Flipped regiospecificity in L434F mutant of 8-lipoxygenase.

Lipoxygenases are non-heme iron containing enzymes that catalyze oxygenation of poly-unsaturated fatty acids in different animal and plant species with extremely high regio- and stereospecificity. Nature employs 8-lipoxygenase to produce 8R-hydroperoxide from the oxygenation of arachidonic acid. A single-point L434F mutation of 8-lipoxygenase alters the regio- and stereospecificity of the final products, with a product ratio of 66 : 34 for 8R- and 12S-hydroperoxide, respectively. A molecular level explanation of this flipped regiospecificity is presented in this work on the basis of molecular dynamics simulations and transition network analysis of oxygen migration in the protein matrix. Phe434 is shown to exist in two conformations, the so-called open and closed conformations. In the closed conformation, the phenyl group of Phe434 shields the C8 site of the substrate, thereby preventing access of the oxygen molecule to this site, which leads to a quenching of the 8R-product. On the other hand, both closed and open conformations of Phe434 allow the oxygen molecule to approach the pro-S face of the C12 site of the substrate, which enhances the propensity of the 12S-hydroperoxide.

Origin of Regio- and Stereospecific Catalysis by 8-Lipoxygenase.

Lipoxygenases (lox's) are a group of non-heme iron containing enzymes that catalyze oxygenation of polyunsaturated fatty acids with precise regio- and stereoselectivities. The origin of regio- and stereospecific catalysis by 8-lox is explored in its wild-type (wt) form and in three mutants (Arg185Ala, Ala592Met, and Ala623His). The catalytic action of this enzyme progresses in two steps, namely, hydrogen abstraction from one double allylic carbon atom of substrate followed by oxygen insertion at the resulting prochiral carbon radical of the substrate. It is shown that the positional specificity of the hydrogen abstraction is a result of conformational dynamics of the bound substrate. While the C10 atom of the substrate is found to be the most probable site of hydrogen abstraction in the wt-lox, hydrogen abstraction from C13 is more favorable in the mutants. The present study discovers the presence of an interconnected network of a three-channel migration pathway operating in the protein matrix for efficient oxygen transport. Each migration channel is bestowed with a pocket at the peripheral region of protein as an oxygen access site, which transfers the oxygen to the active site through a well-connected migration path on a time scale of a few hundred picoseconds. By a careful geometric analysis of the oxygen pockets near the substrate binding cleft, the present study identifies the launching sites for oxygenation at the prochiral carbon centers C8, C11, C12, and C15 and the stereochemistry (R/S) of the corresponding products. It is found that the dominating 8R product in the wt-lox is due to the presence of the aromatic ring pair of Tyr181 and Phe173 acting as a gatekeeper for efficient delivery of oxygen at the pro-R face of C8. The change in the stereochemistry of the products in mutants is explained in terms of dynamic interactions between substrate and the surrounding residues.

Gly188Arg substitution eliminates substrate inhibition in arachidonate 11R-lipoxygenase.

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 µM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance.

Fatty acids modulate the expression of pyruvate kinase and arachidonate-lipoxygenase through PPARγ/CYP2C45 pathway: a link to goose fatty liver.

Cytochrome P-450 2C45 (CYP2C45) is the most highly expressed cytochrome P-450 isoform in chicken liver, and may play an important role in avian liver biology. However, information regarding the function of CYP2C45 in fatty liver is generally limited. The aim of this study was to investigate the role of CYP2C45 during the development of goose fatty liver. Our result indicated that the transcription of CYP2C45, together with PK and ALOX5, was increased in goose liver upon overfeeding for 19 D (P < 0.05). In goose primary hepatocytes, CYP2C45 RNA expression was also upgraded by the treatment with various chemicals like insulin, the fatty acids, and PPAR agonists (P < 0.05). We also found that both CYP2C45 overexpression and troglitazone treatment could increase the expression of pyruvate kinase (PK) and arachidonate 5-lipoxygenase (ALOX5), and furthermore, showed that the up-regulation of PK and ALOX5 induced by troglitazone could be suppressed by small interfering RNAs targeting CYP2C45 (P < 0.05). These findings suggest that fatty acids treatment and the overfeeding can induce the up-regulation of CYP2C45 expression possibly via PPARγ and that the induction of PK and ALOX5 in goose fatty liver is at least partially attributed to fatty acid-induced expression of CYP2C45. Thus, our data provides an insight into the mechanism by which glycolysis and arachidonic acid metabolism are modulated in goose fatty liver.

Production of 8S- and 10S-hydroxy polyunsaturated fatty acids by recombinant Escherichia coli cells expressing mouse arachidonate 8S-lipoxygenase.

OBJECTIVE: To quantitatively hydroxylate 8S- and 10S-positions on polyunsaturated fatty acids by recombinant Escherichia coli cells expressing mouse arachidonate 8S-lipoxygenase (8S-LOX). RESULTS: Hydroxylated products gained from the conversion of arachidonic acid (20:4Δ5Z,8Z,11Z,14Z, AA), eicosapentanoic acid (20:5Δ5Z,8Z,11Z,14Z,17Z, EPA), and (22:6Δ4Z,7Z,10Z,13Z,16Z,19Z, DHA) by recombinant E. coli cells containing 8S-LOX from mouse were identified as 8S-hydroxy-5,9,11,14(Z,E,Z,Z)-eicosatetranoic acid (8S-HETE), 8S-hydroxy-5,9,11,14,17(Z,E,Z,Z,Z)-eicosapentanoic acid (8S-HEPE), and 10S-hydroxy-4,8,12,14,16,19(Z,E,Z,Z,Z,Z)-docosahexaenoic acid (10S-HDoHE), respectively. Under the optimal hydroxylation conditions of pH 7.5, 30 °C, 5% (v/v) ethanol, 15 g cells l-1, and 5 mM substrate, AA, EPA, and DHA were hydroxylated into 4.37 mM 8S-HETE, 3.77 mM 8S-HEPE, and 3.13 mM 10S-HDoHE for 60, 90, and 60 min, with 87, 75, and 63% molar conversions, respectively. CONCLUSION: To the best of our knowledge, this is the first quantitatively biotechnological production of 8S-HETE, 8S-HEPE, and 10S-HDoHE.

Stabilization and improved activity of arachidonate 11S-lipoxygenase from proteobacterium Myxococcus xanthus.

Lipoxygenases (LOXs) catalyze the dioxygenation of PUFAs to produce regio- and stereospecific oxygenated fatty acids. The identification of regio- and stereospecific LOXs is important because their specific products are involved in different physiological activities in various organisms. Bacterial LOXs are found only in some proteobacteria and cyanobacteria, and they are not stable in vitro. Here, we used C20 and C22 PUFAs such as arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid to identify an 11S-specific LOX from the proteobacterium Myxococcus xanthus and explore its in vitro stability and activity. The activity and stability of M. xanthus ARA 11S-LOX as well as the production of 11S-hydroxyeicosatetraenoic acid from ARA were significantly increased by the addition of phosphatidylcholine, Ca2+, and coactosin-like protein (newly identified in the yeast Rhodosporidium toluroides) as stimulatory factors; in fact, LOX activity in the presence of all three factors increased approximately 3-fold. Our results indicate that these stimulatory factors can be used to increase the activity and stability of bacterial LOX and the production of bioactive hydroxy fatty acids, which can contribute to new academic research.

Expression of an 8R-Lipoxygenase From the Coral Plexaura homomalla.

Methods are presented for the use of the coral 8R-lipoxygenase from the Caribbean sea whip coral Plexaura homomalla as a model enzyme for structural studies of animal lipoxygenases. The 8R-lipoxygenase is remarkably stable and can be stored at 4°C for 3 months with virtually no loss of activity. In addition, an engineered "pseudo wild-type" enzyme is soluble in the absence of detergents, which helps facilitate the preparation of enzyme:substrate complexes.

Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes.

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB5, HXD3, HXE3, TrXB5, TrXD3 and TrXE3. We succeed in the biotransformation of PUFAs into eight types of HXs (>35% conversion) and TrXs (>10% conversion) by expressing M. xanthus 12-LOX or 11-LOX with or without EH in Escherichia coli. We determine 11-hydroxy-eicosatetraenoic acid, HXB3, HXB4, HXD3, TrXB3 and TrXD3 as potential peroxisome proliferator-activated receptor-γ partial agonists. These findings may facilitate physiological studies and drug development based on lipid mediators.

Effect of N-Feruloylserotonin and Methotrexate on Severity of Experimental Arthritis and on Messenger RNA Expression of Key Proinflammatory Markers in Liver.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, leading to progressive destruction of joints and extra-articular tissues, including organs such as liver and spleen. The purpose of this study was to compare the effects of a potential immunomodulator, natural polyphenol N-feruloylserotonin (N-f-5HT), with methotrexate (MTX), the standard in RA therapy, in the chronic phase of adjuvant-induced arthritis (AA) in male Lewis rats. The experiment included healthy controls (CO), arthritic animals (AA), AA given N-f-5HT (AA-N-f-5HT), and AA given MTX (AA-MTX). N-f-5HT did not affect the body weight change and clinical parameters until the 14th experimental day. Its positive effect was rising during the 28-day experiment, indicating a delayed onset of N-f-5HT action. Administration of either N-f-5HT or MTX caused reduction of inflammation measured as the level of CRP in plasma and the activity of LOX in the liver. mRNA transcription of TNF-α and iNOS in the liver was significantly attenuated in both MTX and N-f-5HT treated groups of arthritic rats. Interestingly, in contrast to MTX, N-f-5HT significantly lowered the level of IL-1ß in plasma and IL-1ß mRNA expression in the liver and spleen of arthritic rats. This speaks for future investigations of N-f-5HT as an agent in the treatment of RA in combination therapy with MTX.

Cytochrome P450 and Lipoxygenase Metabolites on Renal Function.

Arachidonic acid metabolites have a myriad of biological actions including effects on the kidney to alter renal hemodynamics and tubular transport processes. Cyclooxygenase metabolites are products of an arachidonic acid enzymatic pathway that has been extensively studied in regards to renal function. Two lesser-known enzymatic pathways of arachidonic acid metabolism are the lipoxygenase (LO) and cytochrome P450 (CYP) pathways. The importance of LO and CYP metabolites to renal hemodynamics and tubular transport processes is now being recognized. LO and CYP metabolites have actions to alter renal blood flow and glomerular filtration rate. Proximal and distal tubular sodium transport and fluid and electrolyte homeostasis are also significantly influenced by renal CYP and LO levels. Metabolites of the LO and CYP pathways also have renal actions that influence renal inflammation, proliferation, and apoptotic processes at vascular and epithelial cells. These renal LO and CYP pathway actions occur through generation of specific metabolites and cell-signaling mechanisms. Even though the renal physiological importance and actions for LO and CYP metabolites are readily apparent, major gaps remain in our understanding of these lipid mediators to renal function. Future studies will be needed to fill these major gaps regarding LO and CYP metabolites on renal function.

[Resistance to antiplatelet drugs in patients with cerebrovascular disorders].

This review concerns clinical and laboratory resistance to antiplatelet drugs (aspirin and clopidogrel) in patients with cerebrovascular disorders. Results of certain clinical trials showed that laboratory resistance to antiaggregants is associated with recurrent thromboembolic vascular events. The commonest causes of aspirin resistance are production of arachidonic acid metabolites via the lipoxygenase pathway, poor compliance with the treatment, polymorphism of the genes encoding for cyclooxygenase and glycoprotein (GP) IIb/IIIa, endothelial dysfunction. The causes of clopidogrel resistance include inadequate doses of the drug, its low absorption, poor compliance with the treatment, polymorphism of ADP receptors, GP IIb/IIIa and cytochrome P450 genes, acute coronary syndrome and stroke, metabolic syndrome. Therapeutic efficacy of antiaggregants can be improved by increasing their doses, using membranotropic agents, correcting endothelial dysfunction, etc. Because the apparent variability of antiplatelet drug resistance is currently due to the use of different test-systems by different authors, the evaluation of individual sensitivity to a given drug showing laboratory resistance and the choice of alternative therapy are thus far possible only in the framework of clinical studies. Large-scale prospective multicenter trials of antiplatelet drug resistance are needed along with research for better understanding mechanisms of individual platelet sensitivity and resistance to antiaggregants and developing efficacious methods for their correction.

Applicability of the triad concept for the positional specificity of mammalian lipoxygenases.

The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less space-filling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.

Variation in eicosanoid genes, non-fatal myocardial infarction and ischemic stroke.

OBJECTIVES: Eicosanoids are lipid mediators that may play a role in atherosclerosis. We investigated the association of common genetic variation in prostaglandin H synthase 1 (PTGS1), prostaglandin H synthase 2 (PTGS2), thromboxane A2 synthase (TBXAS1), prostacyclin synthase (PTGIS), prostaglandin E synthase (PTGES), 5-lipoxygenase activating protein (ALOX5AP), 12-lipoxygenase (ALOX12) and 15-lipoxygenase (ALOX15) with the risks of myocardial infarction (MI) and ischemic stroke. A secondary aim was to replicate the interaction of PTGS2 rs20417 (-765G to C) with aspirin use on coronary heart disease risk observed in the Atherosclerosis Risk in Communities Study (ARIC). METHODS: We conducted a case-control study in a large Health Maintenance Organization. Cases were men and women, aged 30-79 years with incident non-fatal myocardial infarction (n=1063) or ischemic stroke (n=469) between January 1995 and December 2004. Controls (n=3462) were randomly selected and frequency matched to cases on age, sex, hypertension and calendar year. RESULTS: Common variation in TBXAS1 and PTGIS was associated with MI risk (p-value for global Chi-square test, 0.01 and 0.03, respectively). Common variation in ALOX5AP, ALOX12, ALOX15, PTGS1, PTGS2 and PTGES was not associated with risks of MI and ischemic stroke. We replicated the observation of the Atherosclerosis Risk in Communities Study and observed an interaction of rs20417 with aspirin use on myocardial infarction risk (p for interaction=0.03). CONCLUSIONS: Study results suggest that variation in TBXAS1 and PTGIS may influence MI risk, and carriers of rs20417C allele might derive greater benefits from aspirin use in primary prevention.

Inducible expression of 15-lipoxygenase-2 and 8-lipoxygenase inhibits cell growth via common signaling pathways.

Human 15-lipoxygenase (LOX)-2 and mouse 8-LOX represent orthologous members of the LOX family but display different positional specificities and tissue distribution. To study the functional role of 15-LOX-2 and 8-LOX in keratinocytes, an inducible Tet-On gene expression system was established in the premalignant mouse keratinocyte cell line 308. Doxycycline (dox)-induced expression of enzymatically active 15-LOX-2 and 8-LOX led to an inhibition of cell growth that was associated with an inhibition of DNA synthesis, as shown by a 15-46% reduction of 5-bromo-2-deoxy-uridine (BrdU) incorporation. The inhibitory effects were increased in the presence of exogenous arachidonic acid. In contrast, addition of linoleic acid or the LOX inhibitor baicalein reversed the growth-inhibitory effects. Treatment of the cells with 15-hydroxyeicosatetraenoic acid (HETE) or 8-HETE resulted in a similar inhibition of BrdU incorporation, whereas 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE, in contrast, had no effects. Dox-induced keratinocytes showed increased levels of reactive oxygen species (ROS). The antioxidant N-acetyl-L-cysteine and a specific inhibitor of p38 mitogen-activated protein kinase, but not of extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase/stress-activated kinases, completely abolished the LOX-induced growth inhibition, indicating a critical role of ROS and p38. Our data suggest that 15-LOX-2 and 8-LOX, although displaying different positional specificity, may use common signaling pathways to induce growth inhibition in premalignant epithelial cells.

Identification and characterization of an arachidonate 11R-lipoxygenase.

11R-Lipoxygenase (11R-LOX) activity has been detected in several marine invertebrates, and here we report the first cloning and expression of the enzyme. The cDNA encoding a protein of 77kDa was isolated by RT-PCR from the soft coral Gersemia fruticosa and expressed in Escherichia coli. Incubations of recombinant enzyme with arachidonic acid yielded a single product, identified by RP-HPLC, GC-MS, and chiral phase-HPLC as 11R-hydroperoxyeicosatetraenoic acid. Other C18, C20, and C22 substrates are also oxygenated, preferentially at the omega10 position. Significantly, both Ca(2+)-ions and a membrane fraction are required for catalytic activity. Calcium effects translocation of the soluble 11R-LOX to the membrane and this association is reversible by Ca(2+) chelation. The enzyme sequence contains some conserved amino acids implicated in calcium activation of mammalian 5-LOX, and with its obligate requirement for membrane interaction the 11R-LOX may thus provide a new model for further analysis of this aspect of lipoxygenase activation.

Insights from the X-ray crystal structure of coral 8R-lipoxygenase: calcium activation via a C2-like domain and a structural basis of product chirality.

Lipoxygenases (LOXs) catalyze the regio- and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 A resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca(2+)-ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca(2+)-regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly(428) (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate.

An antitumorigenic role for murine 8S-lipoxygenase in skin carcinogenesis.

The levels of 8S-lipoxygenase (8S-LOX) expression and of its arachidonic acid metabolite, 8-hydroxyeicosatetraenoic acid (8-HETE), are highly elevated in the early stages of mouse skin carcinogenesis. On the other hand, several reports showing that 8-HETE is also closely associated with keratinocyte differentiation raise a question concerning the role of 8S-LOX/8-HETE in skin carcinogenesis. To address that question, here we conducted a series of gain-of-function studies. Skin targeted loricrin 8S-LOX/C57BL/6J transgenic mice showed a more differentiated epidermal phenotype as well as a 64% reduced papilloma development in a two-stage skin carcinogenesis protocol. Forced expression of 8S-LOX in MT1/2 cells, a murine papilloma cell line, also caused a more differentiated appearance as well as keratin 1 expression. Overexpression of 8S-LOX in CH72 cells, a murine carcinoma cell line, inhibited cell proliferation by 30% in vitro and by 86% in in vivo xenografts. Exogenous addition of 5 muM 8-HETE to CH72 cells caused cell cycle arrest at the G1 phase. Finally, immunohistochemical analyses showed 8S-LOX protein expression was strictly confined to the differentiated compartment of mouse skin and throughout tumorigenesis. Collectively, these data suggest that 8S-LOX plays a role as a prodifferentiating, antitumorigenic, and tumor suppressing gene in mouse skin carcinogenesis.

Arachidonate lipoxygenase (ALOX) and cyclooxygenase (COX) polymorphisms and colon cancer risk.

In the human colon, arachidonic acid is metabolized primarily by cyclooxygenase (COX) and arachidonate lipoxygenase (ALOX) to bioactive lipids, which are implicated in colon cancer risk. Several polymorphisms in ALOX and COX genes have been identified, including G-1752A, G-1699A and Glu254Lys in ALOX5; Gln261Arg in ALOX12; Leu237Met and Val481Ile in COX1; and C-645T and Val511Ala in COX2. Because of the significant role of arachidonic acid metabolism in colon cancer, we hypothesized that these polymorphisms could influence susceptibility to colon cancer. We addressed this hypothesis in African-Americans and Caucasians using colon cancer cases (n = 293) and hospital- (n = 229) and population-based (n = 304) control groups. Polymorphisms did not differ between the control groups (P > 0.05); thus, they are combined for all analyses presented. ALOX5 Glu254Lys and COX2 C-645T and Val511Ala allele frequencies differed between Caucasians and African-American controls (P < 0.001). The ALOX5 -1752 and -1699 polymorphisms were in linkage disequilibrium (P < 0.001) and associated with a decreased risk in Caucasians in ALOX5 haplotype analyses (P = 0.03). Furthermore, an inverse association was observed between A alleles at positions -1752 and -1699 of ALOX5 and colon cancer risk in Caucasians, but not in African-Americans. Caucasians with A alleles at ALOX5 -1752 had a reduced odds of colon cancer versus those with G alleles [odds ratio (OR) (GA versus GG), 0.63; 95% confidence interval (CI), 0.39-1.01; OR (AA versus GG), 0.33; 95% CI, 0.07-1.65, P(trend) = 0.02]. Similar results were observed for ALOX5 G-1699A [OR (GA versus GG), 0.59, 95% CI, 0.37-0.94; OR (AA versus GG), 0.27, 95% CI, 0.06-1.32, P(trend) = 0.01]. Statistically significant associations with colon cancer were not observed for the other polymorphisms investigated. We have shown for the first time that a haplotype containing ALOX5 G-1752A and G-1699A in a negative regulatory region of the promoter may influence colon cancer risk in Caucasians.

Elevated expression of 12/15-lipoxygenase and cyclooxygenase-2 in a transgenic mouse model of prostate carcinoma.

Changes in expression of arachidonic acid (AA) metabolizing enzymes are implicated in the development and progression of human prostate carcinoma (Pca). Transgenic mouse models of Pca that progress from high-grade prostatic intraepithelial neoplasia (HGPIN) to invasive and metastatic carcinoma could facilitate study of the regulation and function of these genes in Pca progression. Herein we characterize the AA-metabolizing enzymes in transgenic mice established with a prostate epithelial-specific long probasin promoter and the SV40 large T antigen (LPB-Tag mice) that develop extensive HGPIN and invasive and metastatic carcinoma with neuroendocrine (NE) differentiation. Murine 8-lipoxygenase (8-LOX), homologue of the 15-LOX-2 enzyme that is expressed in benign human prostatic epithelium and reduced in Pca, was not detected in wild-type or LPB-Tag prostates as determined by enzyme assay, reverse transcription-PCR, and immunohistochemistry. The most prominent AA metabolite in mouse prostate was 12-HETE. Wild-type prostate (dorsolateral lobe) converted 1.6 +/- 0.5% [(14)C]AA to 12-HETE (n = 7), and this increased to 8.0 +/- 4.4% conversion in LPB-Tag mice with HGPIN (n = 13). Quantitative real-time reverse transcription-PCR and immunostaining correlated the increased 12-HETE synthesis with increased neoplastic epithelial expression of 12/15-LOX, the leukocyte-type (L) of 12-LOX and the murine homologue of human 15-LOX-1. Immunostaining showed increased L12-LOX in invasive carcinoma and approximately one-half of metastatic foci. COX-2 mRNA was detectable in neoplastic prostates with HGPIN but not in wild-type prostate. By immunostaining, COX-2 was increased in the neoplastic epithelium of HGPIN but was absent in foci of invasion and metastases. We conclude that (a) AA metabolism in wild-type mouse prostate differs from humans in the basal expression of LOXs (15-LOX-2 in human, absence of its 8-LOX homologue in mouse prostate); (b) increased expression of 12/15-LOX in HGPIN and invasive carcinoma of the LPB-Tag model is similar to the increased 15-LOX-1 in high-grade human Pca; and (c) the LPB-Tag model shows increased COX-2 in HGPIN, and therefore, it may allow additional definition of the role of this enzyme in the subset of human HGPINs or other precursor lesions that are COX-2 positive, as well as investigation of its contribution to neoplastic cell proliferation and tumor angiogenesis in Pca.

Arachidonate 8(S)-lipoxygenase.

The recently identified mouse 8(S)-lipoxygenase almost exclusively directs oxygen insertion into the 8(S) position of arachidonic acid and, with lower efficiency, into the 9(S) position of linoleic acid. The protein of 677 amino acids displays 78% sequence identity to human 15(S)-lipoxygenase-2 which is considered to be its human orthologue. The 8(S)-lipoxygenase gene, Alox15b, consisting of 14 exons and spanning 14.5 kb is located within a gene cluster of related epidermis-type lipoxygenases at the central region of mouse chromosome 11. 8(S)-Lipoxygenase is predominantly expressed in stratifying epithelia of mice, constitutively in the hair follicle, forestomach, and foot-sole and inducible in the back skin with strain-dependent variations. The expression is restricted to terminally differentiating keratinocytes, in particular the stratum granulosum and 8(S)-lipoxygenase activity seems to be involved in terminal differentiation of mouse epidermis. Tumor-specific up-regulation of 8(S)-lipoxygenase expression and activity indicate a critical role of this enzyme in malignant progression during tumor development in mouse skin.