Arachidonic acid metabolism in the human mast cell line HMC-1: 5-lipoxygenase gene expression and biosynthesis of thromboxane

Abstract

Metabolism of arachidonic acid was studied in the unique human mast cell line HMC-1. By HPLC and/or gas chromatography mass spectrometry (GC-MS), 19 oxygenated metabolites were identified, including monohydroxy acids, leukotrienes, prostaglandins, and thromboxane. Intact cells incubated with the calcium ionophore A23187 and arachidonic acid expressed 5-lipoxygenase activity and produced 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolite (745 pmol/10 7 cells) followed by leukotriene (LT) C 4 (245 pmol/10 7 cells) and 11- trans-LTC 4 (74 pmol/10 7 cells). Low but clearly detectable levels of LTB 4 were also observed. The total amounts of 5-LO products were comparable to those obtained with RBL-1 cells and corresponded to approx. 30% of the levels obtained with isolated human polymorphonuclear leukocytes. Time-course experiments revealed that HMC-1 cells contained the enzyme activities required to metabolize LTC 4 into LTD 4 and further into LTE 4. The profile of prostanoids included, prostaglandin (PG) E 2, PGF 2α, and PGD 2, whereas 6-keto-PGF 1α, reflecting prostacyclin formation, could not be detected. Futhermore, we were able to unambiguously establish that HMC-1 cells could produce substantial amounts of thromboxane (TX) A 2, measured as TXB 2 (0.1–2.2 nmol/10 7 cells). Generation of TXA 2 in such quantities, exceeding those of LTC 4, suggests that mast cells may be an important source of thromboxane and points to a possible role for these cells in hemostasis and thrombosis. After approx. 10 passages in culture, 5-lipoxygenase activity in HMC-1 cells drastically declined concomitantly with changes in growth behavior and cell morphology. Analysis by Northern and Western blots revealed that loss of 5-lipoxygenase activity correlated well with a reduced 5-lipoxygenase gene expression at both a transcriptional and translational level. This loss of enzyme activity and gene expression may be related to a genetic abnormality propagated in HMC-1 cells, i.e., a 10;16 translocation, which thus involves the chromosome containing the 5-lipoxygenase gene.