels and cell differentiation were assayed at 72 hr, treatment with 20 nM 1,25-(OH)2D3 for the initial 6 hr increased PKCbeta 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 PKCbeta levels and cell differentiation also was examined. Alone, 5 microM ionomycin promoted few cells (3% CD14 positive) to differentiate. In contrast, cells treated with 5 microM 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 to replace the requirement for the continuous presence of 1,25-(OH)2D3. Chelerythrine chloride (3 microM), 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 PKCbeta 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 PKCbeta levels and also a nongenomic action requiring its continuous presence to promote HL-60 cell differentiation.
Pan
Q
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Department of Pharmacology, School of Medicine, The University of Michigan, Ann Arbor 48109-0632, U.S.A.
Granger
J
J
O'Connell
T D
TD
Somerman
M J
MJ
Simpson
R U
RU
D002117:Calcitriol / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D018922:HL-60 Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D016130:Immunophenotyping; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D016897:Respiratory Burst
D007476:Ionophores; D018950:Lipopolysaccharide Receptors; D015759:Ionomycin; D011493:Protein Kinase C; D002117:Calcitriol