Abstract
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.
Highlights
Multiple Doses of Diacylglycerol and Calcium Ionophore Are Necessary to Activate AP-1 Enhancer Activity and Induce Markers of Macrophage Differentiation*
Adding phorbol esters to U937 leukemic cells stimulates an increase in c-jun and c-fos RNA levels starting within 15 min of addition and decreasing after l-2 h (Fig. 1)
Changes occur in both the 2.7- and 3.4-kb c-jun mRNA over a similar time course. Both the HL-60 and PLB-985 leukemic cell lines (Tucker et al, 1988) which are induced to differentiate into macrophages by phorbol esters yielded similar changes in both of these mRNAs when treated with phorbol esters
Summary
Materials-The diacylglycerol dioctanoylglycerol (DiC8) was purchased from Avanti (Birmingham, AL). ’ The abbreviations used are: DiC8, dioctanoylglycerol; tk, thymidine kinase; CAT, chloroamphenicol acetyltransferase; PMA, phorbol myristate acetate; AP-1, 5’-TGATGCA-3’, AP-1 enhancer; SDS, sodium dodecyl sulfate; EGTA, [ethylenehis (oxyethylenenitrilo)]. Immunoprecipitation-2 x lo cells were incubated in methioninefree medium for % h prior to the addition of 150 &i/ml of [35S]. Staphylococks aureus protein ;\ (Calbiochem), the pellet-was washed, and the comolex was dissociated bv boiling in SDS buffer. 24 h after treatment, cells were pelleted, washed twice with Dulbecco’s phosphate-buffered saline, aid suspended in 100 ~1 of a buffer containing 250 mM Tris-HCl. DH 7.8. 100 we of protein from each time point was added to. The majority of the ethyl acetate was removed by centrifugation under vacuum, and the remaining material was spotted onto a silica thin layer plate. After chromatography in chloroform/methanol (95:1), the plate was dried, and autoradiography was done for 24 h
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