Abstract
The overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) by RBL-2H3 cells was used as the basis for an investigation of the effects of PHGPx on the formation of leukotrienes. The rates of production of leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in cells that overexpressed PHGPx were 8 times lower than those in a control line of cells. The reduction in rates of production of leukotrienes apparently resulted from the increase in the PHGPx activity since control rates of formation of leukotrienes could be achieved in PHGPx-overexpressing cells upon inhibition of PHGPx activity by diethyl malate. The conversion of radioactively labeled arachidonic acid to intermediates in the lipoxygenase pathway, such as 5-hydroxyeicosatetraenoic acid (5-HETE), LTC4, and LTB4, was strongly inhibited in PHGPx-overexpressing cells that had been prelabeled with [14C]arachidonic acid. PHGPx apparently inactivated the 5-lipoxygenase that catalyzed the conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) since 5-HPETE is a common precursor of 5-HETE, LTC4, and LTB4. The rates of formation of LTC4 and LTB4 in PHGPx-overexpressing cells returned to control rates upon the addition of a small amount of 12-HPETE. Flow cytometric analysis revealed that the rapid burst of formation of lipid hydroperoxides induced by A23187 was suppressed in PHGPx-overexpressing cells as compared with the control lines of cells. Subcellular fractionation analysis showed that the amount of PHGPx associated with nuclear fractions from PHGPx-overexpressing cells was 3.5 times higher than that from the control line of cells. These results indicate that PHGPx might be involved in inactivation of 5-lipoxygenase via reductions in levels of the fatty acid hydroperoxides that are required for the full activation of 5-lipoxygenase. Thus, in addition to its role as an antioxidant enzyme, PHGPx appears to have a novel function as a modulator of the production of leukotrienes.
Highlights
Leukotrienes are important mediators both in host defense mechanisms and in inflammatory disease states since they have potent effects on cell migration, muscle contraction, vascular permeability, and the release of lysosomal enzymes [1,2,3]
We are interested in the possible role of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the regulation of leukotriene synthesis since PHGPx is unique as an isozyme of glutathione peroxidase (GPx), being able to interact with the nuclear membrane in which the synthesis of leukotrienes occurs
These results indicate that the level of PHGPx was quite low in the control lines of cells as compared with that of cGPx
Summary
Reagents—Antibodies against PHGPx were prepared previously [28]. Antibodies against 5-lipoxygenase and FLAP were a kind gift from Dr M. The suspension of cells (0.25 ml) was transferred to an electroporation cuvette (0.4-cm gap; Bio-Rad) with a total of 20 g of linearized DNA, which consisted of 18 g of SR␣-PHGPx and 2 g of pSV2neo [33]. The latter plasmid was used to confer resistance to G418 (Geneticin; Life Technologies, Inc.). Immunoblot Analysis—Cell homogenates and nuclear fractions were fractionated by SDS-PAGE on 12.5% acrylamide gels and transferred to PVDF membrane filter (Millipore Co., Bedford, MA) at 50 V for 150 min in 25 mM Tris, 192 mM glycine, 10% (w/v) methanol at 4 °C in a protein transfer system (Bio-Rad), as described previously [35]. Release of radiolabeled arachidonic acid was expressed as the ratio of the radioactivity of arachidonic acid released into the culture medium to the total radioactivity of cells multiplied by 100 to give a percentage
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