Abstract
Cyclooxygenase (COX) has two isoforms, COX-1 and -2, which catalyze the key step in the conversion of cellular arachidonic acid into prostaglandins. In recent years, interest in COX-2 has significantly increased since it has been a target for the development of specific non-steroidal anti-inflammatory drugs. We report that COX-2 expression is up-regulated in phorbol ester (phorbol myristate acetate, PMA)-differentiated human U937 macrophage-like cells stimulated with lipopolysaccharide (LPS), whereas COX-1 is not up-regulated. We show that the LPS-induced up-regulation of COX-2 depends on the activity of the Mg(+2)-dependent phosphatidic acid phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 by bromoenol lactone, propranolol, or ethanol resulted in a decrease in LPS-induced COX-2 mRNA transcript production, COX-2 protein expression, and prostaglandin E(2) release from U937 macrophages. To ensure that these results did not arise because of PMA treatment of the U937 cells, similar experiments were conducted with the P388D(1) cell line, which does not require PMA differentiation. LPS increased the levels of endogenous cellular diacylglycerol (DAG) within 2 min of stimulation. This increase was observed to be sensitive to the PAP-1 inhibitors. Furthermore, phosphatidic acid phosphohydrolase activity assays showed that the bromoenol lactone-sensitive PAP-1 activity was translocated from the cytosolic fraction to the membrane fraction within 2 min of LPS exposure. Finally, DAG add-back experiments demonstrate that LPS-induced COX-2 expression is enhanced by the addition of exogenous DAG.
Highlights
32978 JOURNAL OF BIOLOGICAL CHEMISTRY phages produce and release a host of cytokines and eicosanoids in response to inflammatory stimulation, including prostaglandins (PGs)
To ensure that phosphatidic acid phosphohydrolase (PAP)-1 is present in this cell line, U937 cell lysates were checked for the presence of Lipin 1 by Western
PGE2 Production in a Time-dependent Manner—Because LPSinduced PGE2 release is known to be dependent on the function of the Phospholipase A2 (PLA2) and COX enzymes, we measured their expression over time with LPS stimulation to determine whether changes on SDS-PAGE electrophoresis [28], and we observed a band at this molecular size (Fig. 2D)
Summary
Materials—Human promonocytic leukemia U937 cells and murine P388D1 cells were purchased from American Type Culture Collection (Manassas, VA). IMDM and RPMI 1640 cell culture medium were obtained from Invitrogen. Cell Culture and Stimulation Protocol—The normal growth medium of the U937 cells contained RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. The DAG was initially dissolved in Me2SO, and further diluted into cellular medium contained prior to being dispensed into the medium of the cultured cells. P388D1 cells were maintained at 37 °C in a humidified atmosphere at 90% air and 10% CO2 IMDM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and non-essential amino acids. The membrane was washed three times in PBS containing 0.1% Tween 20 before addition of a rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Alpha Diagnostic) for 1 h.
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