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Am J Physiol Lung Cell Mol Physiol 277: L457-L464, 1999;
1040-0605/99 $5.00
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Vol. 277, Issue 3, L457-L464, September 1999

15-HETE-substituted diglycerides selectively regulate PKC isotypes in human tracheal epithelial cells

Stephen E. Alpert1, Ronald W. Walenga1, Atashi Mandal1, Nicole Bourbon2, and Mark Kester2

1 Pediatric Pulmonary Division, Case Western Reserve University, Cleveland, Ohio 44106; and 2 Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania 17033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human tracheal epithelial (TE) cells selectively incorporate their major lipoxygenase product, 15-hydroxyeicosatetraenoic acid (15-HETE), into the sn-2 position of phosphatidylinositol (PI) (S. E. Alpert and R. W. Walenga. Am. J. Respir. Cell Mol. Biol. 8: 273-281, 1993). Here we investigated whether 15-HETE-PI is a substrate for receptor-mediated generation of 15-HETE-substituted diglycerides (DGs) and whether these 15-HETE-DGs directly activate and/or alter conventional diacylglycerol-induced activation of protein kinase C (PKC) isotypes in these cells. Primary human TE monolayers incubated with 0.5 µM 15-[3H]-HETE or 15-[14C]HETE for 1-2 h were stimulated with 1 nM to 1 µM platelet-activating factor (PAF) for 30 s to 6 min, and the radiolabel in the medium, cellular phospholipids, and neutral lipids was assessed by high-performance liquid and thin-layer chromatography. PAF mobilized radiolabel from PI in a dose-dependent manner (22 ± 5% decrease after 1 µM PAF) without a concomitant release of free intra- or extracellular 15-HETE. 14C-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major 14C band, identified as sn-1,2-15-[14C]HETE-DG, increased transiently in response to PAF. Western blots of freshly isolated and cultured human TE cells revealed PKC isotypes alpha , beta I, beta II, delta , epsilon , and zeta . In vitro, cell-generated sn-1,2-15-[14C]HETE-DG selectively activated immunoprecipitated PKC-alpha and inhibited diacylglycerol-induced activation of PKC-alpha , -delta , -beta I, and -beta II. Our observations indicate that 15-HETE-DGs can modulate the activity of PKC isotypes in human TE cells and suggest an intracellular autocrine role for 15-HETE in human airway epithelia.

15-hydroxyeicosatetraenoic acid; protein kinase C; human airway epithelial cells; signal transduction; monohydroxy-substituted diacylglycerols


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

15-HYDROXYEICOSATETRAENOIC ACID (15-HETE) is the predominant lipoxygenase metabolite of arachidonic acid (AA) generated by human airway epithelial cells (13). Although some studies reported that 15-HETE may have proinflammatory effects in the airway, causing increased mucus secretion (24) and bronchial hyperresponsiveness (18), more recent findings indicate that 15-HETE can downregulate various human neutrophil functions including agonist-induced superoxide anion production (34), integrin-mediated adhesion and transendothelial migration (36), and leukotriene B4 synthesis (5). Free 15-HETE has been detected in human airway lavage fluid (26, 33), suggesting that 15-HETE might serve as a paracrine regulator of airway smooth muscle and/or neutrophils recruited into the airway mucosa. However, a role for 15-HETE within airway epithelial cells themselves has not yet been determined.

The observation that 15-HETE is preferentially incorporated into phosphatidylinositol (PI) in various mammalian cell types (35) has led to studies on whether such incorporation might alter phosphoinositide signal transduction. In the reports cited above (5, 34, 36), changes in neutrophil function induced by exogenous 15-HETE were associated with altered production of inositol trisphosphate and mobilization of intracellular calcium. Recent findings in cells of nonpulmonary origin (6, 8, 19), in conjunction with work by Alpert and Walenga (1, 2), suggest that endogenously generated 15-HETE in human airway epithelial cells might participate in intracellular signaling through the formation of modified diacylglycerols (DAGs). Alpert and Walenga (1, 2) have reported that primary cultured human tracheal epithelial (TE) cells selectively incorporate 15-HETE into the sn-2 position of phosphatidylinositol (PI) and that the increased 15-HETE produced by these cells in response to ozone exposure is retained intracellularly esterified to phospholipids. Legrand et al. (19) demonstrated that esterification of 15-HETE to PI (15-HETE-PI) in bovine endothelial cells can result in the generation of diglycerides (DGs) that contain 15-HETE at the sn-2 position and speculated that such 15-HETE-substituted DGs (15-HETE-DGs) might exhibit an altered ability to activate protein kinase C (PKC). However, in a subsequent study with rat liver epithelial cells (37), a 15-HETE-DG and its sn-2-AA-DG counterpart had similar in vitro activity toward unfractionated total PKC derived from rat brain. In contrast, Cho and Ziboh (6, 8), using guinea pig epidermal cells, observed that DGs containing either 13(S)-hydroxyoctadecadienoic acid (13-HODE) or 15-hydroxyeicosatrienoic acid (15-HETrE) at the sn-2 position had no effect on total epidermal cell PKC activity but inhibited the interaction of a conventional DAG with PKC isotype beta . Similarly, studies from our laboratories with rat mesangial cells (21, 27) indicateed that sn-1 ether-linked DG species do not activate PKC but inhibit DAG-stimulated activation of PKC isotypes alpha , delta , and epsilon . Collectively, these observations suggest several mechanisms by which 15-HETE-DGs might modulate PKC-mediated signal transduction in human TE cells.

In this study, we demonstrated the presence of 15-HETE-DGs in primary cultured human TE cells after short-term incubation with 15-HETE and their increased formation in response to a membrane receptor-coupled agonist, platelet-activating factor (PAF). In vitro, using PKC isotypes recovered from human TE monolayers, we assessed whether these 15-HETE-DGs could stimulate PKC activity and/or alter conventional DAG-induced PKC activation. We found that 15-HETE-DGs selectively activated PKC isotype alpha  and inhibited DAG-stimulated activation of other PKC isotypes. Our observations indicate that 15-HETE-DGs can participate in receptor-induced phosphoinositide signal transduction in human TE cells and might modulate PKC-regulated cell processes by altering PKC-isotype bioactivity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and culture of TE cells. Primary monolayer cultures of human TE cells were established from tracheal segments obtained at autopsy as previously described (1). Protease-dissociated TE cells were plated onto collagen-coated (Vitrogen, Collagen, Palo Alto, CA) 12-well plates (4 cm2/well) or T25 (25-cm2) tissue culture flasks (Costar, Cambridge, MA). Defined serum-free medium (small-airway growth medium, Clonetics, San Diego, CA) was changed daily, and confluent monolayers were achieved in 4-6 days. In experiments in which TE monolayers were used for recovery of PKC isotypes by immunoprecipitation, the cultures were maintained in serum-free medium to avoid potential upregulation of PKC as a result of serum components. In all other studies, on the night before use, the cell culture medium was supplemented with 5% fetal bovine serum as a source of lipids. Such treatment results in cultured cells with a fatty acid composition more similar to that of native airway epithelium (3). Companion cultures established from the same trachea were used in a given experiment.

Incubation of TE monolayers with 15-[3H]HETE or 15-[14C]HETE and stimulation with PAF. Before the addition of radiolabeled 15-HETE, the growth medium was removed and the TE cultures were washed with 37°C Hank's balanced salt solution (HBSS; GIBCO BRL, Life Technologies, Grand Island, NY). Initial experiments assessing agonist-induced mobilization of 15-HETE from PI were conducted with 15-[3H]HETE and 12-well cultures, whereas the larger flask cultures and 15-[14C]HETE were used to detect 15-HETE-DG production. PAF was used as the agonist in these studies because cultured human TE cells have a specific membrane PAF receptor (38), and in most cells, PAF stimulates phospholipase (PL) C-induced turnover of PI (15). Briefly, solutions of 15(S)-[3H]HETE (178 Ci/mmol; New England Nuclear, Boston, MA) were prepared in HBSS-0.1% fatty acid-free bovine serum albumin (BSA; Sigma, St. Louis, MO) with unlabeled 15-HETE (Cayman Chemical, Ann Arbor, MI) to a final concentration of 0.5 µM, and 0.5 ml/well was added to the 12-well cultures for 1 h at 37°C in a humidified 5% CO2-air atmosphere. Alpert and Walenga (1) have previously demonstrated that uptake and incorporation of 15-HETE by human TE cells is rapid and complete by 1 h. After this 1-h incubation, the 15-[3H]HETE medium was removed, and the wells were rinsed with HBSS-0.1% BSA and stimulated with 1 nM to 1 µM PAF (Cayman Chemical) in 1 ml of HBSS-0.1% BSA at 37°C for 10 min. Medium from three similarly treated wells was removed and pooled, and the lipids were extracted with four volumes of chloroform-methanol (2:1) for further studies. After the addition of ice-cold methanol, cells were recovered from the wells by scraping with a rubber policeman, and total cellular lipids were extracted with chloroform-methanol (2:1).

15(S)-[14C]HETE was prepared from [14C]AA (55 mCi/mmol; Amersham, Arlington Heights, IL) with soybean lipoxygenase with minor modifications of described methods (9), and the concentration of 15-[14C]HETE was calculated from the specific activity of the [14C]AA substrate. Confluent T25 flask cultures were incubated with 0.5 mM 15-[14C]HETE in 4 ml of HBSS-0.1% BSA for 1 h followed by a second 1-h incubation with a fresh volume of 15-[14C]HETE. Such successive incubations result in progressively more 15-HETE incorporation into PI (19; Alpert and Walenga, unpublished observations). After the second incubation with 15-[14C]HETE, the medium was removed, the cultures were stimulated with 1 mM PAF in 4 ml of HBSS-0.1% BSA for 30 s to 6 min, and the lipids in the HBSS medium and in cells from individual flask cultures were extracted separately.

A study (5) with human neutrophils suggested that 15-HETE-PI can be mobilized by agonists and released extracellularly. As described in Characterization of putative 15-HETE-glycerolipids and HPLC, we determined whether PAF might cause similar deacylation of 15-HETE from human TE cell phospholipids and release of 15-HETE and/or its beta -oxidation metabolites by analysis of 15-HETE-derived radiolabel in both intra- and extracellular compartments. Walenga and Statt (38) have observed that PAF is a potent stimulus for prostaglandin E2 (PGE2) production in human TE cells, presumably through PLA2-mediated release of membrane stores of AA. In some experiments, PAF-induced PGE2 synthesis by TE monolayers preincubated with 15-[3H]HETE was confirmed by measurement of PGE2 in the medium with an enzyme-linked immunoassay (Cayman Chemical) as previously employed (1, 2).

Characterization of putative 15-HETE-glycerolipids. The chloroform extract of 15-[14C]HETE-labeled human TE cells [containing >98% cell-associated counts/min (1)] was evaporated under nitrogen, and the residue was resuspended in 50 ml of chloroform-methanol (9:1 vol/vol). A portion (5 ml) was removed to determine total cell-associated radiolabel, and the remainder was spotted onto Silica Gel 60 plates (Merck/EM Science, Gibbstown, NJ) to resolve various lipid species with two thin-layer chromatography (TLC) systems. Individual phospholipids, neutral lipids, and free unesterified 15-HETE were resolved with chloroform-methanol-water-NH4OH (65:35:3:2) as described previously (1). The lipids were visualized with toluidino-2-naphthalene-6-sulfonic acid (TNS) spray under ultraviolet light, identified by comparison to authentic standards (Supelco, Bellefonte, PA, and Cayman Chemical), and the radioactivity in each lipid fraction was then measured by liquid scintillation (1). Monoglycerides (MGs), DGs, and triglycerides (TGs) were resolved with benzene-ethyl ether-triethylamine (100:80:1) (11). After autoradiographic detection of 14C-labeled lipids and visualization of authentic standards with TNS, density of the autoradiographic bands was quantitated with a Sci Scan 5000 light-transmission densitometer (US Biochemicals) and OS Image Analysis System software (Oberlin Scientific).

Several radiolabeled spots were detected when lipids from TE monolayers incubated with 15-[14C]HETE were resolved by TLC for MGs, DGs, and TGs (see Fig. 1). To assess whether these lipids might be 15-[14C]HETE-DGs, authentic 15-[14C]HETE-DGs were biosynthesized from cell-generated 15-[14C]HETE-PI by in vitro hydrolysis with PLC. Briefly, the 15-[14C]HETE-PI TLC band was eluted with chloroform-methanol-water (5:5:1), dried, and resuspended in 1 ml of 100 mM sodium borate and 10 mM CaCl2, pH.7.4, containing 2 U of PI-specific PLC (Bacillus cereus, Sigma) (14, 27). After incubation for 2.5 h at 23°C, the reaction was stopped with hexane, and the products were resolved by TLC for MGs, DGs, and TGs.

To confirm that the radiolabel in putative 15-[14C]HETE-DGs was unmodified 15-HETE at the sn-2 position, isolated [14C]DGs were incubated in vitro with a DG lipase (6). [14C]DGs were eluted from TLC silica with chloroform-methanol-water (60:30:5) containing 0.1% sodium borate, pH 7.0, resuspended in 1 ml of 25 mM Tris · HCl, pH 8.2, containing 10 µM CaCl2, and digested with 100 U of DG lipase (Clostridium viscosum, Sigma) for 30 min at 30°C. The reaction was stopped with 10 ml of methanol, and the sample was concentrated and spotted for TLC resolution of MGs, DGs, and TGs in which free 15-HETE remains at the origin. After autoradiography of the developed TLC plate, radiolabel at the origin was further assessed by high-performance liquid chromatography (HPLC).

HPLC. HBSS medium removed from control and PAF-stimulated cultures preincubated with 15-HETE and putative 15-HETE released by DG lipase were subjected to HPLC analysis with a step-gradient elution of acetonitrile, water, and trifluoroacetic acid as previously described in detail (1).

Western blot analysis of PKC isotypes in freshly isolated and cultured human TE cells. A recent study (39) has described the PKC isotypes present in primary cultured human TE cells (19) and bovine bronchial epithelial cells (39). In the latter report, loss of PKC activity was observed in serially passaged cells as well as in primary cells maintained in culture for >2 wk. To determine whether human TE cells grown in our culture conditions retained the PKC isotypes present in native airway epithelium, we assessed PKC isotypes in freshly isolated human TE cells and in primary human TE monolayers <10 days old using methods previously described (21, 27). Freshly isolated human TE cells or confluent TE monolayers were washed in ice-cold phosphate-buffered saline, and the cells were lysed on ice with 0.5 ml of 20 mM HEPES buffer, pH 7.5, containing 40 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 µM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml of leupepetin, 1 µg/ml of pepstatin, and 1 mM benzamidine hydrochloride (21). The cell lysate proteins (12-15 µg) were resolved by SDS-PAGE and electroblotted onto nitrocellulose. The nitrocellulose membranes were incubated for 2 h at 25°C with polyclonal antibodies directed against PKC isotypes alpha , beta I, beta II, delta , epsilon , gamma , or zeta  (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and then incubated for 1 h at 25°C with a secondary antibody, goat anti-rabbit IgG coupled to horseradish peroxidase at a 1:5,000 dilution (Kirkegaard and Perry, Gaithersburg, MD). Enhanced chemiluminescence with horseradish peroxidase substrate (Amersham) was used to reveal positive bands according the manufacturer's instructions. Lysates from rat renal mesangial cells and authentic PKC standards (Santa Cruz Biotechnology) served as positive controls.

Immunoprecipitation of PKC isotypes and assay of PKC activity. The methods used for immunoprecipitation of PKC isotypes and the subsequent assay of PKC activity have been described in detail (21, 27). The specificity of the immunoprecipitation procedure has been verified by Western blotting of the immunocomplexes, and this procedure recovers equal masses of each soluble PKC isotype as determined by visualization of the protein bands on the membranes with Ponceau S (Sigma) (21, 27). Briefly, cell lysates from 25-cm2 flask cultures were cleared of nuclear protein by centrifugation, and polyclonal anti-PKC isotype serum (0.5 µg) was added overnight at 4°C. The immune complexes were subsequently collected with goat anti-rabbit IgG agarose (Sigma). The ability of cell-generated 15-HETE-DGs to activate PKC isotypes recovered from human TE monolayers was assessed with an in vitro reconstitution assay that measures the phosphorylation of histone IIIS (Sigma) as an exogenous substrate. In some experiments, activation of PKC-epsilon was also assessed with a PKC-epsilon substrate (Peptide epsilon , Alexis, San Diego, CA) as the reporter protein (17, 31). The kinase reaction buffer consisted of 50 mM HEPES, pH 7.55, 25 mM beta -glycerophosphate, 75 mM KCl, 1 mM vanadate, 10 mM MgCl2, and 0.1 mM CaCl2. The assay was performed in 50 µl of kinase buffer with 1 µCi of [gamma -32P]ATP (4,500 Ci/mmol; ICN, Costa Mesa, CA) and 20 µM unlabeled ATP, 40 µg/ml of phosphatidylserine (Avanti Polar Lipids, Alabaster, AL), 10 µg/ml of histone IIIS, or PKC-epsilon substrate and 10-7 M 15-HETE-DGs (concentration calculated from the specific activity of 15-[14C]HETE) for 20 min at 30°C. Because various fatty acids have been shown to activate PKC isotypes (29), in some experiments, we assessed whether free 15-HETE alone was capable of stimulating PKC activity. To determine whether 15-HETE-DGs inhibit DAG-stimulated PKC activation, 15-HETE-DGs and conventional DAG (1-palmitoyl-2-oleoyl-sn-glycerol; Avanti) were both added at 10-7 M to the in vitro reconstitution assays. Positive controls for the assay consisted of 10-7 M DAG, and phosphatidylserine was omitted as a negative control for DAG-dependent PKC isotypes. Phosphorylated proteins were resolved by 12% SDS-PAGE and visualized by autoradiography, and the band corresponding to histone IIIS was quantitated by light-transmission densitometry (Macromolecular Core Facility, Milton S. Hershey Medical Center, Hershey, PA).

Data analysis. Data from replicate experiments are expressed as means ± SD. Where appropriate, data were analyzed post hoc by Bonferroni correction after multivariate ANOVA (SigmaStat), and significance of differences among groups was assessed.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Uptake and intracellular distribution of 15-HETE by human TE cells. Consistent with the earlier study by Alpert and Walenga (1), when human TE monolayers were incubated with 0.5 µM 15-[3H]HETE for 1 h, nearly one-third (28.5 ± 3.9%; n = 8 experiments) of the total initial radiolabel was incorporated intracellularly, with the remainder present in medium primarily as beta -oxidation metabolites of 15-HETE. Of the cell-associated radiolabel, 56-65% was present in phospholipids, 25-34% was in neutral lipids, and the remainder was present as free unesterified 15-HETE. As shown in Table 1, 15-HETE was incorporated preferentially into PI, which accounted for ~75% of the total phospholipid counts per minute. Based on the demonstration (1) that human TE cells selectively esterify 15-HETE without modification into the sn-2 position of phospholipids, radiolabeled PI is presumably sn-2-15-[3H]HETE-PI.

                              
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  		Table 1.   PAF-induced mobilization of radiolabel from phospholipids in human TE cells incubated with 15-[3H]HETE

PAF-induced mobilization of cell-associated radiolabel and PGE2 production. When human TE cells prelabeled with 15-[3H]HETE were stimulated with PAF, radiolabel was mobilized selectively from PI in a concentration-dependent manner (Table 1). In eight separate experiments, 1 µM PAF released 22 ± 5% of the PI-associated 15-HETE radiolabel. This decline in PI-radiolabel occurred without a change in total cell-associated counts per minute and without an increase in free intracellular 15-HETE (Table 1) or release of 15-HETE (or its beta -oxidation metabolites) into the medium as determined by HPLC. As shown in Table 1, the net decline in phospholipid-associated 15-HETE radiolabel induced by PAF was almost entirely accounted for by release from PI, without evidence for any redistribution of radiolabel among other phospholipid classes. In these same experiments, PAF caused a dose-dependent increase in PGE2 (5- to 12-fold stimulation after 1 µM PAF; n = 3 experiments; data not shown). Thus, despite receptor-mediated increased production of PGE2, presumably by activation of PLA2 and release of membrane-bound AA, PAF did not cause deacylation of 15-HETE from PI or other lipids.

Characterization of putative 15-HETE-DGs. One possibility for the selective decrease in PI-associated radiolabel after PAF stimulation was PLC-mediated hydrolysis of 15-HETE-PI to 15-HETE-DGs (6, 19). After incubation of TE monolayers with 15-[14C]HETE, TLC resolution of cellular lipids for MGs, DGs, and TGs revealed 14C products that migrated slower than authentic sn-1,2- and sn-1,3-DAG standards (Fig. 1, lane A, bands 1-3), consistent with the presence of a more polar hydroxyl group of 15-HETE in these putative 15-HETE-DGs. To determine whether these 14C products were in fact 15-HETE-DGs, we generated 15-HETE-DGs in vitro by incubating isolated 15-[14C]HETE-PI with PLC. The TLC migration of the slower-migrating PLC hydrolysis product, presumably the sn-1,2-15-[14C]HETE-DG isomer, was identical to the major cell-generated 15-HETE-DG product (Fig. 1, lanes A and C, band 2). A minor cell-generated product comigrated with the presumptive sn-1,3-15-[14C]HETE-DG isomer (Fig. 1, lanes A and C, band 1). [Note: in vitro, nonenzymatic racemization of 1,2-DG to 1,3-DG isomers occurs, and equilibrium favors the latter (23).] An additional minor 14C product present in vivo (Fig. 1, lane A, band 3), moving more slowly than the presumptive sn-1,2-15-[14C]HETE-DG isomer, had no correlate with the in vitro generated PLC hydrolysis products. When the major cell-generated putative 15-[14C]HETE-DG (Fig. 1, band 2) was incubated in vitro with DG lipase, there was a decrease in counts per minute associated with the [14C]DG spot and an appearance of radiolabel at the origin of the TLC plate that comigrated with authentic 15-HETE on HPLC (data not shown). These observations indicate that human TE cells incubated with 15-HETE accumulate a neutral lipid species that appears to be an sn-1,2-15-[14C]HETE-DG derived from 15-[14C]HETE-PI.


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  		Fig. 1.   Autoradiogram of TLC resolution of putative 15-[14C]hydroxyeicosatetraenoic acid (15-[14C]HETE)-diglycerides (DGs) in human tracheal epithelial (TE) cells and products from in vitro incubation of 15-[14C]HETE-phosphatidylinositol (PI) with phospholipase (PL) C. Lane A: total lipid extracts from human TE monolayers incubated with 15-[14C]HETE and resolved for monoglycerides (MGs), DGs, and triglycerides (TGs) as described in METHODS. 15-[14C]HETE-PI was isolated from cellular lipids and left untreated (lane B) or was incubated in vitro with PLC (lane C). Positions of migration of phospholipids (PLs) and authentic MGs, 1,2- and 1,3-diacylglycerols (1,2-DAG and 1,3-DAG, respectively), and TGs are indicated. Bands 1-3, 14C-labeled neutral lipids in cellular extracts migrating slower than sn-1,2- and sn-1,3-DAG standards.

Increased 15-HETE-DGs in PAF-stimulated cells. When human TE monolayers preincubated with 15-[14C]HETE were treated with 1 µM PAF for 30-90 s, the putative major sn-1,2-15-[14C]HETE-DG band consistently increased. Figure 2 shows that the increase in 15-[14C]HETE-DG was transient, with a maximum increase observed 45 s after the addition of PAF followed by a decline to below control levels when assessed 2 or 6 min post-PAF stimulation. These results, in conjunction with the findings in Characterization of putative 15-HETE-DGs, demonstrate that physiological stimuli can lead to a transient increase in the levels of 15-[14C]HETE-DG in human TE cells, consistent with receptor-mediated hydrolysis of 15-[14C]HETE-PI.


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  		Fig. 2.   Kinetics of platelet-activating factor (PAF)-induced production of 15-[14C]HETE-DGs in human TE cells. Human TE monolayer cultures were preincubated with 15-[14C]HETE and then stimulated with 1 µM PAF for 30 s to 6 min, and densitometry measurements were made from autoradiographs of cellular lipids resolved by TLC as in Fig. 1. For each sample in a given experiment, a ratio of absolute densitometry values of the major presumptive 15-[14C]HETE-DGs (Fig. 1, band 2) and [14C]TGs was calculated and normalized to control cultures. Each data point represents mean ± SD of 3-5 values from 5 separate experiments. * P < 0.05 vs. control.

Stimulation of human TE cell PKC isotypes by DAG and 15-HETE-DG. Western blot analysis of freshly isolated human TE cells and TE monolayers maintained in culture for up to 10 days revealed the presence of PKC isotypes alpha , beta I, beta II, delta , epsilon , and zeta  but not gamma  as previously reported (20). The basal enzymatic activity of the immunoprecipitated soluble PKC isotypes recovered from cultured cells, determined in vitro, was 46.4 ± 12.1% PKC-delta , 29 ± 10.9% PKC-alpha , 17.7 ± 3.4% PKC-zeta , 4.2 ± 2.0% PKC-beta II, and 2.8 ± 0.8% PKC-beta I. PKC-epsilon accounted for <1% of total PKC activity in any culture. In preliminary experiments, as expected, a conventional DAG, 1-palmitoyl-2-oleoyl-sn-glycerol, induced an approximately twofold increase in the activity of immunoprecipitated PKC-alpha , -beta I, -beta II, and -delta but not PKC-zeta . Additional experiments with cell-derived 15-HETE-DGs were then conducted with PKC isotypes alpha , delta , and zeta  as major representatives of the conventional, novel, and atypical PKC subtypes, respectively, in human TE cells, accounting for >93% of the immunoprecipitated PKC activity. As shown in Fig. 3, DAG stimulated PKC-alpha and -delta but not PKC-zeta . 15-HETE-DG stimulated the activity of PKC-alpha by approximately twofold, an increase similar to that induced by conventional DAG (Fig. 3A). Increases in the activity of PKC-delta and -zeta were also observed in response to 15-HETE-DG, but these increases were not significant. Free 15-HETE did not stimulate any of the immunoprecipitated PKC isotypes tested.


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  		Fig. 3.   Effects of DAG and 15-HETE-DG on activity of soluble protein kinase (PK) C isotypes from human TE cells. PKC isotypes alpha  (A), delta  (B), and zeta  (C) were immunoprecipitated from human TE cell cultures as described in METHODS. Activity of each PKC isotype was then determined in vitro in presence of 0.1 µM 1-palmitoyl-2-oleoyl-sn-glycerol (DAG), 0.1 µM 15-HETE-DG, 0.1 µM each DAG and 15-HETE-DG (Both), or 0.1 µM unesterified 15-HETE. Controls for each experiment consisted of no addition of DAG. Results are expressed as means ± SD of percent control incorporation of gamma -32PO4 from ATP into histone IIIS; n = 4-6 measurements for each PKC isotype recovered from separate TE cultures. * P < 0.05 vs. control activity. ** P < 0.05 vs. DAG-stimulated activity.

Surprisingly, the activity of PKC-alpha , which was stimulated either by DAG or by 15-HETE-DG alone, was significantly reduced when both DG species were added in combination. DAG-stimulated activation of PKC-delta was also inhibited by the combination of DAG and 15-HETE-DG (Fig. 3). The effect of 15-HETE-DG on the minor isotypes PKC-beta I or -beta II was similar to that observed for PKC-delta ; i.e., 15-HETE-DG did not activate either PKC-beta isotype, but the combination of DAG and 15-HETE-DG inhibited DAG-stimulated PKC-beta I and -beta II activity (data not shown). Thus 15-HETE-DG selectively stimulated the activity of PKC-alpha and inhibited conventional DAG-induced activation of PKC isotypes delta , beta I, and beta II.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human airway epithelial cells express 15-lipoxygenase (4, 28), the enzyme that metabolizes AA to 15-HETE. However, the biological role of 15-HETE in airway epithelial cells has not been established. Alpert and Walenga (1, 2) have previously reported that primary cultured human TE cells esterify 15-HETE exogenously provided or endogenously generated into cellular phospholipids with a high degree of selectivity for the sn-2 position of PI. Here we demonstrate that 15-HETE-PI can serve as a source of 15-HETE-substituted DGs, which differentially regulate the activity of PKC isotypes in these cells. Substantial amounts of 15-HETE-DGs were present in resting TE monolayers after short-term incubation with exogenous 15-HETE, and increased formation of 15-HETE-DGs was detected in response to stimulation with a membrane receptor-coupled agonist. Moreover, agonist-induced generation of 15-HETE-DGs occurred without a concomitant release of free 15-HETE.

PKCs are a family of homologous serine/threonine protein kinases that are activated by membrane-derived lipid second messengers. Presently, there are at least 12 different isoforms of PKC grouped into three classes; conventional (diglyceride and calcium dependent), novel (diglyceride dependent and calcium independent), and atypical (diglyceride independent) (12, 30). For the diglyceride-dependent isotypes, the chain length and the degree of saturation of the fatty acids esterified to the sn-1 or sn-2 position of the glycerol moiety have been shown to affect PKC activation (10, 22). More recent studies have focused on the ability of DAGs containing monohydroxy-substituted fatty acids to activate and/or inhibit specific PKC isotypes. As previously reviewed, sn-2-15-HETE-DG stimulated total unfractionated rat brain PKC to the same extent as did sn-2-AA-DG (37), whereas structurally similar monohydroxy-substituted DGs containing 13-HODE or 15-HETrE (the 15-lipoxygenase products of linoleic and dihomo-gamma -linoleic acids, respectively) had no effect on total guinea pig epidermal PKC activity (composed entirely of PKC-beta and -alpha ) but inhibited DAG-induced stimulation of PKC-beta but not of PKC-alpha (6, 8).

We observed complex interactions between 15-HETE-DGs and a conventional DAG in the activation of PKC isotypes immunoprecipitated from human TE cells. 15-HETE-substituted DGs inhibited DAG-induced stimulation of the major PKC isotypes alpha  and delta . Similar findings were observed for the minor PKC isotypes beta I and beta II. However, in the absence of DAG, 15-HETE-DG stimulated PKC-alpha but not -delta , -beta I, or -beta II. A model for PKC regulation by 15-HETE-DGs can be envisioned based on precedents in the literature. In the studies above (6, 7), and in work from our laboratory (27), 13-HODE-, 15-HETrE- and ether-linked DGs competitively inhibit DAG-activated PKCs by blocking the DG binding site. Lipid-derived cofactors can also inhibit activation of PKCs via mechanisms independent of the DG binding site. For example, AA inhibits ceramide-activated PKC-zeta via a two-site model (25). Binding of AA and DAGs to discrete sites on PKC can also facilitate the formation of stable membrane-bound, intrinsically active forms of PKC (32). To explain the actions of 15-HETE-DG on PKC isotypes, we propose a two-site binding model in which 15-HETE-DG binds to a distinct site expressed on PKC-alpha but not on PKC-beta I, -beta II, or -delta to selectively activate PKC-alpha . However, in the presence of both DAG and 15-HETE-DG binding to distinct sites on PKC-alpha , a synergistic conformational change occurs in rendering the enzyme refractory to the lipid cofactors. Alternatively, this conformational change in PKC-alpha may be a function of 15-HETE-DG altering protein-lipid interactions because unsaturated and oxygenated fatty acid DGs can affect the physical properties of lipid bilayers by altering lateral phase separation, lipid packing, and viscosity (40). Our data also suggest that this putative 15-HETE binding site is not a free fatty acid binding site because free 15-HETE did not activate PKC-alpha . The elucidation of a distinct binding site on PKC-alpha from human TE cells for oxygenated diglycerides is in progress and may explain the observed different effects of 15-HETE-, 13-HODE-, and 15-HETrE-DGs on PKC-alpha between our study and the reports of Cho and Ziboh with guinea pig epidermal cells (6, 8).

The consequences of selective activation and/or inhibition of PKC isotypes by 15-HETE-DGs in human airway epithelial cell function are not known. In studies with human neutrophils, the incorporation of exogenous 15-HETE into PI results in altered phosphoinositide signal transduction and downregulation of several proinflammatory functions (5, 34, 36). In guinea pigs, incorporation of 13-HODE into epidermal phospholipids in vivo was associated with increased levels of 13-HODE-DAG, decreased expression and activity of PKC-beta in epidermal tissue, and resolution of hyperproliferative psoriatic skin lesions (7). In a related study from our laboratories with rat mesangial cells (21), downregulation of receptor-stimulated PKC isotype activities occurred concomitantly with antimitogenic and anti-inflammatory responses. Thus we speculate that the ability of 15-HETE-substituted DGs to differentially regulate PKC isotypes may have important implications for the response of human airway epithelial cells at sites of airway inflammation.

The findings of this study and the earlier investigations by Alpert and Walenga (1, 2) provide further insight into the actions of 15-HETE in the airway. Most studies (16, 18, 24, 34, 36) on the role of 15-HETE in human or animal airways have focused on the effects of extracellular free 15-HETE, presumably generated in part by airway epithelial cells, on airway mucus production, smooth muscle contraction, and inflammatory cell function. This interest in free 15-HETE stems largely from in vitro observations demonstrating that human airway epithelial cells release micromolar concentrations of 15-HETE when provided with a vast excess of exogenous AA (13). However, release of 15-HETE under these conditions most likely is an artifact of the production of 15-HETE to levels that overwhelm the ability of the cells to esterify it into membrane phospholipids. In contrast, our studies suggest an intracellular autocrine role for 15-HETE in human airway epithelia. We have reported that 15-HETE esterified into PI in human TE cells is metabolically stable (half-life ~12 h). Moreover, it is not subject to mobilization by the calcium ionophore A-23187 (1), suggesting that 15-HETE-PI is not a substrate for PLA2 in these cells. Similarly, in the present study, the membrane receptor-coupled agonist PAF did not induce release of intra- or extracellular free 15-HETE despite concomitant evidence for the activation of PLA2. We have also observed that increased 15-HETE produced by human TE monolayers in response to ozone exposure was not released extracellularly but instead was retained intracellularly esterified to phospholipids (2). Collectively, our findings lead us to propose that under most physiological conditions, endogenously generated 15-HETE in human airway epithelial cells is retained intracellularly where it gives rise to 15-HETE-substituted DGs that modulate the activity of select PKC isotypes.


    ACKNOWLEDGEMENTS

We acknowledge the assistance and use of facilities of the Molecular Biology Core of Case Western Reserve University (Cleveland, OH), funded in part by National Cancer Institute Grant CA-43703-07S2, National Institute of Allergy and Infectious Diseases Grant AI-36219, and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-39750.


    FOOTNOTES

This work was by supported by National Heart, Lung, and Blood Institute Grant RO1-HL-51910 (to S. E. Alpert and R. W. Walenga) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53715 (to M. Kester).

Present address of and address for reprint requests and other correspondence: S. E. Alpert, Section of Pediatric Respiratory Medicine, Emory Univ. School of Medicine, 2040 Ridgewood Dr., N.E., Atlanta, GA 30322 (E-mail: sealper{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 29 January 1999; accepted in final form 10 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 277(3):L457-L464
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