Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males.

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.

Hyperforin is a novel type of 5-lipoxygenase inhibitor with high efficacy in vivo.

We previously showed that, in vitro, hyperforin from St. John's wort (Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo. Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B(4) formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo.

Expression of 5-lipoxygenase and 15-lipoxygenase in rheumatoid arthritis synovium and effects of intraarticular glucocorticoids.

INTRODUCTION: It was previously shown that lipoxygenase (LO) pathways are important in the rheumatoid arthritis (RA) inflammatory process and that synovial fluid from RA patients contains high amounts of leukotrienes. We therefore aimed to investigate the 5-LO and 15-LO-1 expression pattern in RA and ostheoarthritis (OA) synovial tissue and to study the effect of intraarticular glucocorticoid (GC) therapy on enzyme expression. METHODS: Expression of LOs was evaluated by immunohistochemistry in RA and OA synovial biopsies. Cellular localization of these enzymes was analyzed by double immunofluorescence. In synovial biopsies from 11 RA patients, 5-LO and 15-LO-1 expression was evaluated before and after triamcinolone hexacetonide knee injection and assessed by image analysis to quantify their expression. We also investigated the presence of 15-LO-1 by immunohistochemistry in synovial fluid (SF) cells as well as their ability to form 15-hydroxyeicosatetraenoic acid (15-HETE) following treatment with arachidonic acid (AA). RESULTS: 5-LO and 15-LO-1 are present in RA and OA synovium, with 5-LO being mostly expressed in lining and sublining macrophages, neutrophils and mast cells and 15-LO-1 mainly in lining macrophages, fibroblasts and sublining endothelial cells. Intraarticular GC treatment resulted in a significant suppression of 5-LO expression, but did not influence the 15-LO-1 enzyme significantly. Also, SF cells express a functional 15-LO-1 and produce 15-HETE when challenged with AA. CONCLUSIONS: These data demonstrate that local therapy with GC decreases 5-LO expression in RA synovium and offer an additional possible mechanism for the efficiency of intraarticular adjuvant therapy in RA.

Novel oxylipins formed from docosahexaenoic acid by potato lipoxygenase--10(S)-hydroxydocosahexaenoic acid and 10,20-dihydroxydocosahexaenoic acid.

Potato tuber lipoxygenase (ptLOX) has been shown to catalyze the aerobic formation of at least four major oxygenated derivatives of DHA. Two of the products--7,17(S)- and 10,17(S)-dihydro(pero)xy-DHA [7,17- and 10,17-diH(P)DHA]--were formed from soybean 15-LOX-derived 17(S)-hydro(pero)xy-DHA [17(S)-H(P)DHA], whereas two novel oxylipin compounds--10(S)-hydro(pero)xy-DHA and 10,20-dihydro(pero)xy-DHA [10(S)-H(P)DHA and 10,20-diH(P)DHA, respectively]--were the major direct products of DHA oxidation by ptLOX. The reactions proceeded relatively slowly but could be stimulated by catalytic amounts of SDS. Micromolar concentrations of 10(S)-HPDHA effectively abolished the kinetic lag period of ptLOX activation. Enzymatic activity with DHA or 17(S)-HPDHA as substrate was about 8% of that with linoleic acid--a standard natural ptLOX substrate--whereas 17(S)-HDHA was converted at a rate of approximately 1%. The enzyme was relatively unstable and quickly inactivated during the reaction with DHA on with 17(S)-HPDHA (first-order kinetic constant of inactivation kin = 1.5 +/- 0.3 min(-1)), but not with 17(S)-HDHA. Both 7,17- and 10,20-diH(P)DHA were clearly products of double oxygenation catalyzed by soybean 15-LOX and/or ptLOX. Our observation that ptLOX could convert 17-HDHA to 10,17-diH(P)DHA indicates that this dihydroxylated derivative of DHA also can be formed via a double lipoxygenation mechanism.

Molecular cloning and functional characterization of mouse coactosin-like protein.

Coactosin was first isolated from Dictyostelium discoideum and, as reported, human coactosin-like protein (CLP) was identified in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. A mouse CLP (mCLP) cDNA clone was identified among EMBL/GenBank EST sequences. The derived amino acid sequence (142 residues) was 95.1% identical with human CLP. Here, we also show that mCLP interacts with actin and 5LO in the two-hybrid system. High-speed cosedimentation assays and GST-binding assays confirmed these protein interactions. In chemical cross-linking experiments, one molecule of mCLP was covalently linked to either one subunit of actin or one molecule of 5LO. The mCLP-F-actin and mCLP-5LO associations were pH-insensitive and Ca(2+)-independent. However, association with actin was best observed at low salt concentrations, while association with 5LO was favored by salt, indicating different binding characteristics.