Cloning of human airway 15-lipoxygenase: identity to the reticulocyte enzyme and expression in epithelium.

Lipoxygenases constitute a family of enzymes which are implicated in a variety of inflammatory disorders including asthma. Although the 15-lipoxygenase has been identified as the major route of arachidonic acid metabolism in human lung, airway epithelial cells, eosinophils, and developing red cells, the localization of the enzyme within lung has not been clearly defined. Furthermore, the existence of isoforms of 15-lipoxygenase in different tissues has recently been proposed. To address these issues, we isolated a 2.6-kb cDNA encoding human airway 15-lipoxygenase from a human bronchus cDNA library using a previously characterized reticulocyte 15-lipoxygenase cDNA as a probe. The airway 15-lipoxygenase sequence was found to be identical to that of the reticulocyte-derived clone. Immunocytochemical studies using an antibody to human recombinant 15-lipoxygenase specifically localizes the enzyme to the basal and ciliated cells of the trachea, bronchi, and bronchioles. Staining for 15-lipoxygenase is not present in the airway secretory epithelial cells, epithelial cells located in the gas-exchanging regions of lung, vascular structures, or inflammatory cells. Taken together these results suggest that the 15-lipoxygenase of human lung is identical to that of the reticulocyte enzyme and is preferentially expressed in airway epithelium.

Cloning and expression of an airway epithelial 12-lipoxygenase.

Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function. To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe. The resulting 2.9-kb cDNA, the identity of which was confirmed by expression of active catalytic function in Escherichia coli has a 2.0-kb open reading frame encoding a protein of 75,000 kDa and includes 5 bp of 5'-untranslated region and 0.9 kb of 3'-untranslated region. On Northern blots, the 12-lipoxygenase cDNA hybridized to one band (3.5 kb) of bovine tracheal epithelium RNA. Polyclonal antibodies that recognize human tracheal 15-lipoxygenase cross-reacted on immunoblots to the expressed bovine tracheal 12-lipoxygenase. Further, the deduced amino acid sequence is 86% identical (93% similar) to human 15-lipoxygenase but 64% identical to human platelet 12-lipoxygenase, suggesting that the bovine tracheal enzyme is the homologue of the human 15-lipoxygenase. This is the first sequence of an epithelial lipoxygenase from any species. A comparison of the bovine sequence with other lipoxygenase sequences shows that there are only four amino acids which are conserved differences between a 12-lipoxygenase and a 15-lipoxygenase. We hypothesize that these four amino acids may be responsible for the positional specificity of the enzyme.

Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase.

Arachidonate 15-lipoxygenase (arachidonate:oxygen 15-oxidoreductase, EC 1.13.11.33) is a lipid-peroxidating enzyme that is implicated in oxidizing low density lipoprotein to its atherogenic form. Monocyte/macrophage 15-lipoxygenase is present in human atherosclerotic lesions. To pursue a basis for induction of the enzyme, which is not present in blood monocytes, the ability of relevant cytokines to regulate its expression was investigated. Interleukin 4 (IL-4), among 16 factors tested, specifically induced 15-lipoxygenase mRNA and protein in cultured human monocytes. Interferon gamma and hydrocortisone inhibited this induction. High-performance liquid chromatography analysis of lipid extracts from IL-4-treated monocytes detected 15-lipoxygenase products esterified to the cellular membrane lipids, indicating enzymatic action on endogenous substrates. Stimulation of IL-4-treated monocytes with calcium ionophore or opsonized zymosan A enhanced the formation of 15-lipoxygenase products. These data identify IL-4 and interferon gamma as physiological regulators of lipoxygenase expression and suggest an important link between 15-lipoxygenase function and the immune/inflammatory response in atherosclerosis as well as other diseases.

Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related.

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.

Molecular cloning and primary structure of human 15-lipoxygenase.

A full-length cDNA encoding 15-lipoxygenase has been isolated from a human reticulocyte cDNA library. The predicted primary structure of the enzyme exhibits a sequence similarity of 61% and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygenases are aligned, there are two distinct regions of significant sequence identity including a cluster of five histidine residues conserved in all three lipoxygenases. Because histidines can serve as ligands for the enzymatically active iron, this region may be critical to enzymatic function. These results provide a basis for exploring functional domains of lipoxygenases.

Sodium-pump density of cells from dog tracheal mucosa.

Uptake of tritiated ouabain by cells isolated from dog tracheal epithelium showed two components: a saturable component with a Km of 5.1 X 10(-8) M and a maximal uptake of 8.3 X 10(5) molecules/cell and a nonsaturating component of uptake that was linear with concentration. Several criteria indicated that the saturable uptake component represented binding to the Na+-K+-ATPase. To estimate the average surface area per cell, a known number of cells were pelleted and weighed, and the average surface area was calculated, assuming the cells to be perfectly spherical. The validity of this assumption was confirmed by comparing the calculated surface areas of cells in isotonic and hypotonic media. From the values for maximal saturable uptake and average surface area, a pump density of approximately 2,400 sites/micron2 was calculated. Given that the apical membrane lacks Na pumps and accounts for only approximately 5% of the total surface area, this value corresponds to the pump density of the basolateral cell membrane. The pump densities of ciliated, goblet, and basal cells were compared by autoradiography. The three cell types had approximately the same density of pump sites.

Effects of "loop" diuretics on ion transport by dog tracheal epithelium.

The "loop" diuretics MK-196, bumetanide, piretanide, and furosemide are all potent inhibitors of Cl transport by the dog's tracheal epithelium. In short-circuited tissues, the drugs caused significant decreases in both unidirectional Cl fluxes and in the net flux of Cl toward the lumen; the change in net Cl flux was not significantly different from the change in short-circuit current. The drugs had no effect on active Na absorption. All drugs caused a significant fall in tissue conductance. All drugs, except MK-196, were more potent from the serosal bath; MK-196 was equipotent from either side of the tissue. In experiments with isolated cells, the diuretics caused no significant changes in intracellular Na and K concentrations, a fall in intracellular Cl concentration, and approximately equal falls in Na and Cl influxes. These results suggest that the site of action of these drugs is on a basolateral linked Na-Cl entry process. Additional evidence for such a linked entry process was provided by experiments in which removal of Cl reduced Na influx and removal of Na reduced Cl influx.

Ion contents and other properties of isolated cells from dog tracheal epithelium.

We have developed a preparation of isolated cells from dog tracheal mucosa. The three major cell types of the intact epithelium (ciliated, secretory, and basal) are present in approximately the same proportions as in the intact tissue. These cells show high viability as judged by exclusion of vital dyes, high O2 consumption, and incorporation of amino acids into protein. The intracellular ion contents (in mmol/l cell H2O) are [K]i, 150; [Na]i, 20; and [Cl]i, 50. Ouabain (10(-4) M) causes a rise in [Na]i and a reciprocal loss of intracellular K.