leukotriene-a4 and ubenimex

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.

Topics: Aminopeptidases; Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Differentiation; Cell Nucleus; Cell Proliferation; Cytoplasm; Epoxide Hydrolases; Humans; Leucine; Leukotriene A4; Leukotriene B4; Male; Mice; Protease Inhibitors; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Inbred F344; Respiratory Mucosa; Subcellular Fractions; Tissue Distribution

Topics: Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Arachidonate 5-Lipoxygenase; Cell Transformation, Neoplastic; Cyclooxygenase Inhibitors; Disease Models, Animal; Epoxide Hydrolases; Esophageal Neoplasms; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Leucine; Leukotriene A4; Neoplasms; Signal Transduction; Up-Regulation

Esophageal adenocarcinoma (EAC) is increasing at the most rapid rate of any cancer in the United States. An esophagogastroduodenal anastomosis (EGDA) surgical model in rats mimics human gastroesophageal reflux and results in EAC. Leukotriene A4 hydrolase (LTA4H), a protein overexpressed in EAC in this model, is a rate-limiting enzyme in the biosynthesis of leukotriene B4 (LTB4), a potent inflammatory mediator. We used this model and human EAC and non-tumor tissues to elucidate the expression pattern of LTA4H and to evaluate it as a target for chemoprevention.. LTA4H expression was examined by western blotting and immunohistochemistry. The functional role of LTA4H in carcinogenesis was investigated by use of an LTA4H inhibitor, bestatin, in the rat EGDA model. All statistical tests were two-sided.. LTA4H was overexpressed in all 10 rat EACs examined, compared with its level in normal rat tissue; it was also overexpressed in four of six human EAC tumor samples, compared with its level in adjacent non-tumor tissue. In tissue sections from 20 EGDA rats and 92 patients (86 with EAC, one with dysplasia, and five with columnar-lined esophagus), LTA4H was expressed in infiltrating inflammatory cells and overexpressed in the columnar cells of preinvasive lesions and cancers, especially in well-differentiated EACs, as compared with the basal cells of the normal esophageal squamous epithelium. Bestatin statistically significantly inhibited LTB4 biosynthesis in the esophageal tissues of EGDA rats (without bestatin = 8.28 ng/mg of protein; with bestatin = 4.68 ng/mg of protein; difference = 3.60, 95% CI = 1.59 to 5.61; P = .002) and reduced the incidence of EAC in the EGDA rats from 57.7% (15 of 26 rats) to 26.1% (6 of 23 rats) (difference = 31.6%, 95% CI = 0.3% to 56.2%; P = .042).. LTA4H overexpression appears to be an early event in esophageal adenocarcinogenesis and is a potential target for the chemoprevention of EAC.

Topics: Adenocarcinoma; Anastomosis, Surgical; Animals; Antibiotics, Antineoplastic; Blotting, Western; Chemoprevention; Disease Models, Animal; Duodenum; Epoxide Hydrolases; Esophageal Neoplasms; Esophagus; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Incidence; Inflammation; Leucine; Leukotriene A4; Rats; Stomach; Treatment Outcome; Up-Regulation

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).

Topics: Amino Acid Sequence; Aminopeptidases; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Epoxide Hydrolases; Humans; Hydrogen Bonding; Inflammation; Leucine; Leukotriene A4; Models, Molecular; Molecular Sequence Data; Multienzyme Complexes; Protein Structure, Secondary; Protein Structure, Tertiary; Thermolysin; Ytterbium; Zinc

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the proinflammatory mediator leukotriene B4. In previous studies with site-directed mutagenesis on mouse leukotriene A4 hydrolase, we have identified Tyr-383 as a catalytic amino acid involved in the peptidase reaction. Further characterization of the mutants in position 383 revealed that [Y383H], [Y383F], and [Y383Q] leukotriene A4 hydrolases catalyzed hydrolysis of leukotriene A4 into a novel enzymatic metabolite. From analysis by high performance liquid chromatography, gas chromatography/mass spectrometry of material generated in the presence of H216O or H218O, steric analysis of the hydroxyl groups, treatment with soybean lipoxygenase, and comparison with a synthetic standard, the novel metabolite was assigned the structure 5S, 6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5S,6S-DHETE). The kinetic parameters for the formation of 5S,6S-DHETE and leukotriene B4 were found to be similar. Also, both activities were susceptible to suicide inactivation and were equally sensitive to inhibition by bestatin. Moreover, from the stereochemical configuration of the vicinal diol, it could be inferred that 5S, 6S-DHETE is formed via an SN1 mechanism involving a carbocation intermediate, which in turn indicates that enzymatic hydrolysis of leukotriene A4 into leukotriene B4 follows the same mechanism. Inasmuch as soluble epoxide hydrolase utilizes leukotriene A4 as substrate to produce 5S,6R-DHETE, our results also suggest a functional relationship between leukotriene A4 hydrolase and xenobiotic epoxide hydrolases.

Topics: Aminopeptidases; Animals; Chromatography, High Pressure Liquid; Epoxide Hydrolases; Hydroxyeicosatetraenoic Acids; Leucine; Leukotriene A4; Leukotriene B4; Mass Spectrometry; Metalloproteins; Mice; Models, Chemical; Multienzyme Complexes; Mutagenesis, Site-Directed; Spectrophotometry, Ultraviolet; Tyrosine; Zinc

Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of the unstable epoxide intermediate LTA4 into the proinflammatory substance LTB4 and also exhibits an amidase/peptidase activity toward synthetic substrates. Based on proposed reaction mechanisms for other zinc hydrolases, we have synthesized inhibitors of LTA4 hydrolase and evaluated their effects on the formation of LTB4 from LTA4 using both purified enzyme and intact polymorphonuclear leukocytes. The two most effective inhibitors, an alpha-keto-beta-amino ester (compound IV) and a thioamine (compound VIII), exhibited IC50 values of 1.9 +/- 0.9 and 0.19 +/- 0.12 microM (mean +/- SD, n = 4), respectively. Compounds IV and VIII were also potent inhibitors of LTB4 biosynthesis in ionophore stimulated polymorphonuclear leukocytes with IC50 < 200 nM. At higher concentrations, the biosynthesis of 5-hydroxy-eicosatetraenoic acid was also inhibited with IC50 approximately 10 microM for both substances. In contrast, leukocyte 15-lipoxygenase and platelet LTC4 synthase activity were not inhibited by these substances at the highest concentrations tested, 50 and 10 microM, respectively. Compounds IV and VIII thus exhibit selectivity among enzyme activities in the arachidonic acid cascade. In conclusion, we describe two compounds that are among the most potent and selective inhibitors of LTA4 hydrolase and LTB4 biosynthesis by intact polymorphonuclear leukocytes, described thus far.

Topics: Angiotensin-Converting Enzyme Inhibitors; Calcimycin; Captopril; Enzyme Inhibitors; Epoxide Hydrolases; Humans; Ionophores; Kinetics; Leucine; Leukotriene A4; Leukotriene B4; Neutrophils; Protease Inhibitors

Leukotriene B4 formation can take place by cell interaction between keratinocytes and neutrophils. Thus, keratinocytes without proven 5-lipoxygenase activity can transform neutrophil-derived leukotriene A4 into leukotriene B4. The purpose of the present study was to investigate whether human epidermis is able to transform leukotriene A4 sequentially into the peptide leukotrienes (LTC4, LTD4 and LTE4). Epidermis isolated using the suction blister technique or keratomed skin specimens were incubated with either neutrophils or exogenously added leukotriene A4. Peptide leukotrienes were determined by integrated optical density after RP-HPLC separation, and the identity of leukotrine C4 was confirmed by (1) the retention time similarity with authentic leukotriene C4; (2) the UV spectrum determined with an on-line diode array detector; and (3) conversion by gamma-glutamyl transpeptidase of the peak coeluting with authentic leukotriene C4 into a new peak coeluting with authentic leukotriene D4. The results of this study showed that while human epidermis cannot form detectable amounts of peptide leukotrienes by itself, it can transform exogenous leukotriene A4 into peptide leukotrienes. Furthermore, coincubation of human epidermis and neutrophils resulted in a marked increase (90%) in peptide leukotriene formation when compared with neutrophils alone, indicating that human epidermis can transform neutrophil-derived leukotriene A4 into peptide leukotrienes. These results indicate that human skin contains leukotriene C4 synthase activity capable of producing significant amounts of leukotriene C4 from leukotriene A4, and that the keratinocytes may play a more active role in peptide leukotriene formation in the skin than previously thought.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biotransformation; Captopril; Chromatography, High Pressure Liquid; Epidermis; Ethacrynic Acid; gamma-Glutamyltransferase; Humans; Keratinocytes; Leucine; Leukotriene A4; Leukotriene B4; Leukotriene C4; Leukotrienes; Neutrophils; Peroxidase

The covalent coupling of [3H]LTA4 to human leukocyte LTA4 hydrolase is inhibited in a concentration-dependent fashion by pre-incubation with bestatin. This inhibition correlated with the concentration-dependent inhibition by bestatin of LTB4 and LTB5 synthesis by LTA4 hydrolase. Epibestatin, a diastereomer of bestatin, neither inhibited LTB4 or LTB5 production by LTA4 hydrolase nor prevented the covalent coupling of [3H]LTA4 to the enzyme. In contrast, captopril inhibited both LTB4 synthesis by LTA4 hydrolase and covalent coupling of [3H]LTA4 to the enzyme.

Topics: Captopril; Chromatography, Gel; Epoxide Hydrolases; Humans; Leucine; Leukocytes; Leukotriene A4; Leukotriene Antagonists; Leukotriene B4; Leukotrienes