1 Pediatric Pulmonary Division, Case Western Reserve University, Cleveland, Ohio 44106; and 2 Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania 17033
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ABSTRACT |
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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 ,
I,
II,
,
, and
. In
vitro, cell-generated
sn-1,2-15-[14C]HETE-DG
selectively activated immunoprecipitated PKC-
and inhibited diacylglycerol-induced activation of PKC-
, -
, -
I,
and -
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
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INTRODUCTION |
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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 . 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
,
, and
. 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 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.
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METHODS |
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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 ![]() |
RESULTS |
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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 -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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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 -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.
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DISCUSSION |
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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--linoleic acids,
respectively) had no effect on total guinea pig epidermal PKC activity
(composed entirely of PKC-
and -
) but inhibited DAG-induced
stimulation of PKC-
but not of PKC-
(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 and
. Similar findings were observed for the
minor PKC isotypes
I and
II. However, in
the absence of DAG, 15-HETE-DG stimulated PKC-
but not -
, -
I, or -
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-
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-
but not on PKC-
I,
-
II, or -
to selectively activate PKC-
. However,
in the presence of both DAG and 15-HETE-DG binding to distinct sites on
PKC-
, a synergistic conformational change occurs in rendering the
enzyme refractory to the lipid cofactors. Alternatively, this
conformational change in PKC-
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-
. The elucidation of a distinct binding site on PKC-
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-
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- 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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