Pan Q, Granger J, O'Connell T D, Somerman M J, Simpson R U
Department of Pharmacology, School of Medicine, The University of Michigan, Ann Arbor 48109-0632, U.S.A.
Biochem Pharmacol. 1997 Oct 15;54(8):909-15. doi: 10.1016/s0006-2952(97)00286-4.
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.
激素1,25 - 二羟基维生素D3 [1,25-(OH)2D3]可促进包括HL - 60早幼粒细胞白血病细胞在内的多种细胞类型的分化。现已确定蛋白激酶Cβ（PKCβ）在HL - 60细胞成熟为单核细胞/巨噬细胞表型的过程中起关键作用。在本研究中，我们调查了PKCβ水平及其激活在1,25-(OH)2D3介导的HL - 60细胞分化中的重要性。48小时时，1,25-(OH)2D3促进的细胞分化有39±3%（平均值±标准误）的硝基蓝四氮唑（NBT）阳性，72小时时为35±2%的NBT阳性和70%的CD14阳性。因此，20 nM 1,25-(OH)2D3处理促进细胞分化的作用在48 - 72小时达到最大。当在72小时检测PKCβ水平和细胞分化时，最初6小时用20 nM 1,25-(OH)2D3处理可使PKCβ水平升高175%，但对细胞分化影响很小（7±2%的NBT阳性；11%的CD14阳性）。我们还研究了钙离子载体离子霉素对PKCβ水平和细胞分化的影响。单独使用时，5μM离子霉素仅促使少数细胞（3%的CD14阳性）分化。相比之下，先用20 nM 1,25-(OH)2D3预处理6小时后，再用5μM离子霉素处理66小时，结果产生了34±5%的NBT阳性细胞和73%的CD14阳性细胞。从数量上看，这种分化诱导与持续用1,25-(OH)2D3处理的培养物中观察到的情况相同（35±2%的NBT阳性；70%的CD14阳性）。因此，离子霉素似乎可以替代持续存在1,25-(OH)2D3的需求。氯化白屈菜红碱（3μM），一种特异性PKC抑制剂，可阻断单独由1,25-(OH)2D3促进的分化（抑制率为82±2%）或与离子霉素先后处理促进的分化（抑制率为86±3%）。综上所述，我们的数据表明，1,25-(OH)2D3提高PKCβ水平和激活PKC的能力被用于促进HL - 60细胞分化。这些数据进一步表明，1,25-(OH)2D3具有增加PKCβ水平的基因组作用，也具有需要其持续存在以促进HL - 60细胞分化的非基因组作用。
