Effect of differentiation on platelet-activating factor metabolism in HL-60 cells LYNN L. STOLL, NAGENDER R. YERRAM and ARTHUR A. SPECTOR* Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA * Author for correspondence Summary The formation and metabolism of l-O-alkyl-2-acetyl- sn-glycerol (AAG), a protein kinase C (PKC) activator formed from platelet-activating factor (l-O-alkyl-2- acetyl-sn-glycero-3-phosphocholine; PAT), was stud- ied in HL-60 cells to determine whether differen- tiation may influence this process. HL-60 cells differ- entiated to macrophages (HL-6O/M0) with a phorbol ester convert added [ 3 H]PAF to AAG; 22% of the incorporated radioactivity is converted to AAG within 15 s. By contrast, neither undifferentiated HL- 60 cells (HL-60/U) nor HL-60 cells differentiated to granulocytes (HL-60/GN) with retinoic acid produce AAG from PAF. The HL-6O/M0 rapidly convert radiolabeled AAG to 1-O-alkyl-sn-glycerol and, sub- sequently, to two other unidentified metabolites. However, some apparently unmodified AAG persists in the cell lipids for at least 6 h. The HL-60 subtypes which do not convert PAF to AAG can nevertheless catabolize AAG; HL-60/U and HL-60/GN produce alkylglycerol and the other AAG metabolites. These findings demonstrate that differentiation can alter the processing of PAF in a human leukocyte cell line. Furthermore, they suggest that PAF may produce at least some of its biological effects in macrophages by conversion to AAG. Key words: diacylglycerol, protein kinase C, ether lipids. Introduction Platelet-activating factor (l-O-alkyl-2-acetyl-sra-glycero- 3-phosphocholine; PAF) is an ether lipid (Hanahan, 1986; Snyder, 1987, 1990; Winslow and Lee, 1987) formed by activated blood cells such as neutrophils (Camussi et al. 1981; Ludwig et al. 1984) and macrophages (Arnoux et al. 1980; Roubin and Benveniste, 1985). PAF also is syn- thesized by endothelial cells in response to a number of agonists (Prescott et al. 1984; Camussi et al. 1983; Bussolino et al. 1986). PAF produces a wide range of biological responses in many different target cells, including changes in cytosolic free calcium and calcium efflux (Brock and Gimbrone, 1986), altered vascular permeability (Humphrey et al. 1982, 1984; Handley et al. 1984), stimulation of vascular smooth muscle cell prolifer- ation (Stoll and Spector, 1989), and activation of the proto- oncogenes c-fos and c-jun (Squinto et al. 1989). We have recently observed the rapid formation of a biologically active PAF metabolite, l-O-alkyl-2-acetyl-sn- glycerol (AAG), in vascular smooth muscle cells that are exposed to PAF (Stoll et al. 1989). While a role for AAG in the complete biosynthesis pathway for PAF has previously been reported (Snyder et al. 1986; Lee et al. 1990), its possible role as a mediator of PAF responses has not been considered previously. The AAG produced from PAF by vascular smooth muscle cells is a biologically active diacylglycerol analog that persists in the lipids of these cells for at least several hours. It activates protein kinase C (PKC) and produces the same pattern of effects on vascular smooth muscle cell proliferation as does PAF Journal of Cell Science 100, 145-152 (1991) Printed in Grent Britain © The Company of Biologists Limited 1991 (Stoll et al. 1989). This raises the question of whether AAG may have some relationship to the differentiation process. The HL-60 human promyelocytic leukemia cell line is a model system that is widely used to study the effects of differentiation on metabolic processes (Collins, 1987; Leglise et al. 1988). Undifferentiated HL-60 cells (HL- 60/UD) are immature cells that retain the capacity to differentiate into either granulocytes (HL-60/GN) or monocyte/macrophages (HL-60/M</>), depending on the chemical inducer. Vallari et al. (1990) have shown that HL-60/UD do not express high-affinity PAF receptors but readily metabolize PAF. In addition, McNamara et al. (1984) have reported that synthetic AAG at concentrations of 10-30 /iM stimulates differentiation of HL-60/UD to the macrophage-like phenotype. To obtain a more complete understanding of the role of AAG in differentiation, we have investigated the conversion of PAF to AAG in the various phenotypes of the HL-60 system. Materials and methods Cell culture HL-60 cells were obtained from the American Type Culture Collection (ATCC CCL 240). Cultures were maintained in RPMI- 1640 (GIBCO, Grand Island, NY) supplemented with 10 * fetal bovine serum (HyClone, Logan, UT), MEM Nonessential Amino Acids (GIBCO), MEM Vitamin Solution (GIBCO), 2mM L- glutamine (Sigma, St Louis, MO), 15 mM 4-(2-hydroxyethyl)-l- piperazine ethane sulfonic acid (Hepes; Sigma); and 50/<gml~' gentamicin (Schering, Kenilworth, NJ). Cultures were grown at 37 °C in a humidified atmosphere containing 5% CO2, and were subcultured twice weekly. For experiments, cells were differen- 145