Potential role of phospholipase A 2 in HL-60 cell differentiation to macrophages induced by protein kinase C activation ( 2-lysophosphatidylcholine / cis-unsaturated fatty acid / phorbol ester / diacylglycerol )

Content Provider	Semantic Scholar
Author	Asaoka, Yoshinori Nishizuka, Yasutomi
Copyright Year	2005
Abstract	2-Lysophosphatidylcholine and cis-unsaturated fatty acids such as linoleic and linolenic acids, which are the products of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2 (EC 3.1.1.4), significantly potentiate the differentiation ofHL-60 cells to macrophages that is induced by either a membrane-permeant diacylglycerol or a phorbol ester. The cell differentiation was assayed by measuring the expression ofCD11b, one of the cell surface markers of macrophages, and also by the appearance ofphagocytic activity. Snake venom phospholipase A2 added directly to the cells is also active for this potentiation. Neither lysophosphatidylcholine, fatty acid, nor phospholipase A2 is active unless a membrane-permeant diacylglycerol or a phorbol ester is present. The results presented provide further evidence that activation of phospholipase A2 may be intimately related to the signal transduction pathway through protein kinase C. The HL-60 cell line is frequently used as a model system to investigate the mechanism of cell differentiation, since retinoic acid and several other chemicals lead the cells to produce granulocytes, whereas phorbol 12-myristate 13-acetate (PMA) generates macrophages from these cells (for review, see ref. 1). A single dose of PMA can induce the differentiation of HL-60 cells to macrophages. On the other hand, a single dose of a membrane-permeant diacylglycerol (DAG), 1,2-dioctanoylglycerol (1,2-DiC8), is normally insufficient to induce this differentiation because it is rapidly metabolized and disappears from the cells (2-5). Repeated additions of 1,2-DiC8, however, can induce the cell differentiation, as measured by the expression of CD11b, a cell surface marker of macrophages (5), suggesting that sustained activation of protein kinase C (PKC) is essential for causing this cellular response (6, 7). The formation of DAG from receptormediated hydrolysis of inositol phospholipids is usually transient, and recent evidence suggests that, at a later phase in cellular responses, DAG is produced from the signal-induced hydrolysis of phosphatidylcholine (PtdCho), and this reaction is initiated presumably by phospholipase D activation (for reviews, see refs. 8-11). We have previously reported (12, 13) that 2-lysophosphatidylcholine (lysoPtdCho) and cis-unsaturated fatty acids, both of which can be produced from PtdCho hydrolysis by phospholipase A2 (EC 3.1.1.4), significantly enhance T-lymphocyte activation and platelet release reaction, which are induced by 1,2-DiC8 and PMA. The hydrolysis of various membrane phospholipids, therefore, appears to play roles in eliciting short-term as well as long-term cellular responses such as release reaction and cell proliferation. The present studies were undertaken, with HL-60 cells as an experimental system, to demonstrate further that phospholipase A2 actiThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. vation may intensify the role of PKC in signal transduction, eventually leading to cell differentiation. MATERIALS AND METHODS Materials. HL-60 cells donated by J. Minowada (Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Okayama, Japan) were maintained at a cell density between 0.1 and 1.0 x 106 cells per ml as a suspension in RPMI 1640 medium supplemented with 5% fetal bovine serum (Flow Laboratories) at 37°C in a humidified 5% CO2 atmosphere. Chemicals. A membrane-permeant DAG, 1,2-DiC8, was obtained from Nacalai Tesque (Kyoto). This preparation consists of approximately 95% DL-1,2-DiC8 and 5% 1,3-DiC8 as estimated by thin-layer chromatography. PMA was a product of LC Services (Woburn, MA). Lysophospholipids, fatty acids, and Crotalus adamanteus phospholipase A2 were obtained from Sigma. Phospholipase A2 activity was determined by assaying with dipalmitoylphosphatidylcholine as a substrate. One unit of phospholipase A2 was defined as the amount of enzyme that released 1 nmol of palmitic acid per min. Other chemicals were obtained from commercial sources. Determination of CD11b Expression. HL-60 cells were stimulated and incubated as specified in each experiment. After incubation, the cells were collected, washed, resuspended in phosphate-buffered saline (PBS) containing 20% human serum at a density of 4.0 x 106 cells per ml, and allowed to stand for 30 min at 4°C to saturate Fc binding sites. After three washes, the cells (4.0 x 106 cells per ml) were treated for 30 min at 4°C with an appropriately diluted mouse monoclonal antibody against CD11b (Immunotech, Marseille, France) or with mouse IgG as a control. After three washes, the cells (4.0 x 106 cells per ml) were incubated for 30 min at 4°C with a fluorescein-conjugated goat affinitypurified F(ab')2 fragment that recognizes mouse IgG (Cappel Laboratories) and then washed and suspended in PBS. The fluorescence was determined by analysis of 5000 cells with a flow cytometer (Cyto ACE-150, Japan Spectroscopic, Tokyo). Assay of Phagocytosis. The phagocytic activity was determined by measuring the uptake of fluorescent microspheres (Fluoresbrite Carboxylate Microspheres, 1.75 ,um in diameter, Polysciences), as described by Blair et al. (14). The cells (2.0 x 105 per ml) were stimulated as specified in each experiment in the presence of 5.0 x 105 fluorescent microspheres per ml. After a 24-hr incubation, cells were washed, and fluorescent intensity was determined with the flow
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