William F, Wagner F, Karin M, Kraft A S
Division of Hematology/Oncology, University of Alabama, Birmingham 35294.
J Biol Chem. 1990 Oct 25;265(30):18166-71.
In contrast to phorbol esters, multiple doses of diacylgycerols are needed to differentiate U937 human monoblastic leukemic cells to a macrophage-like phenotype. Although both of these agents similarly activate protein kinase C in vitro, it is not known why these agents appear to have differing biologic effects. One possibility is that they regulate gene transcription in slightly different ways. Regulation of gene transcription by phorbol esters is complex and involves the stimulation of the transactivating proteins Jun and Fos which form dimers and bind to the AP-1 enhancer elements (5'-TGAGTCA-3'). To understand whether diacylglycerols regulate gene transcription similarly to phorbol esters and to examine whether activation of AP-1 enhancer activity is correlated with differentiation, we have treated U937 human monoblastic leukemic cells with these agents and examined activation of transcription from AP-1 enhancer elements. We find that, although a single dose of diacylglycerol, like phorbol esters, is sufficient to elevate mRNA levels of both the c-jun and c-fos protooncogenes, in contrast to phorbol esters there is no increase in either Jun protein or activation of AP-1 enhancer activity. However, multiple doses of this agent given over 24 h stimulate repeated elevations in c-jun and c-fos mRNA, increases in Jun protein, and enhancer activation. Treatment of U937 cells with ionomycin, a calcium ionophore, also stimulates an increase in c-jun mRNA, but neither activates AP-1 enhancer activity nor stimulates differentiation of these cells. However ionomycin functions to enhance the effects of diacylglycerols both on transcriptional activation and U937 differentiation. These results suggest a complex regulation of AP-1 enhancer activity in U937 cells by diacylglycerols involving both transcriptional and post-transcriptional regulatory mechanisms. Maximal activation of AP-1 enhancer elements, and not changes in jun and fos mRNA, is correlated with increases in markers of U937 differentiation. These changes may be important in the early events leading to differentiation of hematopoietic cells.
与佛波酯不同，需要多剂量的二酰基甘油才能将U937人单核细胞白血病细胞诱导分化为巨噬细胞样表型。尽管这两种试剂在体外均能类似地激活蛋白激酶C，但尚不清楚为何这些试剂似乎具有不同的生物学效应。一种可能性是它们以略有不同的方式调节基因转录。佛波酯对基因转录的调节很复杂，涉及对反式激活蛋白Jun和Fos的刺激，它们形成二聚体并与AP-1增强子元件（5'-TGAGTCA-3'）结合。为了了解二酰基甘油是否与佛波酯类似地调节基因转录，并研究AP-1增强子活性的激活是否与分化相关，我们用这些试剂处理了U937人单核细胞白血病细胞，并检测了AP-1增强子元件的转录激活情况。我们发现，虽然单剂量的二酰基甘油与佛波酯一样，足以提高原癌基因c-jun和c-fos的mRNA水平，但与佛波酯不同的是，Jun蛋白没有增加，AP-1增强子活性也没有激活。然而，在24小时内多次给予该试剂会刺激c-jun和c-fos mRNA反复升高、Jun蛋白增加以及增强子激活。用离子霉素（一种钙离子载体）处理U937细胞也会刺激c-jun mRNA增加，但既不激活AP-1增强子活性，也不刺激这些细胞的分化。然而，离子霉素的作用是增强二酰基甘油对转录激活和U937分化的影响。这些结果表明，二酰基甘油对U937细胞中AP-1增强子活性的调节很复杂，涉及转录和转录后调控机制。AP-1增强子元件的最大激活，而不是jun和fos mRNA的变化，与U937分化标志物的增加相关。这些变化可能在导致造血细胞分化的早期事件中很重要。
