Epidermal growth factor upregulates
-adrenergic receptor
signaling in a human salivary cell line
Chih-Ko
Yeh1,2,
Tazuko K.
Hymer1,
April L.
Sousa1,
Bin-Xian
Zhang3,4,
Meyer D.
Lifschitz3,4, and
Michael S.
Katz1,4
1 Geriatric Research, Education and Clinical Center,
and 3 Research Service, South Texas Veterans Health Care
System, Audie L. Murphy Division, San Antonio 78229-4404; and
Departments of 2 Dental Diagnostic Science and
4 Medicine, University of Texas Health Science Center at
San Antonio, San Antonio, Texas 78229-3900
 |
ABSTRACT |
The effects of epidermal growth
factor (EGF) on the
-adrenergic receptor-coupled adenylyl cyclase
system were studied in a human salivary cell line (HSY). The
-adrenergic agonist isoproterenol (10
5 M) stimulated
adenylyl cyclase activity by ~2-fold, and the isoproterenol response
was increased 1.8-fold after prolonged (48 h) exposure to EGF (5 × 10
10 M). In contrast, enzyme activation via
stimulatory prostaglandin receptors and by agents acting on nonreceptor
components of the adenylyl cyclase system was not enhanced by EGF.
-Adrenergic receptor density, assessed by binding of the
-adrenergic receptor antagonist
(
)-[125I]iodopindolol, was increased threefold after
EGF treatment. Competition binding studies with unlabeled antagonists
selective for
1- and
2-adrenergic
receptor subtypes indicated that the increase in (
)-[125I]iodopindolol binding sites induced by EGF
reflected an increased number of
2-adrenergic receptors.
Likewise, Northern blot analysis of RNA from EGF-treated cells revealed
selective induction of
2-adrenergic receptor mRNA, which
was blocked by the RNA synthesis inhibitor actinomycin D. The increase
in
-adrenergic receptor density produced by EGF was unaltered after
phorbol ester-induced downregulation of protein kinase C (PKC).
Enhancement of isoproterenol-responsive adenylyl cyclase activity and
phosphorylation of mitogen-activated protein kinase (MAPK) by EGF were
both blocked by the MAPK pathway inhibitor PD-98059. The results
suggest that in HSY cells EGF enhances
-adrenergic responsiveness by
upregulating
2-adrenergic receptor expression at the
transcriptional level. Moreover, the stimulatory effect of EGF on
2-adrenergic receptor signaling appears to be mediated
by the MAPK pathway and independent of PKC activation.
adenylyl cyclase; G protein-coupled receptor; signal transduction; mitogen-activated protein kinase; protein kinase C
 |
INTRODUCTION |
SALIVARY GLAND
FUNCTION is tightly regulated by the autonomic nervous
system through the actions of neurotransmitters on G protein-coupled
receptor signaling pathways (3). In general, cholinergic-muscarinic and
-adrenergic receptor agonists induce secretion of salivary fluid and electrolytes via activation of the
intracellular calcium ([Ca2+]i) cascade
(3). On the other hand,
-adrenergic receptor agonists stimulate the secretion of salivary proteins by activation of adenylyl
cyclase and the generation of intracellular cAMP (3). Stimulation of the
-adrenergic receptor-coupled adenylyl cyclase pathway also causes transcriptional and posttranslational modifications of salivary gland proteins (52). In vivo, the
-adrenergic agonist isoproterenol has been demonstrated to induce
hypertrophy and hyperplasia of salivary glands (10, 50).
In a number of cell types,
-adrenergic receptor activation of
adenylyl cyclase is modulated by systemic hormones as well as local
growth factors and cytokines (21, 29, 51, 54). Whether
similar modulation of
-adrenergic receptor signaling might play a
regulatory role in salivary cell secretion and growth has not been well characterized.
Epidermal growth factor (EGF) is a multifunctional factor produced in
abundance by rodent salivary glands; lesser amounts of EGF have also
been identified in human salivary cells (33). The actions
of EGF are mediated by a receptor tyrosine kinase linked to multiple
intracellular signaling events such as activation of protein kinase C
(PKC) and the extracellular signal-regulated kinase
(ERK)/mitogen-activated protein kinase (MAPK) cascade (14, 37). Interactions, or "cross talk," between EGF-induced
signals and G protein-coupled receptor signaling pathways are
recognized in a variety of tissues (18, 19, 44, 47).
Earlier studies showed that EGF administration to rats modulates
muscarinic receptor activation of salivary secretion (46).
Using a ductal cell line (HSY) from human parotid (57), we
recently demonstrated (60) inhibition of muscarinic
receptor-mediated [Ca2+]i mobilization by
EGF. In contrast, EGF regulation of signaling events mediating
-adrenergic receptor-linked functions in salivary cells has not been
described previously. In the present study we have examined the effect
of EGF on the
-adrenergic receptor-stimulated adenylyl cyclase
system in HSY cells. Our results indicate that EGF enhances
-adrenergic receptor signaling via an increase in
-adrenergic
receptor expression at the transcriptional level. Moreover, the
stimulatory action of EGF on
-adrenergic responsiveness appears to
be mediated by activation of ERK/MAPK but not PKC.
 |
MATERIALS AND METHODS |
Materials.
Recombinant human (rh) EGF, insulin-like growth factor-I (rhIGF-I), and
transforming growth factor-
(rhTGF-
) were purchased from Promega
(Madison, WI); platelet-derived growth factor (rhPDGF-AB) was from
Peprotech (Rocky Hill, NJ). (
)-[125I]iodopindolol
(2,200 Ci/mmol) was obtained from NEN Life Science Products (Boston,
MA). Dulbecco's modified Eagle's medium (DMEM) was from Life
Technologies (Gaithersburg, MD). (
)-Isoproterenol (+) bitartrate
salt, phorbol 12-myristate 13-acetate (PMA),
3-isobutyl-1-methylxanthine (IBMX), prostaglandin E2
(PGE2), cholera toxin, actinomycin D, and other chemicals
were purchased from Sigma (St. Louis, MO). CGP-20712A methanesulfonate
was obtained from Research Biochemicals International (RBI; Natick,
MA), and ICI-118,551 hydrochloride was a gift from Tocris (Bristol,
UK). 2-(2'-Amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD-98059) was
purchased from RBI, and forskolin was from Calbiochem (San Diego, CA).
Rabbit antibodies against p44/p42MAPK and
phospho-p44/p42MAPK were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA) and Cell Signaling Technology (Beverly,
MA), respectively; goat anti-rabbit IgG Fc conjugated to horseradish
peroxidase was from Jackson Immuno-Research Laboratories (West Grove, PA).
Cell culture.
The HSY cell line, which was originally established by Yanagawa et al.
(57), was kindly provided by Dr. James Turner [National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health, Bethesda, MD]. Cells were plated at a density of
~2 ×104 cells/cm2 in 48-well culture plates
(for adenylyl cyclase assay) or 100-mm culture dishes (for
-adrenergic receptor binding assay and Northern and immunoblot
analyses) and cultured in DMEM supplemented with 10% fetal calf serum
(FCS) and penicillin (100 U/ml)-streptomycin (100 µg/ml) at 37°C in
a humidified 5% CO2 atmosphere incubator. Unless otherwise
specified cells were grown to near confluence at 72 h. EGF and
other growth factors (TGF-
, PDGF, or IGF-I) were generally added to
the culture medium containing 10% FCS 48 h before confluence,
i.e., 24 h after plating. Under these conditions EGF caused a
small but significant increase in cell number (~16% increase;
P < 0.005) at 72 h.
Adenylyl cyclase assay.
Adenylyl cyclase activity was measured in intact HSY cells cultured for
72 h. Enzyme activity was determined as the conversion of
[3H]ATP to [3H]cAMP after cellular
incorporation of [3H]adenine, as described previously
(20). Briefly, cells were incubated with
[3H]adenine (1 µCi/well) in DMEM for 2 h at 37°C
and then exposed to adenylyl cyclase-stimulating agents (isoproterenol,
PGE2, or forskolin) for 10 min in the presence of the
phosphodiesterase inhibitor IBMX (0.5 mM). For measurements of cholera
toxin-stimulated adenylyl cyclase activity, cholera toxin (1 µg/ml) was present during the 2-h incubation with
[3H]adenine. The reaction was terminated with ice-cold
trichloroacetic acid (0.12 mM), and the [3H]cAMP product
was isolated by two-column chromatography (19). Cell
numbers were determined in separate cultures grown under conditions
identical to those used for cells undergoing enzyme assay. The
adenylyl cyclase activity is expressed as counts per minute (cpm)
of [3H]cAMP per 103 cells per 10-min
incubation period.
-Adrenergic receptor binding assay.
-Adrenergic receptor binding in membrane preparations from HSY cells
cultured for 72 h was measured by an equilibrium binding assay
using the radiolabeled
-adrenergic receptor antagonist (
)-[125I]iodopindolol (28). Membranes
(43,000 g pellets) were prepared as described previously
(60), and 50-100 µg of membrane protein were
incubated with (
)-[125I]iodopindolol in 125-250
µl of reaction buffer [15.2 mM Na-HEPES (pH 7.5), 115 mM NaCl, 0.66 mM L-ascorbic acid] for 30 min at 30°C. Reactions were
terminated by adding 4 ml of wash buffer [10 mM Tris (pH 7.5), 154 mM
NaCl] at room temperature, and membrane-bound radioligand was
collected on Whatman glass fiber filters (GF/C) with a Brandel Cell
Harvester (Biomedical Research and Development Laboratories,
Gaithersburg, MD). Nonspecific binding of
(
)-[125I]iodopindolol was defined as the amount of
radioligand bound in the presence of an excess (10
4 M) of
the
-adrenergic agonist (
)-isoproterenol. Specific binding was
70-90% of the total binding at radioligand concentrations in the
range of the dissociation constant (Kd). Protein
concentration was determined by the method of Bradford
(8).
Saturation binding curves were constructed by measuring specific
binding of (
)-[125I]iodopindolol at eight
concentrations of radioligand in the range of 0.01-0.5 nM.
Competition binding studies were performed by measuring the binding of
(
)-[125I]iodopindolol (at a concentration approximating
the Kd) in the presence of 17-19
concentrations of nonlabeled (
)-isoproterenol (a nonselective
-adrenergic receptor agonist; Ref. 36), ICI-118,551 (a
selective
2-adrenergic receptor antagonist; Ref.
6), or CGP-20712A (a selective
1 adrenergic
receptor antagonist; Ref. 15) in the range of
10
11 to 10
4 M.
Northern blot analysis.
Total RNA was isolated from HSY cells by the guanidinium
thiocyanate-phenol-chloroform extraction method with TRI Reagent (Molecular Research Center, Cincinnati, OH; Refs. 12,
13). RNA samples (20 µg) were electrophoresed on 1.2%
agarose-formaldehyde gels and transferred to nitrocellulose membranes
(Micron Separations, Westborough, MA). The membranes were
prehybridized, hybridized with 32P-labeled
1- or
2-adrenergic receptor cDNA insert,
and washed as described previously (59). Each membrane was
then exposed to a PhosphorImager screen for 1-10 days, and
hybridization signals were quantified with a Molecular Dynamics Storm
860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). As a control
for RNA loading and transfer, the membranes were stripped and reprobed
with a 5'-end labeled antisense 18S ribosomal RNA oligonucleotide
(5'-GCCGTGCGTACTTAGACATGCATG), followed by washing and exposure to
PhosphorImager screens for 30 min.
The
1-adrenergic receptor cDNA, a PstI
fragment of the rat
1-adrenergic receptor gene in
pGEM3Zf(+), was a generous gift from Dr. C. A. Machida (Oregon
Regional Primate Research Center, Beaverton, OR; Ref. 49).
The
2-adrenergic receptor probe, a NcoI/SalI fragment of human
2-adrenergic receptor cDNA in the expression vector
pBC12MI, was kindly provided by Dr. R. J. Lefkowitz (Duke
University, Durham, NC; Ref. 32). cDNA inserts were
labeled with [
-32P]dCTP by using the Rediprime kit
from Amersham Pharmacia Biotech (Piscataway, NJ). Total RNA samples
from rat hippocampus and COS-7 cells (American Type Culture Collection,
Manassas, VA) were used as positive controls for
1- and
2-adrenergic receptor mRNAs, respectively.
Immunoblot analysis of ERK/MAPK.
Immunoblot analysis was performed as described previously, with minor
modification (60). HSY cells cultured for 24 h were washed three times with cold PBS and lysed in a buffer containing 50 mM
Tris · HCl (pH 7.4), 150 mM NaCl, 10% NP-40, 2 mM EDTA, 0.5 mM EGTA, 2.5 µg/ml leupeptin, 10 µg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate
(Na3VO4). After centrifugation of cell lysates
at 8,000 g for 2 min at 4°C, supernatant proteins (50 µg) were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH). The membranes were immunoblotted with
p44/p42MAPK or phospho-p44/p42MAPK primary
antibody (1:1,000) and a secondary horseradish peroxidase-conjugated antibody (1:1,000). MAPKs were visualized by an enhanced
chemiluminescence system (ECL Plus, Amersham Pharmacia Biotech) and
quantified with ImageQuant computer software (version 5; Molecular
Dynamics, Sunnyvale, CA).
Data analysis.
Data from multiple experiments are expressed as means ± SE.
Statistical significance of single comparisons was determined with
Student's t-test. Multiple comparisons were performed by analysis of variance (ANOVA) followed by Sidak's multiple-comparison test. Scatchard analysis of (
)-[125I]iodopindolol
saturation binding curves was used to determine
-adrenergic receptor
density (Bmax) and Kd. Competition
curves describing (
)-[125I]iodopindolol binding in the
presence of increasing concentrations of (
)-isoproterenol,
ICI-118,551, and CGP-20712A were analyzed with a weighted, nonlinear
least-squares curve-fitting program (28).
 |
RESULTS |
Enhancement of isoproterenol-stimulated adenylyl cyclase activity
by EGF.
The effect of EGF on
-adrenergic-responsive adenylyl cyclase
activity in HSY cells is shown in Fig. 1.
The
-adrenergic agonist isoproterenol (10
5 M)
stimulated adenylyl cyclase activity by about twofold relative to
unstimulated (basal) enzyme activity. The isoproterenol response was
markedly increased (1.8 ± 0.1-fold) by treatment of cells with
EGF (5 × 10
10 M, 48 h), even though the basal
level of adenylyl cyclase activity was slightly reduced (~12%) by
EGF treatment. Enhancement of isoproterenol-stimulated adenylyl cyclase
activity was dependent on EGF concentration and time of incubation with
the growth factor (Fig. 2). The
stimulatory effect of EGF was maximal at ~5 × 10
10 M (3 ng/ml), with half-maximal effect at 1 × 10
10 M (Fig. 2A). The response to
isoproterenol increased within 24 h of EGF treatment, with
progressive enhancement observed through 48 h (Fig.
2B).

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Fig. 1.
Effect of epidermal growth factor (EGF) on
isoproterenol-stimulated adenylyl cyclase activity. HSY cells were
treated with EGF (+EGF; 5 × 10 10 M) or left
untreated ( EGF) for 48 h and then assayed for isoproterenol
(10 5 M)-stimulated adenylyl cyclase activity as described
in MATERIALS AND METHODS. Values represent means ± SE
of isoproterenol-stimulated (+Iso) and unstimulated, or basal ( Iso),
adenylyl cyclase activities from 28 experiments. *P < 0.05, **P < 0.0001 vs. EGF.
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Fig. 2.
Concentration dependence and time course of EGF effect on
isoproterenol-stimulated adenylyl cyclase activity. A: HSY
cells were treated with increasing concentrations of EGF for 48 h.
Values represent means ± SE of isoproterenol (10 5
M)-responsive (+Iso) and basal ( Iso) adenylyl cyclase activity
from 8 experiments. B: cells were incubated with 5 × 10 10 M EGF for 2-48 h before confluence or without
EGF (time 0). Values represent means ± SE of
isoproterenol (10 5 M)-stimulated (+Iso) and basal ( Iso)
adenylyl cyclase activity from 4 experiments.
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To examine the specificity of EGF action on isoproterenol-stimulated
adenylyl cyclase, we compared isoproterenol responses in HSY cells
treated with EGF and other growth factors (TGF-
, PDGF, and IGF-I).
TGF-
acts by binding to EGF receptors (11), whereas
PDGF and IGF-I activate distinct receptor tyrosine kinases. TGF-
(3 ng/ml), like EGF, enhanced isoproterenol responsiveness in HSY cells
(1.4 ± 0.1-fold, n = 17; P < 0.001). In contrast, PDGF (10 ng/ml) and IGF-I (50 ng/ml) had no effect
on isoproterenol-stimulated adenylyl cyclase activity (data not shown).
The concentrations of PDGF and IGF-I used in these experiments have
been reported to elicit biological responses in a variety of cell
types. The results implicate a specific stimulatory effect of EGF
receptor activation on isoproterenol-responsive adenylyl cyclase in HSY cells.
Adenylyl cyclase activation via stimulatory receptors other than
-adrenergic receptors was not enhanced by EGF. In the current study
we found that adenylyl cyclase in HSY cells is linked not only to
-adrenergic receptors but also to stimulatory receptors for
prostaglandins. However, unlike the
-adrenergic response, stimulation of adenylyl cyclase by PGE2 was unchanged after
EGF treatment (Fig. 3). We also
determined whether EGF modified non-receptor-mediated activation of
adenylyl cyclase in response to cholera toxin, which stimulates enzyme
activity by catalyzing ADP-ribosylation of the stimulatory
Gs
protein (17), and forskolin, which
exerts a direct stimulatory effect on adenylyl cyclase
(48). EGF had no effect on cholera toxin-stimulated enzyme
activity. In contrast, a significant reduction (~20%,
P < 0.002) in the forskolin response was observed
after EGF treatment, suggesting an inhibitory effect of the growth
factor at the level of the adenylyl cyclase enzyme (Fig. 3).

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Fig. 3.
Effects of EGF on prostaglandin receptor and non-receptor-mediated
activation of adenylyl cyclase. HSY cells were treated with EGF (+EGF;
5 × 10 10 M) or left untreated ( EGF) for 48 h
and then assayed for adenylyl cyclase responses to prostaglandin
E2 (PGE2, 10 6 M;
n = 5 experiments), cholera toxin (1 µg/ml;
n = 4), and forskolin (10 4 M;
n = 21) as described in MATERIALS AND
METHODS. Values represent mean ± SE enzyme activities;
*P < 0.002 vs. EGF. Unstimulated, or basal,
activities with and without EGF treatment were also measured in each
experiment; the effect of EGF on basal activity was similar to that
shown for a larger number of experiments in Fig. 1.
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Effects of EGF on
-adrenergic receptors.
Because EGF preferentially increased
-adrenergic responsive adenylyl
cyclase activity, we examined the effects of EGF on
-adrenergic
receptors assessed by binding of the
-adrenergic receptor antagonist
(
)-[125I]iodopindolol to HSY cell membrane
preparations. Scatchard analysis of
(
)-[125I]iodopindolol saturation binding curves
revealed a single class of binding sites with high affinity
(Kd = 9.6 ± 1.7 × 10
11 M) for the radioligand. In untreated HSY cells the
density (Bmax) of
-adrenergic receptors was 4.4 ± 0.8 fmol/mg protein. EGF caused a threefold increase in
Bmax (12.9 ± 2.5 fmol/mg protein; P < 0.001 vs. untreated cells) without affecting receptor binding affinity (Kd = 11.1 ± 3.2 × 10
11 M; Fig. 4).

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Fig. 4.
Effect of EGF on -adrenergic receptor binding. Cell membranes
were obtained from HSY cells treated with EGF (+EGF; 5 × 10 10 M) or left untreated ( EGF) for 48 h. Membrane
receptor binding was measured with the -adrenergic receptor
antagonist ( )-[125I]iodopindolol as the radioligand as
described in MATERIALS AND METHODS. A: a
representative experiment showing saturation binding curves with and
without EGF treatment (left) and Scatchard analysis used to
determine receptor density (Bmax) and dissociation constant
(Kd) (right). B: mean ± SE values of Bmax (left) and
Kd (right) from 13 experiments. *
P < 0.001 vs. EGF.
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Modulation of
-adrenergic receptor binding by EGF was further
characterized in studies measuring competition for
(
)-[125I]iodopindolol binding sites by unlabeled
isoproterenol and antagonists selective for
2- and
1-adrenergic receptor subtypes. The results of the
competition binding studies and curve-fitting analysis of binding data
are presented in Fig. 5 and Table 1,
respectively. As in a variety of other
tissues, competition studies with HSY cell membranes demonstrated
binding of the nonselective
-adrenergic agonist isoproterenol to
both high- and low-affinity binding sites. Treatment of HSY cells with
EGF resulted in a threefold increase (P < 0.02) in the
number of receptor sites binding isoproterenol with high affinity; an
apparent increase in the number of low-affinity receptors was not
statistically significant (Fig. 5A; Table 1). Both high- and
low-affinity binding sites for the
2-selective adrenergic receptor antagonist ICI-118,551 were also identified in HSY
cell membranes (Fig. 5B). In contrast, only a single class of low-affinity binding sites for the
1-selective
adrenergic receptor antagonist CGP-20712A was detected (Fig.
5C). The sites binding ICI-118,551 with high affinity,
presumably receptors of the
2-adrenergic subtype, were
increased about threefold (P < 0.01) in number by EGF
treatment. Growth factor-induced increases in low-affinity binding for
both
1- and
2-selective antagonists were
not statistically significant. The dissociation constants of high- and
low-affinity binding sites for isoproterenol, ICI-118, 551, and
CGP-20712A were unaltered by EGF (Table 1). Together with the
(
)-[125I]iodopindolol saturation binding data, the
results of the competition studies suggest that increased high-affinity
binding of (
)-[125I]iodopindolol and isoproterenol in
EGF-treated HSY cells reflects at least in part an increased number of
receptors of the
2-adrenergic subtype.

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Fig. 5.
Competition for ( )-[125I]iodopindolol
binding sites by isoproterenol, ICI-118,551, and CGP-20712A: effects of
EGF. Cell membranes were obtained from HSY cells treated with EGF
(+EGF; 5 × 10 10 M) or left untreated ( EGF) for
48 h. Binding of ( )-[125I]iodopindolol to cell
membranes was assayed in the presence of increasing concentrations of
unlabeled isoproterenol (A), ICI-118,551 (B), and
CGP-20712A (C) as described in MATERIALS AND
METHODS. Composite curves are presented with mean ± SE
values from 4-7 experiments.
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The
1- and
2-adrenergic receptor
subtypes, like many G protein-coupled receptors, are subject to
well-recognized regulatory processes in which the number of cell
surface receptors decreases progressively over time of exposure to
agonist (53). In view of the stimulatory action of EGF on
-adrenergic receptor number in HSY cells, we performed studies to
determine whether agonist-induced reduction of membrane receptor
content might be altered by growth factor treatment. Figure
6 shows that exposure of HSY cells to isoproterenol (10
5 M) caused a time-dependent decrease in
(
)-[125I]iodopindolol binding to cell membranes, with a
>40% decline in radioligand binding occurring by 30 min and >80%
loss of binding by 24 h. Treatment of cells with EGF had no
significant effect on isoproterenol-induced decreases in
(
)-[125I]iodopindolol binding. Thus EGF increases
-adrenergic receptor binding in HSY cells by regulatory processes
that appear to be unrelated to those induced by
-adrenergic agonist.

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Fig. 6.
Isoproterenol-induced loss of
( )-[125I]iodopindolol binding to membranes from
EGF-treated and untreated HSY cells. HSY cells cultured for a total of
72 h were treated with EGF (+EGF; 5 × 10 10 M)
or left untreated ( EGF) for 48 h as described in MATERIALS
AND METHODS; cells were preincubated with isoproterenol
(10 5 M) for 0-24 h before the end of the 72-h
culture period. Specific binding of
( )-[125I]iodopindolol to cell membrane preparations was
measured at a saturating concentration (0.25 nM) of radioligand.
Binding data are expressed as % of control binding measured in the
absence of isoproterenol preincubation [100% binding at time
0 = 4.9 ± 0.4 fmol/mg protein ( EGF), 14.4 ± 2.1 fmol/mg protein (+EGF); n = 10 experiments]. Values
are means ± SE from 3-10 experiments. No significant effect
(P > 0.05) of EGF on %binding was observed at any
time of isoproterenol preincubation.
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We next examined the effect of EGF on expression of
1-
and
2-adrenergic receptor subtypes at the mRNA level.
Treatment of HSY cells with EGF for periods of 1-24 h produced a
biphasic increase in the levels of
2-adrenergic receptor
mRNA assessed by Northern blot analysis. As shown in Fig.
7,
2-adrenergic receptor
mRNA levels increased transiently by threefold at 1 h of EGF
treatment; after a return to the basal level of expression at 4 h,
receptor mRNA levels rose again by twofold at 8 h and remained
elevated through 24 h of EGF exposure. In contrast, treatment of
HSY cells with EGF for periods up to 24 h had no effect on
1-adrenergic receptor mRNA expression, with the
exception of a relatively small increase (40%) in mRNA levels observed
at 1 h (Fig. 7). The increase in
2-adrenergic
receptor mRNA induced by 1 h of EGF treatment was blocked by
actinomycin D, an inhibitor of RNA synthesis (Fig. 8). These findings suggest that although
both
1- and
2-adrenergic receptor
subtypes are expressed in HSY cells, EGF selectively stimulates
2-adrenergic receptor expression at the level of
transcription.

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Fig. 7.
Effects of EGF on 1- and
2-adrenergic receptor mRNA levels. HSY cells were
cultured for 24 h and then treated with EGF (+EGF, 5 × 10 10 M) or left untreated ( EGF) for an additional
1-24 h in continuous culture. Total RNA was isolated and subjected
to Northern blot analysis with 1- and
2-adrenergic receptor ( 1-AR and
2-AR, respectively) cDNA probes; blots were reprobed
with antisense 18S ribosomal RNA (rRNA) oligonucleotide to control for
RNA loading and transfer (see MATERIALS AND METHODS).
A: representative Northern blots showing 2-AR
and 1-AR mRNA levels, with corresponding levels of 18S
rRNA. Samples of total RNA from COS-7 cells and rat hippocampus (HC)
were used as positive controls for 2-AR and
1-AR mRNAs, respectively. B:
2-AR and 1-AR mRNA levels normalized to
the corresponding levels of 18S rRNA. Values are means from 2 experiments.
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Fig. 8.
Inhibition of EGF-induced 2-adrenergic
receptor expression by actinomycin D. HSY cells cultured for 24 h
were preincubated with or without actinomycin D (ActD, 1 µg/ml) for
15 min, followed by addition of EGF (5 × 10 10 M) or
vehicle for 1 h. Total RNA was isolated and subjected to Northern
blot analysis with 2-AR cDNA probe and antisense 18S
rRNA oligonucleotide as described in MATERIALS AND METHODS.
In each sample the receptor mRNA level was normalized to the
corresponding level of 18S rRNA. The effects of treatment with EGF
and/or actinomycin D were in turn expressed as the ratio of normalized
receptor mRNA levels in treated and control (untreated) cells, with
control mRNA set at unity. A: a representative Northern blot
showing 2-AR mRNA levels with corresponding levels of
18S rRNA. Total RNA from COS-7 cells was used as a positive control for
2-AR mRNA. B: ratio of normalized
2-AR mRNA levels in treated and control cells. Values
are means ± range from 2 experiments.
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Effects of EGF-induced signals on
-adrenergic receptor-coupled
adenylyl cyclase.
The EGF receptor tyrosine kinase modulates cellular function by
activating a variety of intracellular signaling cascades including the
PKC and ERK/MAPK pathways (14, 37, 38). Previous studies indicate that activation of PKC regulates
-adrenergic-responsive adenylyl cyclase activity in several cell types through effects on
-adrenergic receptor expression, receptor coupling to G protein, and
activity of the adenylyl cyclase enzyme (7, 34, 42). We
therefore examined whether EGF-induced enhancement of
-adrenergic receptor signaling in HSY cells may be dependent on activation of PKC.
PKC was downregulated by pretreatment of cells with the phorbol ester
PMA (10
5 M) for 4-6 h before addition of EGF.
Exposure of HSY cells and other cell types to high concentrations
(
10
6 M) of PMA for periods of >1 h results in nearly
complete loss of PKC activity and/or immunoreactive PKC isoforms
(30, 58, 60). As shown in Fig.
9, EGF caused a twofold increase in
isoproterenol-responsive adenylyl cyclase activity irrespective of
whether cells were pretreated with PMA; PMA also had no effect on the
threefold increase in
-adrenergic receptor density induced by EGF.
These findings suggest that activation of PKC is not required for the
stimulatory effect of EGF on
-adrenergic receptor signaling.
Interestingly, PMA itself produced twofold increases in both
isoproterenol responsiveness and
-adrenergic receptor density with
or without addition of EGF. Moreover, PMA and EGF in combination
augmented the isoproterenol response and
-adrenergic receptor
density to a much greater extent (4- and >6-fold increases,
respectively) than did either agent alone (Fig. 9). Thus PMA and EGF
appear to exert independent modulatory influences on the
-adrenergic
receptor-coupled adenylyl cyclase system in HSY cells.

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Fig. 9.
Enhancement of -AR signaling by EGF: effects of
pretreatment with phorbol 12-myristate 13-acetate (PMA). HSY cells
cultured for 24 h were incubated with or without PMA
(10 5 M) for 4-6 h, followed by addition of EGF
(5 × 10 10 M) or vehicle for 44 h in the
continued presence of PMA. Cells were then assayed for isoproterenol
(10 5 M)-stimulated and unstimulated (basal) adenylyl
cyclase activities or used to obtain membranes for determination of
-AR density (Bmax) by Scatchard analysis of
( )-[125I]iodopindolol saturation binding curves (see
MATERIALS AND METHODS). Open bars, means ± SE of
isoproterenol-stimulated adenylyl cyclase activities from 11 experiments. Hatched bars, means ± SE of -AR density
(Bmax) from 5 experiments. EGF and PMA individually
increased values of isoproterenol-stimulated adenylyl cyclase activity
and Bmax relative to values in untreated cells
(P < 0.01); isoproterenol responsiveness and
Bmax in cells treated with both EGF and PMA were greater
than in cells treated with either agent alone (P < 0.01). PMA had no effect on basal adenylyl cyclase activity (data not
shown).
|
|
Other experiments were performed to examine whether the ERK/MAPK
pathway may be involved in regulation of
-adrenergic receptor signaling by EGF. The effect of EGF on isoproterenol-stimulated adenylyl cyclase activity was determined in HSY cells pretreated with
PD-98059, a synthetic inhibitor of the ERK/MAPK pathway. The inhibitor
abolished the increase in isoproterenol-responsive enzyme activity
produced by EGF. In control experiments EGF was observed to induce
phosphorylation of ERK in HSY cells, and this action of EGF was
inhibited by PD-98059 (Fig. 10). The
results suggest that EGF-induced enhancement of
-adrenergic receptor signaling is mediated by activation of the ERK/MAPK pathway.

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Fig. 10.
Effects of PD-98059 on EGF-induced enhancement of
isoproterenol-stimulated adenylyl cyclase activity and phosphorylation
of extracellular signal-regulated kinase (ERK). A: HSY cells
cultured for 24 h were treated with PD-98059 (+PD-98059; 2.5 × 10 5 M) or left untreated ( PD-98059) for 30 min,
followed by addition of EGF (+EGF, 5 × 10 10 M) or
vehicle ( EGF) for 48 h. Cells were then assayed for
isoproterenol (10 5 M)-stimulated and unstimulated (basal)
adenylyl cyclase activities as described in MATERIALS AND
METHODS. Values represent means ± SE of
isoproterenol-stimulated adenylyl cyclase activity from 15 experiments.
*P < 0.0001 vs. EGF. PD-98059 had no effect on basal
enzyme activity (data not shown). B: cells cultured for
24 h were treated with PD-98059 (2.5 × 10 5 M)
or left untreated as in A. Phosphorylation of ERK
(p44/p42MAPK) was determined after 10-min incubation with
or without EGF (5 × 10 10 M) by immunoblot analysis
as described in MATERIALS AND METHODS. In this
representative experiment PD-98059 inhibited EGF-induced
phosphorylation of p44/p42MAPK by 41%; when added at a
higher concentration (10 4 M), PD-98059 caused 84%
inhibition of ERK phosphorylation (data not shown).
|
|
 |
DISCUSSION |
The results of this study demonstrate that EGF enhances
-adrenergic receptor-mediated activation of adenylyl cyclase in a human salivary gland cell line (HSY) (Fig. 1). The stimulatory action
of EGF is directed specifically at the
-adrenergic receptor signaling pathway, because prostaglandin receptor-responsive adenylyl cyclase activity is unaffected by growth factor treatment. Moreover, EGF has no stimulatory effect on enzyme activation by agents acting directly on Gs
or adenylyl cyclase (cholera toxin and
forskolin, respectively; Fig. 3). Receptor binding experiments,
including competition studies using antagonists selective for
2- and
1-adrenergic receptor subtypes,
confirm that increased
-adrenergic responsiveness in EGF-treated HSY
cells reflects a threefold increase in the number of membrane receptors
of the
2-adrenergic subtype. In our studies no
high-affinity binding sites of the
1-adrenergic subtype
were detected in either control or EGF-treated cells (Figs. 4 and 5;
Table 1).
Our work is the first to report that in HSY cells
-adrenergic
receptors are predominantly of the
2-subtype.
Competition binding studies of
-adrenergic receptors in other cell
lines established from human and rat salivary glands have similarly identified a predominance of
2-adrenergic receptors
(43). Receptors of the
2-subtype are also
demonstrable in rat parotid glands, in which the
-adrenergic
receptor population is primarily of the
1-subtype
(1, 25, 27). Although secretory responses of salivary
glands to
-adrenergic agonists are generally thought to be mediated
by receptors of the
1-subtype, a number of studies have
implicated a role for
2-adrenergic receptors in
regulation of both salivary secretion and growth (4, 24).
Whether modification of
2-adrenergic receptor content,
as we have observed in HSY cells treated with EGF, may in turn modulate
salivary gland function remains to be clarified.
Agonist-induced reduction of membrane receptor content is
characteristic of
-adrenergic and other G protein-coupled receptors. Interestingly, the two subtypes of the
-adrenergic receptor undergo distinct patterns of agonist-induced regulation. Losses of receptor binding are of greater magnitude, and occur earlier after agonist exposure, for the
2- than the
1-adrenergic receptor subtype (53). In HSY
cells the marked decline in membrane
-adrenergic receptor binding
following agonist exposure closely resembles that previously reported
for the
2-adrenergic receptor subtype (53).
The upregulation of
2-adrenergic receptors we have
observed in EGF-treated HSY cells is not related to altered mechanisms of agonist-induced receptor regulation, because losses of receptor binding following isoproterenol exposure were unaffected by EGF pretreatment (Fig. 6). In contrast to this finding, EGF treatment of
vascular smooth muscle cells has been shown to increase the cell
surface content of the multifunctional receptor low-density lipoprotein
receptor-related protein by altering receptor distribution and
recycling (56).
EGF-induced upregulation of
-adrenergic receptor content in HSY
cells is likely to result, at least in part, from an increase in
2-adrenergic receptor mRNA expression. Northern blot
analyses revealed a biphasic increase in levels of
2-adrenergic receptor subtype mRNA after addition of
EGF, with two- to threefold elevations of receptor mRNA levels
occurring at 1 h and again at 8-24 h of EGF exposure (Fig.
7). The time required for expression and membrane targeting of
increased numbers of
2-adrenergic receptors presumably accounts for the finding that EGF enhances isoproterenol-responsive adenylyl cyclase activity only after prolonged incubation periods (
24
h) (Fig. 2). The increase in
2-adrenergic receptor mRNA observed at 1 h of EGF treatment was found to be blocked by the RNA synthesis inhibitor actinomycin D; in the absence of growth factor,
receptor transcript levels were unaffected by actinomycin D added for
1 h (Fig. 8). These results suggest that EGF upregulates expression of the
2-adrenergic receptor subtype by a
mechanism involving increased transcription of the receptor gene. Our
experiments do not exclude the possibility of an effect of EGF on
2-adrenergic receptor mRNA stability over longer periods
of growth factor exposure. In earlier studies EGF was shown to reduce
the expression of other G protein-coupled receptors, i.e., luteinizing
hormone/chorionic gonadotropin receptors in Leydig tumor cells and
angiotensin II receptors in vascular smooth muscle cells, through
inhibitory effects on receptor gene transcription (40,
41). Interestingly, the 5'-flanking region of the human and/or
rat
2-adrenergic receptor gene contains recognition
sites for several transcription factors (nuclear factor-
B, Sp1, cAMP
response element-binding protein) implicated in the cellular actions of
EGF (26). However, additional studies will be required to
clarify whether EGF regulates expression of the
2-adrenergic receptor gene by transcriptional or
posttranscriptional mechanisms.
Regulation of
-adrenergic receptor expression by EGF had not been
described before the current study in HSY cells. Moreover, only limited
observations relating to long-term effects of EGF on adenylyl cyclase
activation via
-adrenergic or other stimulatory G protein-coupled
receptors have been reported in other cell types. In an earlier study,
prolonged (24-48 h) incubation of rat granulosa cells with EGF was
found to reduce follicle-stimulating hormone activation of adenylyl
cyclase (31). Work from our laboratory (19)
showed that exposure of a rat clonal osteoblast-like cell line
(UMR-106) to EGF for 48 h decreased adenylyl cyclase stimulation by isoproterenol and parathyroid hormone (PTH) but not
PGE2. Although we did not determine the effect of
EGF on
-adrenergic receptor binding in UMR-106 cells, a subsequent
study by others demonstrated reduced numbers of PTH receptors in
UMR-106 cells treated for 22 h with EGF (5). In this
study PTH receptor binding was found to be distributed heterogeneously
among morphologically distinct populations of UMR-106 cells. Of note,
EGF appeared to amplify a proliferating pool of UMR-106 cells
containing reduced numbers of PTH receptors and at the same time
deplete a quiescent population of receptor-enriched cells
(5). The distribution of
-adrenergic receptors among
UMR-106 or HSY cell populations has not been analyzed. Nonetheless, the
contrasting effects of EGF on isoproterenol-responsive adenylyl cyclase
activities in the two cell types suggest at least some degree of tissue
specificity in the stimulatory action of EGF on
-adrenergic receptor
expression observed in HSY cells. Decreased forskolin stimulation of
adenylyl cyclase activity in both HSY and UMR-106 cells, and reduced
basal enzyme activity in HSY cells, after long-term treatment with EGF
may reflect an inhibitory effect of the growth factor on one or more
adenylyl cyclase isozymes common to a number of tissues (Figs. 1 and 3; Refs, 19, 22). It should be emphasized that EGF also causes rapid (i.e., occurring within minutes) changes of adenylyl cyclase activities in a variety of in vitro systems, including rat parotid cell membranes (39, 47). These short-term effects of EGF, which are
generally thought to be mediated by Gs and/or the
inhibitory Gi protein, are distinct from the changes in G
protein-coupled receptor expression and adenylyl cyclase activation
occurring in cells after prolonged exposure to EGF.
A number of observations confirm that the stimulatory effect of EGF on
-adrenergic receptor expression in HSY cells is mediated by
signaling events of the EGF receptor tyrosine kinase pathway. The
concentration range over which EGF increases isoproterenol-stimulated adenylyl cyclase activity (Fig. 2) is characteristic of growth factor
actions at the EGF receptor (19). The finding that
isoproterenol responsiveness was increased by both EGF and TGF-
,
which act by binding to the EGF receptor, but not by agonists (PDGF,
IGF-1) binding to other receptor tyrosine kinases further implicates involvement of the EGF receptor tyrosine kinase in growth factor regulation of
-adrenergic receptor function in HSY cells. In preliminary experiments (not shown) we have also detected the expression of immunoreactive EGF receptors in HSY cells by immunoblot analysis. Because the EGF receptor tyrosine kinase activates the ERK/MAPK signaling cascade in many tissue types including the HSY cell
line (60), we examined whether ERK/MAPK may play a role in
the modulation of
-adrenergic responsiveness by EGF. The ERK/MAPK
pathway inhibitor PD-98059 abolished the increase in
isoproterenol-stimulated adenylyl cyclase activity caused by EGF;
EGF-induced phosphorylation of ERK/MAPK was also inhibited by PD-98059
(Fig. 10). Thus the regulatory action of EGF on the expression of
functional
-adrenergic receptors appears to be exerted at least
partly via EGF receptor tyrosine kinase activation of the ERK/MAPK
signal transduction pathway. Recent studies have suggested that the EGF
receptor may also translocate to the nucleus and act as a transcription
factor (35). It is not known whether this alternate
signaling pathway might occur in salivary cells and play a modulatory
role in
-adrenergic receptor expression.
The EGF receptor tyrosine kinase is also coupled to activation of PKC
in numerous tissues. In our studies EGF-induced increases in
isoproterenol-responsive adenylyl cyclase activity and
-adrenergic receptor number were unchanged after downregulation of PKC by prolonged
(4-6 h) exposure of HSY cells to phorbol ester (Fig. 9). This
finding suggests that activation of PKC does not play any significant
role in the modulatory action of EGF on
-adrenergic receptor
expression. Treatment with phorbol ester alone was found to increase
the expression of functional
-adrenergic receptors (Fig. 9),
although under the experimental conditions used it could not be
determined whether this effect of phorbol ester was attributable to
initial activation or subsequent downregulation of PKC. Phorbol ester-induced activation of PKC was reported previously to decrease expression of
3-adrenergic receptor mRNA, but not
1- and
2-adrenergic receptor transcripts,
in murine 3T3-F442A adipocytes and also
1-adrenergic
receptor gene transcription in rat C6 glioma cells (16,
34). Whereas
2-adrenergic receptor binding was
said to be downregulated in phorbol ester-treated C6 cells, supportive receptor binding data were not presented (34). PKC exerts
a complex array of regulatory effects on the functions and distribution of the EGF receptor (2). As indicated above, however, any
possible changes in EGF receptor action induced by either activation or downregulation of PKC were no longer apparent in HSY cells after 4- to
6-h exposure to phorbol ester. Because PMA in combination with EGF
produced such a marked increase (>6-fold) in
-adrenergic receptor
content (Fig. 9), additional studies are warranted to define further
the signaling processes by which PKC and EGF receptor tyrosine kinase
may function as independent yet interactive determinants of
-adrenergic receptor expression in HSY cells.
In recent years it has become increasingly clear that activation of G
protein-coupled receptors regulates not only classic effectors of
signal transduction (e.g., adenylyl cyclase and phospholipase C) but
also mitogenic signaling cascades (e.g., ERK and other MAPK pathways)
involved in cell growth and differentiation. Many G protein-coupled
receptors, including
2-adrenergic receptors, exert
proliferative effects via "transactivation" of EGF and other tyrosine kinase receptors coupled to the ERK/MAPK pathway (18, 44). The growth-promoting effects of isoproterenol on rat
parotid acinar cells have long been thought to involve activation of
the EGF receptor (45). Interestingly, growth of rat
parotid glands after chronic administration of isoproterenol in vivo is
accompanied by an increase in the number of parotid membrane
2-adrenergic receptors (with a corresponding decline in
1-adrenergic receptor content) (25). This
observation is consistent with possible involvement of the
2-adrenergic receptor subtype in the control of salivary
cell growth (24); a similar growth-promoting role for the
2-adrenergic receptor has been demonstrated in rat FRTL5 thyroid cells (23). Together with previous data, then, our
studies of HSY cells suggest a reciprocal relationship between
2-adrenergic and EGF receptors, in which upregulation of
2-adrenergic receptors induced by the EGF receptor
tyrosine kinase signaling pathway could in turn promote salivary cell
growth via EGF receptor transactivation. In this regard it should be
emphasized that activation of the EGF receptor plays a critical role in
salivary gland morphogenesis (9, 55). Also, the HSY cell
line used in our experiments is thought to originate from salivary
intercalated duct cells, which may act as progenitors in the
replenishment of both acinar and duct cells. Thus EGF-induced
upregulation of
-adrenergic receptor signaling, as observed in HSY
cells, could conceivably contribute to EGF-dependent processes involved
in salivary gland development and regeneration.
In summary, the present study demonstrates for the first time that in a
human salivary cell line (HSY) EGF enhances
-adrenergic-responsive adenylyl cyclase activation via an increase in
2-adrenergic receptor expression at the transcriptional
level. We previously showed (60) that, in contrast to
-adrenergic receptor signaling, the muscarinic receptor-mediated
calcium mobilization pathway in HSY cells is inhibited by EGF. The
effects of EGF on both
-adrenergic and muscarinic receptor signaling
pathways appear to be mediated by the ERK/MAPK cascade and potentiated
by distinct modulatory actions of PKC. Our findings provide the basis
for further investigation of mechanisms by which cross talk between EGF
receptor tyrosine kinase and G protein-coupled receptor signaling
pathways may function in the regulation of salivary cell secretion and growth.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Paramita Ghosh for help with immunoblot analysis of
ERK/MAPK.
 |
FOOTNOTES |
This work was supported by NIDCR Grants DE-10756 and DE-12188 (to C.-K.
Yeh) and medical research funds from the Department of Veterans Affairs
(to B.-X. Zhang and M. S. Katz).
Address for reprint requests and other correspondence:
M. S. Katz, Geriatric Research, Education and Clinical
Center (182), South Texas Veterans Health Care System,
Audie L. Murphy Division, 7400 Merton Minter Blvd., San Antonio,
TX 78229-4404 (E-mail: katz{at}uthscsa.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.
First published January 22, 2003;10.1152/ajpcell.00343.2002
Received 23 July 2002; accepted in final form 10 January 2003.
 |
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