Pulmonary Division, Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
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ABSTRACT |
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Protein kinase
C (PKC)-activated signal transduction pathways regulate cell growth and
differentiation in many cell types. We have observed that interleukin
(IL)-1 upregulates
2-adrenergic receptor
(
2-AR) density and
2-AR mRNA in human
airway epithelial cells (e.g., BEAS-2B). We therefore tested the
hypothesis that PKC-activated pathways mediate IL-1
-induced
-AR
upregulation. The role of PKC was assessed from the effects of
1) the PKC activator phorbol 12-myristate 13-acetate (PMA)
on
-AR density, 2) selective PKC inhibitors (calphostin C
and Ro-31-8220) on
-AR density, and 3) IL-1
treatment
on the cellular distribution of PKC isozymes. Recombinant human IL-1
(0.2 nM for 18 h) increased
-AR density to 213% of control
values (P < 0.001). PMA (1 µM for 18 h) increased
-AR density to 225% of control values (P < 0.005),
whereas Ro-31-8220 and calphostin C inhibited the IL-1
-induced
upregulation of
-AR in dose-dependent fashion. PKC isozymes detected
by Western blotting included
,
II,
, µ,
, and
/
.
IL-1
increased PKC-µ immunoreactivity in the membrane fraction and
had no effect on the distribution of the other PKC isozymes identified.
These data indicate that IL-1
-induced
-AR upregulation is
mimicked by PKC activators and blocked by PKC inhibitors and appears to
involve selective activation of the PKC-µ isozyme. We conclude that
signal transduction pathways activated by PKC-µ upregulate
2-AR expression in human airway epithelial cells.
cytokines; gene expression; airway inflammation; signal
transduction; interleukin-1; protein kinase C
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INTRODUCTION |
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INTERLEUKIN
(IL)-1, a pleiotypic cytokine released into the airway in asthma
(1, 2, 7), produces a variety of effects on respiratory
tract cells (6, 8, 12, 23). In particular, IL-1
profoundly affects the expression and function of the
2-adrenergic receptor (
2-AR) system
expressed by airway epithelial and smooth muscle cells (6, 8,
23). For example, IL-1
enhances the expression of
2-AR protein and mRNA in airway epithelial cells (8). At the same time, IL-1
decreases the
responsiveness of these cells to a
2-AR agonist.
IL-1 binds to a discrete receptor on the cell surface and activates
a spectrum of second messenger pathways composed of G proteins, a
variety of tyrosine and serine/threonine kinases, and transcription
factors (4). The signal transduction pathways activated by
IL-1
are cell-type dependent (4, 5). In some cell
types, the effects of IL-1
are mediated by activation and translocation of protein kinase C (PKC) (3, 18). PKC is a threonine/serine kinase that, when activated, plays a key regulatory role in a variety of cellular functions such as stimulation or inhibition of growth, changes in morphology, and modulation of gene
expression (3, 18, 21). In particular, PKC activation mediates a variety of effects important for the differentiated function
of airway epithelial cells (11-13, 16, 24).
The purpose of this study was to test the hypothesis that PKC-activated
signal transduction pathways mediate IL-1-induced upregulation of
2-AR gene expression. We also sought to characterize the
spectrum of PKC isozymes expressed by human airway epithelial cells.
The role of PKC was assessed from the effects of 1) the PKC
activator phorbol 12-myristate 13-acetate (PMA) on
-AR density; 2) PKC inhibitors, e.g., calphostin C and Ro-31-8220, on
IL-1
-induced
-AR upregulation; and 3) IL-1
on the
cellular distribution of PKC isozymes. The PKC isozymes expressed by
airway epithelial cells and their pattern of cellular distribution were
assessed by immunoblotting of cytosolic and membrane fractions.
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METHODS |
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Cell culture. Experiments were performed on cultured BEAS-2B cells, a transformed normal human airway epithelial cell line (gift of Dr. Curtis Harris, National Cancer Institute, Bethesda, MD) (20). Cells (passages 18-32) were grown in RPMI 1640 medium (Sigma, St. Louis, MO) plus 10% FCS in 5% CO2 at 37°C until 90-100% confluent (5-6 days).
At confluence, 0.2 nM IL-1-AR density.
-AR density was determined in cell suspensions by radioligand
binding with the use of the
-AR subtype nonselective antagonist [125I]iodopindolol ([125I]PIN; specific
activity 2,200 Ci/mmol; NEN Life Sciences, Boston, MA) as previously
described (8-10, 19). A single concentration of
[125I]PIN that saturated specific binding sites
(i.e., ~300 pM) was used. Cell aliquots (~100,000 epithelial cells)
were delivered to polypropylene tubes containing 10 mM
Tris · HCl, 2 mM MgCl2 buffer, pH 7.4, and
[125I]PIN. Separate tubes containing these reactants plus
40 µM alprenolol, a
-AR subtype nonselective antagonist, were used
to determine nonspecific binding. Tubes were then vortexed and
mechanically shaken for 120 min at 28°C. Incubations were
quenched by the addition of ice-cold 10 mM Tris · HCl-2 mM
MgCl2 buffer followed by filtration on Whatman GF/B glass
fiber filters with a cell harvester filtration unit (Brandel Biomedical
Research and Development Laboratories, Gaithersburg, MD). Activity was
measured in triplicate tubes with a gamma counter (LKB Wallac model
1282, 78% efficiency). Specific binding was taken as the difference
between total and nonspecific binding.
Western blot for PKC immunoreactivity. Cells (8-10 × 106) were scraped from the culture dish with a rubber policeman and placed in 800-1,000 µl of ice-cold cell lysis buffer [20 mM Tris · HCL, pH 7.5, 5 mM EGTA, 5 µg/ml of 4-(2-aminoethyl) benzenesulfonyl fluoride, 5 µg/ml of leupeptin, 1 µg/ml of aprotinin, and 1 µg/ml of pepstatin A]. Cell lysates were then homogenized by ultrasonic probe (3 × 15-s pulses). The cell lysate was centrifuged at 1,200 g for 20 s to eliminate unbroken cells. A portion of the supernatant was removed as a source of whole cell lysate, and the remainder was centrifuged at 100,000 g for 1 h at 4°C. The resulting clear supernatant was collected and taken as the cytosolic fraction. Pellets solubilized in cell lysis buffer were then taken as the membrane fraction. Protein concentration was assayed with the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA).
Protein (20-40 µg) from whole cell, cytosolic, and membrane fractions was electrophoresed on a 7.5% SDS-PAGE gel in Tris-glycine buffer at pH 8.3. Separated proteins were then transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA) at 1-A constant current (Hoefer model PS250; Bio-Rad) for 1 h in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Rat brain and Jurkat cell lysates (5 µg) were used as PKC isozyme standards for all isoform-specific antibodies. Rat brain lysates were used with antibodies against PKC-µ, -Reagents. Calphostin C was purchased from Calbiochem (La Jolla, CA). Ro-31-8220 was a gift from Dr. Christopher Hill (Roche Products, Welwyn Garden City, Herts, UK). PMA, trypsin-EDTA, FCS, and RPMI 1640 medium were purchased from Sigma. [125I]PIN was purchased from NEN/Life Sciences.
Data analysis.
Group values are presented as means ± SE. -AR density is
expressed as receptor sites per cell or as a percentage of control value when vehicle-treated cells were taken as control. Significance of
differences in normalized responses of epithelial cells was assessed by
the Mann-Whitney rank sum test. Significance between group means was
accepted at the P < 0.05 level.
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RESULTS |
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IL-1 (0.2 nM) and PMA (1 µM) produced significant increases
in
-AR density after 18 h of exposure
(P < 0.005 for both; Fig. 1) and a trend for
-AR density to
increase 6 but not 2 h postexposure (data not shown). In addition,
in pulse-chase experiments, PMA exposure for 2 and 6 h produced
increases in
-AR density at 18 h similar to those observed with
a full 18 h of PMA exposure. These latter experiments suggest that
increasing the duration of PKC activation beyond 2 h does not
enhance the magnitude of
-AR gene expression.
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The PKC inhibitors calphostin C and Ro-31-8220 both inhibited
IL-1-induced
-AR upregulation in a dose-dependent fashion (P < 0.001 for both; Figs.
2 and 3).
Ro-31822 had no effect at concentrations of 0.1 and 1 µM but
profoundly decreased IL-1
expression at a concentration of 10 µM
(Fig. 2). In contrast, calphostin C inhibited
-AR upregulation at
concentrations of 1 and 3 µM (Fig.
4). In contrast to the effect of
PKC inhibitors, indomethacin at a concentration of 10 µM had no
effect on
-AR density (P > 0.5; data not shown).
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PKC isozymes expressed in airway epithelial cells.
BEAS-2B cells expressed a spectrum of PKC isozymes (Table
1). Isozymes detected included species
from the conventional (,
II), novel (
, µ), and atypical
(
,
/
) categories. The PKC isozymes
I,
, and
were not
detected. We did not test for PKC-
.
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DISCUSSION |
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In a previous study, Kelsen et al. (8) showed that
IL-1 increased
2-AR gene expression in human airway
epithelial (BEAS-2B) cells. IL-1
increased
2-AR mRNA
and
2-AR protein monotonically over time and in biphasic
fashion with increasing dose. Increases in
2-AR protein
were maximum at ~36 h and were blocked by the protein synthesis
inhibitor cycloheximide, indicating a requirement for new protein
synthesis. In this study, we assessed the role of PKC in mediating the
IL-1
-induced increase in
2-AR gene expression. The
role of PKC was assessed from the response to 1) phorbol
esters and 2) the PKC inhibitors calphostin C and Ro-31-8220
and from 3) the effect of IL-1
on the cellular
distribution of PKC isozymes expressed by these cells.
The response to PMA has been widely used as a probe for PKC-mediated
effects (3, 18, 21). In this study, PMA increased -AR
expression and mimicked the effects of IL-1
. Furthermore, the two
PKC inhibitors inhibited the
-AR response to IL-1
in a
dose-dependent fashion and achieved virtually complete inhibition. Finally, IL-1
treatment of airway epithelial cells selectively increased PKC-µ immunoreactivity in the membrane fraction. Taken together, these several lines of evidence strongly support the notion
that the effects of IL-1
are mediated by PKC activation, probably
via the PKC-µ isotype. Of note, we did not test for the PKC-
subtype, and the antibody for PKC-
was not selective, so possible
effects of IL-1
on these two isotypes cannot be ruled out.
Of note, upregulation of -AR expression was first evident at 6 h and increased progressively over 18 h. Accordingly, increases in
-AR density were considerably slower than activation of PKC (<2 h),
probably reflecting the slower time course required for synthesis of
new receptor protein and its insertion into the cell membrane. In
addition, in pulse-chase experiments, upregulation of
-AR expression
was maximal with 2 h of PMA exposure, suggesting that increasing
the duration of PKC activation beyond 2 h did not enhance the
magnitude of
-AR gene expression.
In contrast to the effects of PKC inhibitors, the COX inhibitor
indomethacin had no effect on the response to IL-1, suggesting that
arachidonic acid-derived mediators play no role in the response to
IL-1
in airway epithelial cells.
Our study of PKC isozyme expression in a transformed airway epithelial
cell line immortalized by SV40 adenovirus transfection closely
resembles results obtained in primary cultures of both human and bovine
airway epithelial cells (13, 15, 16, 24). For example, a
study by Liedkte et al. (15) reported that primary cultures of human airway epithelial cells express PKC isozymes from the
conventional, novel, and atypical groups (i.e., ,
II,
,
,
and
) as is the case in BEAS-2B cells. Wyatt et al.
(24) detected PKC-
, -
II, -
, and -
in primary
cultures of bovine airway epithelial cells. Unlike the studies of
Liedkte et al. (15) and Wyatt et al. (24), we
did not detect PKC-
, perhaps because our studies were performed in a
transformed cell line. Unlike the present study, neither Liedkte et al.
(15) nor Wyatt et al. (24) tested for
,
µ, or
/
isotypes, which are expressed in BEAS-2B cells.
In agreement with our study, Liedkte et al. (15) observed
a differential cellular distribution of the isozymes. PKC- was confined to the cytosol, whereas PKC-
, -
, and -
/
were
evenly distributed between the cytosolic and particulate fractions and PKC-µ and -
II were found primarily in the membrane fraction.
PKC is a threonine/serine kinase that, when activated, plays a key regulatory role in a variety of cellular functions such as stimulation or inhibition of growth, changes in morphology, and modulation of gene expression (3, 18, 21). In particular, PKC activation has been observed to mediate a variety of effects important for the differentiated function of airway epithelial cells. For example, PKC-activated pathways are involved in mucin secretion (11), activation of the chloride channel (16), and the sodium-potassium-chloride cotransporter at the basolateral membrane (13, 16).
In addition, the PKC signal transduction pathway is involved in the
airway epithelial cell response to injury and repair and modulates the
airway epithelial response to proinflammatory cytokines. For example,
PKC mediates the airway cell migration response to fibronectin
(24) and the shedding of high-affinity tumor necrosis factor (TNF)- receptors in response to IL-1
and phorbol esters (12). TNF-
receptor shedding mediated by PKC activation
presumably serves to downregulate the airway epithelial cell response
to TNF-
.
Our observation that IL-1 may selectively activate a subset of the
repertoire of PKC isozymes is in keeping with previous observations in
a variety of other cell types and in airway epithelial cells in
particular (6, 13-15, 18, 21). For example, in human
airway epithelial cells, PKC-
and -
are selectively activated in
response to the
1-AR agonist methoxamine, which
activates Na-K-2Cl basolateral to apical cotransport (13,
15). Moreover, antisense mRNA against PKC-
, but not PKC-
,
inhibits the response to methoxamine, suggesting that methoxamine acts
solely through this isozyme (13).
Our data indicate that the PKC signal transduction pathway is involved
in the regulation of expression of the 2-AR gene, a gene
vitally important in airway homeostasis and airway epithelial cell
function, including mucin production and salt and water exchange (17). In the clinical setting, increased concentrations of
IL-1
are present in the airway secretions of patients with asthma
and are thought to promote the inflammatory process in this disease. In
fact, constitutive activation of alveolar macrophage PKC is suggested
in patients with asthma (22). These latter observations and the results of the present study suggest that the PKC signal transduction pathway plays an extensive role in regulating the phenotypic behavior of a variety of respiratory cell types.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. G. Kelsen, Rm. 761 Parkinson Pavilion, Temple Univ. Hospital, 3401 N. Broad St., Philadelphia, PA 19140 (E-mail: kelsen{at}vm.temple.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. Section 1734 solely to indicate this fact.
Received 24 February 2000; accepted in final form 14 September 2000.
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