Vol. 281, Issue 5, L1271-L1278, November 2001
Nuclear factor-
B augments
2-adrenergic receptor expression in human airway
epithelial cells
Mark O.
Aksoy,
Wei
Bin,
Yi
Yang,
Duan
Yun-You, and
Steven G.
Kelsen
Division of Pulmonary, Allergy, and Critical Care Medicine,
Department of Medicine, Temple University School of Medicine,
Philadelphia, Pennsylvania 19140
 |
ABSTRACT |
Interleukin
(IL)-1
increases 2-adrenergic receptor
(2-AR) mRNA and density by protein kinase C
(PKC)-dependent mechanisms in human airway epithelial cells. The
present study examined the role of several nuclear transcription
factors in the PKC-activated upregulation of 2-AR
expression. BEAS-2B cells were exposed to the PKC activator phorbol
12-myristate 13-acetate (PMA; 0.1 µM for 2-18 h). PMA had no
effect on activator protein (AP)-2 or cAMP response element binding
protein DNA binding activity but markedly increased nuclear factor
(NF)-
B and AP-1 binding as assessed by electrophoretic gel mobility
shift assay. PMA also increased the activity of a 2-AR
promoter-luciferase reporter construct in transiently transfected
cells. These effects were inhibited by the PKC inhibitors Ro-31-8220
and calphostin C. Furthermore, with increasing Ro-31-8220,
2-AR promoter-reporter activity correlated closely with
both NF-B and AP-1 activities (r > 0.89 for both). Finally, the selective NF-B inhibitor MG-132 dose dependently reduced NF-B binding and 2-AR promoter activity but
increased AP-1 binding. We conclude that PKC-induced upregulation of
2-AR expression in human airway epithelial cells appears
to be mediated, at least in part, by increases in NF-B activity.
airway epithelium; activator protein-1; nuclear factor-B; phorbol 12-myristate 13-acetate; protein kinase C
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INTRODUCTION |
THE PLEIOTROPIC
CYTOKINE interleukin (IL)-1 enhances expression of the human
2-adrenergic receptor (2-AR) in human
airway and alveolar epithelial cells (13, 20, 29) in a
dose- and time-dependent fashion. This effect of IL-1 on
2-AR expression appears to be mediated by an increase in
2-AR gene transcription because IL-1 enhances
steady-state 2-AR mRNA levels. However, the signal
transduction pathway(s) mediating the IL-1-induced upregulation of
the 2-AR gene is not well understood.
IL-1 is known to activate a variety of inflammatory and immune
response genes by pathways that include activation of protein kinase
(PK) C (21), release of arachidonic acid metabolites, and
production of nitric oxide. Recently, our laboratory
(3) has shown that activation of PKC is necessary
and sufficient for 2-AR upregulation in airway
epithelial cells. Specifically, IL-1 enhances the activation of the
PKC-µ isozyme, and the effect of IL-1 on 2-AR
expression can be abolished by selective PKC inhibitors.
The present study examined the pathways involved in IL-1-mediated
2-AR expression in human airway epithelial cells
downstream of PKC activation. PKC pathways are known to activate the
nuclear transcription factors nuclear factor (NF)-B, activator
protein (AP)-1, and AP-2 (11, 12). Accordingly, we
examined the effects of PKC activation on these transcription factors
in human airway epithelial cells and their potential roles in enhancing
2-AR gene expression. Finally, because the nuclear
transcription factor cAMP response element binding protein (CREB) is
known to upregulate 2-AR gene expression and is the
major homeostatic mechanism by which catecholamines regulate
2-AR transcription, we also examined the effects of PKC
activation on CREB activity. The role of these transcription factors in
PKC-mediated 2-AR gene expression was assessed from the
effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) and
selective PKC and NF-B inhibitors on 1) the DNA-binding
activity of NF-B, AP-1, AP-2, and CREB as assessed by
electrophoretic mobility shift assay (EMSA) and 2) the
activity of a full-length 2-AR promoter-luciferase
reporter construct in transiently transfected human airway epithelial
(BEAS-2B) cells.
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MATERIALS AND METHODS |
Cell culture.
Experiments were performed on cultured human airway epithelial cells
(BEAS-2B) (25). This cell line has been used by our laboratory (14) to examine the regulation of expression
and function of the 2-AR system and by others
(23) to study the regulation of cell calcium and
eicosanoid metabolism.
Cells were cultured in 100-mm flasks or in 6-well plates with RPMI 1640 medium plus 10% fetal bovine serum (FBS). Cultures were grown at
37°C in 5% CO2-95% air until confluent (5-7 days). All experiments were performed in this medium except for studies on
promoter activity, which were performed in DMEM without serum.
Treatment protocols.
Confluent cells were exposed to fresh medium containing PMA (0.1-1
µM; Sigma-Aldrich, St. Louis, MO) for 2-18 h at 37°C. Control cells received an equal volume of the solvent vehicle, DMSO. When used,
the PKC inhibitors Ro-31-8220 (0.1-30 µM) and calphostin C
(0.1-3 µM; Calbiochem, San Diego, CA), the NF-B inhibitor
MG-132 (0.03-3 µM; BIOMOL, Plymouth Meeting, PA), or vehicle
were added 30 min before PMA. The final DMSO concentration did not
exceed 0.3% in any well. Cells were harvested by exposure to
trypsin-EDTA at 37°C, and cell number was determined with a Coulter
counter or hemacytometer. Viability was determined by trypan blue
exclusion. Cell number ranged from 1-5 × 106
cells/well, and viability was >88% for all experiments.
Gel shift assay.
Cells were lysed by thorough mixing with nuclear extraction buffer (100 µl/106 cells) containing 20 mM HEPES, pH 7.5, 200 mM KCl,
20% (vol/vol) glycerol, 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40. After incubation
on ice for 30 min, lysates were microcentrifuged at 16,000 rpm for 5 min at 4°C. Supernatants containing the nuclear fraction were stored at 
80°C until needed.
The EMSAs used consensus oligonucleotides (Promega, Madison, WI) for
the following nuclear transcription factors: NF-B,
5'-AGTTGAGGGGACTTTCCCAGGC-3'; AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3'; AP-2,
5'-GATCGAACTGACCGCCCGCGGCCCGT-3', and CREB,
5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'.
Oligonucleotides were end labeled with 32P in a reaction
containing 3.5 pmol of oligonucleotide, 10 U of T4 polynucleotide
kinase, and [
-32P]ATP (3,000 Ci/mmol) in 70 mM
Tris · HCl, pH 7.6, 10 mM MgCl2, and 5 mM DTT.
After 30 min of incubation at 37°C, EDTA was added (final
concentration 50 mM) to stop the reaction, and the volume was increased
to 100 µl with Tris-EDTA buffer.
The EMSA reaction contained 2-10 µg of nuclear protein in 10 mM
Tris · HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 0.5 mM
EDTA, 0.5 mM DTT, 4% glycerol, and 0.05 mg/ml of poly(dI-dC). In some
reactions, 50-fold excess cold oligonucleotide (3.5 pmol) was added to
determine specificity of binding. After 10 min at 25°C, 1 µl (0.07 pmol) of labeled oligonucleotide was added, and the mixtures were
further incubated for 30 min at 25°C. In some assays, antibodies were present to detect transcription factor supershifts. The antibodies used
were goat polyclonal anti-NF-B p50, rabbit polyclonal anti-NF-B p65, rabbit polyclonal anti-c-Jun/AP-1, and mouse monoclonal anti-CREB1 (Santa Cruz Biotechnology, Santa Cruz, CA). All antibodies were added
10 min after the labeled oligonucleotide at final dilutions of 1:5 and
1:20.
EMSA mixtures were then immediately subjected to gel electrophoresis on
4.5% polyacrylamide gels at 100 V in 0.5× Tris-borate-EDTA buffer.
Gels were dried and exposed overnight to X-ray film at 70°C or to a
phosphorimager screen (Fuji Photofilm) at 25°C. The observed bands
were scanned by computer and quantitated densitometrically with the
Scion Image program (Scion Software).
2-AR promoter-reporter construct.
The 2-AR promoter (1,550 bp) was inserted into a pGL3-basic vector
containing a luciferase reporter gene (Promega). In brief, genomic DNA
was isolated from BEAS-2B cells, and 1,550 bp of the 2-AR promoter
upstream of the coding region (15) were amplified by PCR
with primers modified to insert SacI restriction sites at
each end of the promoter fragment. Purified promoter and pGL3 vector
were then cleaved with SacI followed by ligation of the promoter into the vector.
The construct was transformed into competent INVaF'
Escherichia coli cells (Invitrogen) grown on agar plates in
the presence of neomycin. DNA from antibiotic-resistant colonies was
isolated as minipreps and examined by agarose gel electrophoresis and
DNA sequencing. Colonies containing the construct with promoter in the
proper orientation were then subcultured, and the amplified construct
was harvested as a maxiprep for use in transfection experiments.
Transfection protocol.
BEAS-2B cells at 70-90% confluence were transfected with the
construct by calcium phosphate precipitation. To control for transfection efficiency with minimal trans effects on
2-AR promoter activity, cells were cotransfected with
the promoterless pRL-null vector (Promega) that codes for
Renilla luciferase. The pRL-null vector produces a
detectable luminescence signal but uses a different substrate to
generate light, thereby allowing the activity of the two luciferase
constructs to be assessed independently.
Before transfection, the medium was changed to DMEM plus 10% FBS. An
aliquot of a DNA-calcium phosphate solution containing 3 µg of
2-AR-pGL3, 2 µg of pRL-null covector, and 3 µg of
inert plasmid DNA (pGEM) at 20 µg total DNA/ml was added to each
well. Wells receiving inert DNA only (8 µg of pGEM) were used as
experimental blanks. Cells were transfected overnight for 15-18 h,
washed once in fresh medium, and then transferred to DMEM without FBS
for 24 h. They were then pretreated with inhibitors (Ro-31-8220,
calphostin C, and MG-132) for 30 min followed by the addition of PMA
for a final 6-h period. Control cells received DMSO solvent vehicle. After treatment, the cells were washed with PBS and lysed with lysis
buffer (Promega) followed by mechanical scraping. The resultant lysates
were microcentrifuged for 30 s to remove debris and were then
frozen at 70°C until needed. The lysates were assayed for luciferase activity in a luminometer (Berthold Lumat LB9501) with the
dual assay system from Promega, which allows rapid sequential measurement of both pGL3 and pRL luciferase phosphorescence. To determine promoter activity, experimental blank values were first subtracted from pGL3 and pRL sample readings. The pGL3 readings were
next normalized to changes in transfection efficiency to obtain a net
measure of promoter activity, as follows
where pGL3x* is the calculated promoter
activity for sample x in luminescence units,
pGL3x and pRLx are sample
readings, and pRL is the mean for all pRL readings in the experiment.
2-AR binding.
The density of -AR on BEAS-2B cells was determined as previously
described (14) with a saturating concentration of
[125I]iodopindolol (NEN-Life Sciences, Boston, MA).
Briefly, 105 cells were divided into aliquots into tubes
containing 10 mM Tris · HCl, pH 7.2, plus 450 pM
[125I]iodopindolol and were incubated at 28°C for
2 h on a shaking platform. Nonspecific binding was determined by
the addition of 40 µM alprenolol to some tubes. The cells were
harvested with a Brandel cell harvester onto GF/B Whatman glass fiber
filters, washed several times with the Tris buffer, and dried, and the radioactivity was determined in a gamma counter.
Data analysis.
Group data are reported as means ± SE and represent data from at
least three experiments. Statistical significance of differences in
sample means was determined by paired Student's t-test. In all cases, statistical significance was accepted at the P < 0.05 level. Curve fitting of dose-response and time-course data was performed by linear regression with first- or second-order polynomial fits.
| |
RESULTS |
Effect of PMA in BEAS-2B cells.
NF-B and AP-1 binding were detected constitutively in BEAS-2B cells
and was increased considerably by PMA treatment (0.1 µM) at both 2 and 18 h (Fig. 1, A and
B). In PMA-treated cells, NF-B binding activity was time
dependent and was greater at 2 than at 18 h. In contrast, AP-1
activity was similar at both time points (Fig.
2). For the group as a whole
(n = 7 experiments), NF-B increased to ~370% of
control levels at 2 h (P = 0.01) and to ~200%
of control levels at 18 h (P = 0.0024). AP-1
increased to ~350% of control levels at 2 h (P = 0.0005) and to ~320% of control levels at 18 h
(P = 0.0005; Fig. 2).
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Fig. 1.
Effects of phorbol 12-myristate 13-acetate (PMA; 0.1 µM
for 2 or 18 h) on nuclear factor (NF)-B (A) and
activator protein (AP)-1 (B) binding activity in BEAS-2B
cells are shown in representative autoradiographs. Samples in
lane 2 (A and B) show effects of
50-fold excess unlabeled consensus oligonucleotide, which blocks
binding by the labeled probe and, hence, indicates binding specificity.
+, Presence; , absence.
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Fig. 2.
Effects of PMA (0.1 µM) treatment on NF-B and AP-1
activity in BEAS-2B cells are shown. Group data are means ± SE;
n = 7 experiments. PMA increased NF-B and AP-1
activity at both 2 and 18 h. However, NF-B activity was greater
at 2 than at 18 h, in contrast to AP-1 in which values were
similar at the 2 time points.
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In PMA-treated cells, the NF-B-oligonucleotide complex underwent a
supershift in the presence of antibodies against p50 and p65 (Fig.
3), indicating the presence of the
p50/p65 heterodimer. Likewise, a supershift of the AP-1-oligonucleotide
complex occurred in the presence of anti-c-Jun/AP-1 (Fig. 3).
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Fig. 3.
Electrophoretic mobility shift assay (EMSA) showing supershift of
NF-B (left) and AP-1 (right) oligonucleotides
by antibodies for p50 and p65 or c-Jun. BEAS-2B cells were treated with
PMA for 18 h. Antibodies were diluted 1:20 (lanes 3, 5, and 8) or 1:5 (lanes 4, 6, and 9).
Supershift of oligonucleotide complexes occurred in response to all 3 antibodies.
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Both CREB DNA binding and phosphorylated CREB immunoreactivity
(assessed by Western blot) were observed constitutively but did not
increase in response to PMA (data not shown). Cells treated with IBMX
(0.5 mM) and dibutyryl cAMP (1 mM) for 18 h demonstrated increased
CREB activity and, hence, served as positive controls. No detectable
AP-2 binding was observed in either control or PMA-treated BEAS-2B cells.
PMA (0.1 µM for 6 h) also significantly increased
2-AR promoter activity to 182 ± 12% (SE) of
control levels (P = 0.01; n = 7 experiments).
Effect of Ro-31-8220 and calphostin C
in BEAS-2B cells.
The PKC inhibitor Ro-31-8220 (0.3-30 µM) dose dependently
inhibited NF-B and AP-1 binding activities (n = 6 experiments; Figs. 4, A and
B, and 5) and
2-AR promoter activity (n = 5 experiments; Fig. 6). Furthermore,
changes in 2-AR promoter activity induced by Ro-31-8220
correlated closely with changes in NF-B (r = 0.97) and AP-1 levels (r = 0.89).
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Fig. 4.
Effect of the protein kinase C (PKC) inhibitor Ro-31-8220 on
NF-B and AP-1 activity. BEAS-2B cells were treated with PMA for 2 or
18 h. Representative autoradiographs show dose-dependent
inhibition of NF-B (A) and AP-1 (B) by
Ro-31-8220 at both time points. Samples in lane 2 (A and B) show effects of 50-fold excess
unlabeled consensus oligonucleotide, which blocked binding
by the labeled probe and, hence, indicates binding specificity.
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Fig. 5.
Effect of Ro-31-8220 on NF-B ( ) and
AP-1 ( ) activity in PMA-treated (0.1 µM for 18 h) BEAS-2B cells is shown. Group data are means ± SE;
n = 6 experiments. Ro-31-8220 dose dependently
inhibited both NF-B and AP-1 activity.
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Fig. 6.
Effect of Ro-31-8220 on 2-adrenergic
receptor promoter-luciferase reporter activity in PMA-treated (0.1 µM
for 6 h) BEAS-2B cells. Data are means ± SE of 5 experiments. y-Axis, luciferase reporter activity given in
luminescence units as %control. y-Value at 0 concentration
of Ro-31-8220 indicates effect of PMA alone.
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Similar results were obtained with a second PKC inhibitor, calphostin
C. PMA (0.1 µM for 18 h)-evoked promoter activity (141 ± 6% of control level) was reduced to 74 ± 11% by calphostin C (1 µM; P = 0.004; n = 6 experiments).
Calphostin C (1 µM) also reduced NF-kB binding activity in PMA (0.1 µM for 18 h)-stimulated cells from 158 ± 30 to 58 ± 14% of control levels (P = 0.015; n = 4 experiments).
Effect of the NF-B inhibitor
MG-132 in BEAS-2B cells.
To examine the respective influences of NF-B and AP-1 on
2-AR promoter activity, the effects of the NF-B
inhibitor MG-132 were examined. MG-132 (0.03-1 µM) dose
dependently inhibited PMA-induced increases in NF-B DNA binding
activity but, surprisingly, increased AP-1 binding (n = 4 experiments; Figs. 7 and
8). MG-132 also monotonically inhibited PMA-induced increases in 2-AR promoter
activity (n = 4 experiments; Fig.
9). Reductions in 2-AR
promoter activity produced by MG-132 treatment correlated closely with
simultaneous changes in NF-B activity (r = 0.96) but
not in AP-1 activity (r = 0.36). MG-132 dose
dependently decreased -AR density from 201 ± 45% of control
levels at 0.1 µM MG-132 to 130 ± 22% of control levels at 10 µM MG-132 (n = 4 experiments).
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Fig. 7.
Effect of the NF-B inhibitor MG-132 on NF-B and AP-1 activity
in PMA-treated (18-h) BEAS-2B cells. Representative autoradiograph
shows a dose-dependent reduction in NF-B but not in AP-1 activity.
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Fig. 8.
Effect of MG-132 on NF-B () and AP-1
() activity in PMA-treated (0.1 µM for 18 h) BEAS-2B
cells is shown. Group data are means ± SE; n = 4 experiments. MG-132 dose dependently inhibited NF-B but not AP-1
activity.
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Fig. 9.
Effect of MG-132 on 2-AR
promoter-luciferase reporter activity in PMA-treated (0.1 µM for
6 h) BEAS-2B cells. Data are means ± SE of 4 experiments.
y-Axis, luciferase reporter activity given in luminescence
units expressed as %control. MG-132 dose dependently inhibited
luciferase reporter activity.
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DISCUSSION |
Our laboratory (13) has previously shown that IL-1
dose dependently upregulates 2-AR density in human
airway epithelial cells (BEAS-2B cells). Upregulation by IL-1 was
accompanied by an increase in 2-AR mRNA, suggesting
regulation of gene expression at the transcriptional level. Similar
findings for IL-1 have been reported in A549 lung cells (20,
29). More recently, our laboratory (3) has shown
that activation of the PKC pathway is necessary and sufficient for
IL-1-induced 2-AR upregulation. This upregulation is
completely blocked by the PKC inhibitors calphostin C and Ro-31-8220.
Furthermore, IL-1 specifically activates the PKC-µ isozyme
(3).
In the present study, we sought to determine the nuclear transcription
factor(s) downstream of PKC activation that upregulates 2-AR expression. Initial studies by our laboratory
focused on NF-B (11) and AP-1 (12) for two
reasons. First, both transcription factors are activated by
PKC-dependent pathways. For example, PKC mediates NF-B
translocation/activation in ANG II-stimulated rat cardiomyocytes
(26) and tumor necrosis factor-
-stimulated NF-B
activity and cyclooxygenase 2 expression in human lung epithelial cells
(5). PKC appears to activate NF-B by phosphorylation of
IB kinase (16, 31). PKC-mediated activation of AP-1 has been reported in hydrogen peroxide-stimulated rat aorta vascular smooth muscle cells (27) and mouse osteoblastic MC3T3-E1
cells (30).
Second, the 2-AR promoter contains putative binding
sites for both NF-B and AP-1. An atypical AP-1 site is present at
1,230 bp, upstream from the coding region (15). A
binding site closely matching the NF-B consensus sequence that
selectively binds NF-B has been reported in the rat
2-AR gene promoter (10). Sequences of the
human 2-AR gene promoter show an atypical NF-B
binding site in the same 5'-flanking region from 442 to 433 bp
(7, 15).
AP-1 and NF-B were examined over an 18-h period. In BEAS-2B cells
exposed to PMA, EMSAs showed increased DNA binding by AP-1 and NF-B
that could be blocked by 50-fold excess cold oligonucleotide, indicating specificity of binding. Both transcription factors rose
significantly by 2 h. AP-1 levels were maintained over 2-18 h, whereas NF-B levels declined partway toward basal level. NF-B activity is controlled partly in negative feedback fashion by changes
in expression of IB- (11). Therefore, a decline in NF-B activity after 2 h as observed in the present study may be
due in part to increased IB- synthesis. Such autoregulation of
NF-B has been reported in Jurkat cells (28) and HeLa S3 cells (1). Alternatively, exposure to PMA maintained for
18 h may have induced downregulation of PKC, with a subsequent
decrease in stimulus intensity for NF-B activation
(21). However, the relative lack of change in AP-1
activity with maintained (i.e., 18-h) exposure to PMA argues against
this latter possibility. Lack of change in AP-1 over the same time
course may be explained by the absence of a negative feedback loop for
this transcription factor.
NF-B exists as a variety of homo- and heterodimers. Antibodies
against the p50 and p65 subunits of NF-B demonstrated supershifting of the oligonucleotide-protein complex, indicating that at least some
of the DNA-bound NF-B was of the p50/p65 heterodimer, the principal
form. Likewise, supershifting of the oligo-AP-1 complex by anti-c-Jun
implicated the c-Jun isoform as an AP-1 constituent. However, given
that we only tested these subunits, we cannot rule out the presence of
additional NF-B dimers or of alternative Jun isoforms (e.g., Jun B
and Jun D) in the AP-1 complex.
PMA exposure increased the activity of a 2-AR
promoter-luciferase reporter construct transiently transfected into
BEAS-2B cells. The inserted full-length promoter spanned 1,500 bp of
the 5'-flanking region of the 2-AR coding region and
contained the putative AP-1 and NF-B binding sites mentioned
previously. The increase in promoter activity induced by PMA supports
the notion that upregulation of 2-AR density by IL-1
is at least partly transcriptionally mediated.
The selective PKC inhibitor Ro-31-8220 (22), a
staurosporine analog, produced a dose-dependent decrease in both
NF-B and AP-1 DNA binding and in 2-AR promoter
activity, confirming a dependence on PKC. Moreover, inhibition of
2-AR promoter activity by Ro-31-8220 correlated
closely with inhibition of NF-B and AP-1 binding, which suggests
that either or both factors play a role in activation of the
2-AR promoter by PMA.
The proteasome inhibitory peptide MG-132 is a potent inhibitor of
NF-B (18, 32, 34). In our study, MG-132 completely inhibited PMA-induced NF-B binding in BEAS-2B cells. In contrast, AP-1 binding actually increased as MG-132 concentrations rose. MG-132
also exerted a monotonic dose-dependent inhibition of
2-AR promoter activity that correlated closely with the
inhibition of NF-B activity. The above observations strongly suggest
that the potentiation of 2-AR promoter activity by PMA
is mediated, at least in part, by NF-B.
However, it should be pointed out that MG-132 may affect the
degradation of proteins other than NF-B (e.g., the IL-2 receptor complex, c-Jun, and the -opioid receptor) by inhibition of
proteasome activity (17, 19, 35). The present results with
the use of MG-132, therefore, are supportive but not definitive
evidence implicating NF-B in mediating 2-AR promoter
activity. Additional experiments are required in this regard.
Of interest, inhibition of NF-B by MG-132 was associated with an
increase in AP-1 binding activity. Several possible mechanisms may
explain this phenomenon. First, MG-132 may inhibit c-Jun degradation directly (19). Second, proteasome inhibitors may inhibit
proteasome-associated RNase activity and thereby stabilize c-Jun mRNA
and, hence, increase AP-1 expression (8).
CREB enhances transcription of the 2-AR gene in response
to agonists that act through the PKA signal transduction pathway (6). Any increases in CREB activity induced by PMA could
conceivably have explained the upregulation of 2-AR gene
expression. However, in this study, PMA had no effect on CREB DNA
binding activity or on its level of phosphorylation, suggesting that
the CREB system does not play a major role in the PKC-mediated
activation of the 2-AR promoter.
In summary, the present study indicates that the nuclear transcription
factor NF-B plays an essential role in mediating PKC-induced 2-AR gene activation in human airway epithelial cells.
Several lines of evidence support this conclusion. First, PMA increased NF-B DNA binding as well as the activity of a full-length
2-AR promoter-luciferase reporter construct. Second,
both parameters were reduced by the PKC inhibitors Ro-31-8220 and
calphostin C. Third, MG-123-induced inhibition of
2-AR promoter activity correlated closely with
reductions in NF-B binding activity.
In contrast, the ability of MG-123 to inhibit 2-AR
promoter activity while enhancing AP-1 DNA binding strongly suggests
that AP-1 per se does not mediate PKC-induced 2-AR gene
activation. However, the possibility that AP-1 exerts a facilitative
role in conjunction with NF-B in enhancing 2-AR gene
activation cannot be excluded by our data.
NF-B regulates the expression of a variety of genes such as adhesion
molecules (E-selectin, intercellular adhesion molecule-1, vascular cell
adhesion molecule), inflammatory mediators (inducible nitric oxide
synthase, cyclooxygenase 2), and cytokines and chemokines (tumor
necrosis factor-, IL-1, IL-6, and IL-8) (4, 5, 11, 24, 33,
34). To our knowledge, the present study is the first to
demonstrate that NF-B is involved in the regulation of expression of
the human 2-AR gene, which plays an important role in
the regulation of airway caliber and the airway response to
catecholamines. A recent study (9) in asthmatic subjects showed an increase in NF-B activity in airway epithelial cells and
macrophages compared with that in nonasthmatic subjects. These observations suggest that NF-B may be particularly important in the
regulation of the human 2-AR gene in the setting of
airway disease (2).
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ACKNOWLEDGEMENTS |
We acknowledge the technical assistance of Vondell Burke in
performing gel shift assays and the technical advice of Dr. Farhad Imani in developing the gel shift assay protocol.
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FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant R01-HL-52700-02.
Address for reprint requests and other correspondence: S. G. Kelsen, 761 Parkinson Pavilion, Temple Univ. Hospital, 3401 North 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 13 March 2001; accepted in final form 20 July 2001.
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REFERENCES |
1.
Arenzana-Seisdedos, F,
Thompson J,
Rodriguez MS,
Bachelerie F,
Thomas D,
and
Hay RT.
Inducible nuclear expression of newly synthesized IB negatively regulates DNA-binding and transcriptional activities of NF-B.
Mol Cell Biol
15:
2689-2696,
1995[Abstract].
2.
Barnes, PJ,
and
Adcock IM.
Transcription factors and asthma.
Eur Respir J
12:
221-234,
1998[Abstract].
3.
Bin, W,
Aksoy MO,
Yang Y,
and
Kelsen SG.
IL-1 enhances 2-adrenergic receptor expression in human airway epithelial cells by activating PKC.
Am J Physiol Lung Cell Mol Physiol
280:
L675-L679,
2001[Abstract/Free Full Text].
4.
Chen, CC,
Rosenbloom CL,
Anderson DC,
and
Manning AM.
Selective inhibition of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression by inhibitors of IB phosphorylation.
J Immunol
155:
3538-3545,
1995[Abstract].
5.
Chen, CC,
Sun YT,
Chen JJ,
and
Chiu KT.
TNF--induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C- 2, protein kinase C-, tyrosine kinase, NF-B-inducing kinase, and IB kinase 1/2 pathway.
J Immunol
165:
2719-2728,
2000[Abstract/Free Full Text].
6.
De Cesare, D,
and
Sassone-Corsi P.
Transcriptional regulation by cyclic AMP-responsive factors.
Prog Nucleic Acid Res Mol Biol
64:
343-369,
2000[ISI][Medline].
7.
Emorine, LJ,
Marullo S,
Delavier-Klutchko C,
Kaveri SV,
Durieu-Trautmann O,
and
Strosberg AD.
Structure of the gene for human 2-adrenergic receptor: expression and promoter characterization.
Proc Natl Acad Sci USA
84:
6995-6999,
1987[Abstract/Free Full Text].
8.
Haas, M,
Page S,
Page M,
Neumann FJ,
Marx N,
Adam M,
Loms Zeigler-Heitbrock HW,
Neumeier D,
and
Brand K.
Effect of proteasome inhibitors on monocytic IB- and - depletion, NF-B activation, and cytokine production.
J Leukoc Biol
63:
395-404,
1998[Abstract].
9.
Hart, LA,
Krishnan VL,
Adcock IM,
Barnes PJ,
and
Chung KF.
Activation and localization of transcription factor, nuclear factor-B, in asthma.
Am J Respir Crit Care Med
158:
1585-1592,
1998[Abstract/Free Full Text].
10.
Jiang, L,
Gao B,
and
Kunos G.
DNA elements and protein factors involved in the transcription of the 2-adrenergic receptor gene in rat liver. The negative regulatory role of C/EBPa.
Biochemistry
35:
13136-13146,
1996[Medline].
11.
Jobin, C,
and
Sartor RB.
The IB/NF-B system: a key determinant of mucosal inflammation and protection.
Am J Physiol Cell Physiol
278:
C451-C462,
2000[Abstract/Free Full Text].
12.
Karin, M,
Liu Zg,
and
Zandi E.
AP-1 function and regulation.
Curr Opin Cell Biol
9:
240-246,
1997[ISI][Medline].
13.
Kelsen, SG,
Anakwe OA,
Aksoy MO,
Reddy PJ,
and
Dhaneshekaran ND.
Interleukin-1 alters 2-adrenergic receptor-adenylyl cyclase system function in human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
273:
L694-L700,
1997[Abstract/Free Full Text].
14.
Kelsen, SG,
Anakwe OA,
Aksoy MO,
Reddy PJ,
Dhanesekaran ND,
Penn R,
and
Benovic JL.
Chronic effects of catecholamines on the 2-adrenoreceptor system in cultured human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
272:
L916-L924,
1997[Abstract/Free Full Text].
15.
Kobilka, BK,
Frielle T,
Dohlman HG,
Bolanowski MA,
Dixon RAF,
Keller P,
Caron MG,
and
Lefkowitz RJ.
Delineation of the intronless nature of the genes for the human and hamster 2-adrenergic receptor and their putative promoter regions.
J Biol Chem
262:
7321-7327,
1987[Abstract/Free Full Text].
16.
Lallena, MJ,
Diaz-Meco MT,
Bren G,
Paya CV,
and
Moscat J.
Activation of IB kinase by protein kinase C isoforms.
Mol Cell Biol
19:
2180-2188,
1999[Abstract/Free Full Text].
17.
Li, JG,
Benovic JL,
and
Liu-Chen LY.
Mechanisms of agonist-induced downregulation of the human -opioid receptor: internalization is required for downregulation.
Mol Pharmacol
58:
795-801,
2000[Abstract/Free Full Text].
18.
Li, L,
Hamilton RF, Jr,
and
Holian A.
Effect of acrolein on human alveolar macrophage NF-B activity.
Am J Physiol Lung Cell Mol Physiol
277:
L550-L557,
1999[Abstract/Free Full Text].
19.
Masaki, R,
Saito T,
Yamada K,
and
Ohtani-Kaneko R.
Accumulation of phosphorylated neurofilaments and increase in apoptosis-specific protein and phosphorylated c-Jun induced by proteasome inhibitors.
J Neurosci Res
62:
75-83,
2000[ISI][Medline].
20.
Nakane, T,
Szentendrei T,
Stern L,
Virmani M,
Seely J,
and
Kunos G.
Effects of IL-1 and cortisol on 2-adrenergic receptors, cell proliferation, and differentiation in cultured human A549 lung tumor cells.
J Immunol
145:
260-266,
1990[Abstract].
21.
Nishizuka, Y.
Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J
9:
484-496,
1995[Abstract].
22.
Nixon, JS,
Bishop J,
Bradshaw D,
Davis PD,
Hill CH,
Elliott LH,
Kumar H,
Lawton G,
Lewis EJ,
Mulqueen M,
Westmacott D,
Wadsworth J,
and
Wilkinson SE.
The design and biological properties of potent and selective inhibitors of protein kinase C.
Biochem Soc Trans
20:
419-425,
1992[ISI][Medline].
23.
Noah, TL,
Paradiso AM,
Madden MC,
McKinnon KP,
and
Devlin RB.
The response of a human bronchial epithelial cell line to histamine: intracellular calcium changes and extracellular release of inflammatory mediators.
Am J Respir Cell Mol Biol
5:
484-492,
1991.
24.
Parikh, AA,
Salzman AL,
Fischer JE,
and
Hasselgren PO.
IL-6 production in human intestinal epithelial cells following stimulation with IL-1 is associated with activation of the transcription factor NF-B.
J Surg Res
69:
139-144,
1997[ISI][Medline].
25.
Reddel, RR,
Ke Y,
Gerwin BI,
McMenamin MG,
Lechner JF,
Su RT,
Brash DE,
Park JB,
Rhim JS,
and
Harris CC.
Transformation of human bronchial epithelial cells by infection with SV40 and adenovirus-12 SV40 hybrid virus or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes.
Cancer Res
48:
1904-1909,
1988[Abstract/Free Full Text].
26.
Rouet-Benzineb, P,
Gontero B,
Dreyfus P,
and
Lafuma C.
Angiotensin II induces nuclear factor-B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway.
J Mol Cell Cardiol
32:
1767-1778,
2000[ISI][Medline].
27.
Stauble, B,
Boscoboinik D,
Tasinato A,
and
Azzi A.
Modulation of activator protein-1 (AP-1) transcription factor and protein kinase C by hydrogen peroxide and D--tocopherol in vascular smooth muscle cells.
Eur J Biochem
226:
393-402,
1994[ISI][Medline].
28.
Sun, SC,
Ganchi PA,
Ballard DW,
and
Greene WC.
NF-kB controls expression of inhibitor IB: evidence for an inducible autoregulatory pathway.
Science
259:
1912-1915,
1993[Abstract/Free Full Text].
29.
Szentendrei, T,
Lazar-Wesley E,
Nakane T,
Virmani M,
and
Kunos G.
Selective regulation of 2-adrenergic receptor gene expression by interleukin-1 in cultured human lung tumor cells.
J Cell Physiol
152:
478-485,
1992[ISI][Medline].
30.
Takeshita, A,
Chen Y,
Watanabe A,
Kitano S,
and
Hanazawa S.
TGF- induces expression of monocyte chemoattractant JE/monocyte chemoattractant protein 1 via transcriptional factor AP-1 induced by protein kinase in osteoblastic cells.
J Immunol
155:
419-426,
1995[Abstract].
31.
Trushin, SA,
Pennington KN,
Algeciras-Schimnich A,
and
Paya CV.
Protein kinase C and calcineurin synergize to activate IB kinase and NF-B in T-lymphocytes.
J Biol Chem
274:
22923-22931,
1999[Abstract/Free Full Text].
32.
Wang, XC,
Jobin C,
Allen JB,
Roberts WL,
and
Jaffe GJ.
Suppression of NF-B-dependent proinflammatory gene expression in human RPE cells by a proteasome inhibitor.
Invest Ophthalmol Vis Sci
40:
477-486,
1999[Abstract/Free Full Text].
33.
Xie, QW,
Kashiwabara Y,
and
Nathan C.
Role of transcription factor NF-B/Rel in induction of nitric oxide synthase.
J Biol Chem
269:
4705-4708,
1994[Abstract/Free Full Text].
34.
Yamashita, N,
Koizumi H,
Murata M,
Mano K,
and
Ohta K.
Nuclear factor kappa B mediates interleukin-8 production in eosinophils.
Int Arch Allergy Immunol
120:
230-236,
1999[ISI][Medline].
35.
Yu, A,
and
Malek TR.
The proteasome regulates receptor-mediated endocytosis of interleukin-2.
J Biol Chem
276:
381-385,
2001[Abstract/Free Full Text].
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