Simpson R U, O'Connell T D, Pan Q, Newhouse J, Somerman M J
Department of Pharmacology, the University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
J Biol Chem. 1998 Jul 31;273(31):19587-91. doi: 10.1074/jbc.273.31.19587.
It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKCalpha, PKCbeta, PKCbetaI, and PKCbetaII on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)2D3 (20 nM for 48 h) was inhibited similarly in cells treated with PKCbeta antisense (30 microM) 24 h prior to or at the same time as hormone treatment (86 +/- 9% inhibition; n = 4 versus 82 +/- 8% inhibition; n = 4 (mean +/- S.E.), respectively). In contrast, cells treated with PKCbeta antisense 24 h after 1, 25-(OH)2D3 were unaffected and fully differentiated. PKCalpha antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCbetaI- and PKCbetaII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCbetaII antisense (30 microM) completely blocked CD14 expression induced by 1, 25-(OH)2D3, whereas PKCbetaI antisense had little effect. Interestingly, PKCbetaII antisense blocked differentiation by 87 +/- 7% (n = 2, mean +/- S.D.) but had no effect on 1,25-(OH)2D3 inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)2D3 on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCbetaII expression.
现已认识到蛋白激酶C（PKC）在1,25 - 二羟基维生素D3（1,25 - (OH)2D3）促进HL - 60细胞分化中起关键作用。在本研究中，检测了针对PKCalpha、PKCbeta、PKCbetaI和PKCbetaII的硫代磷酸酯反义寡核苷酸对HL - 60早幼粒细胞分化和增殖的影响。通过非特异性酯酶活性、硝基蓝四氮唑还原以及CD14表面抗原表达来确定细胞分化。在用激素处理（1,25 - (OH)2D3，20 nM，处理48小时）之前24小时或与激素处理同时用PKCbeta反义寡核苷酸（30 microM）处理的细胞中，1,25 - (OH)2D3促进的分化受到类似抑制（抑制率分别为86 +/- 9%；n = 4和82 +/- 8%；n = 4（平均值 +/- 标准误））。相反，在1,25 - (OH)2D3处理24小时后用PKCbeta反义寡核苷酸处理的细胞未受影响且完全分化。PKCalpha反义寡核苷酸未阻断1,25 - (OH)2D3对HL - 60细胞分化的促进作用。接下来，检测了PKCbetaI和PKCbetaII特异性反义寡核苷酸阻断1,25 - (OH)2D3促进细胞分化的能力。PKCbetaII反义寡核苷酸（30 microM）完全阻断了1,25 - (OH)2D3诱导的CD14表达，而PKCbetaI反义寡核苷酸几乎没有作用。有趣的是，PKCbetaII反义寡核苷酸使分化受到87 +/- 7%的阻断（n = 2，平均值 +/- 标准差），但对1,25 - (OH)2D3抑制细胞增殖没有影响。这些结果表明，通过阻断PKCbetaII表达可区分1,25 - (OH)2D3对HL - 60细胞分化和增殖的影响。
