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INTRODUCTION |
Cellular responses to a variety of external stimuli alter the
proliferation or differentiation status via receptor-mediated intracellular signaling cascades. Plasma membrane receptors coupled to
heterotrimeric G proteins (G protein-coupled receptor) a protein family with ~1,000 members, mediate a large variety of important physiological actions induced by hormones, neurotransmitters, and
sensory signals.
1-Adrenergic receptors
(
1-ARs)1 are
members of the superfamily of G protein-coupled receptor and mediate
the effects of the sympathetic nervous system, especially those effects
related to the regulation of cellular hypertrophy and proliferation
(1-3). There is substantial evidence indicating that stimulation of
1-ARs by catecholamines generally enhances growth-related gene expression and cell growth in a variety of cells
including cardiac myocytes, vascular smooth muscle cells, hepatocytes,
and also adipocytes (4-6).
The
1-ARs initiate their physiological effects by
activating phospholipase C at the cell membrane, resulting in
production of inositol 1,4,5-trisphosphate. Inositol
1,4,5-trisphosphate, in turn, mobilizes intracellular calcium and
diacylglycerol, which activates protein kinase C (7, 8). More recently,
the
1-ARs were found to activate a variety of other
effectors such as the mitogen-activated protein kinase (MAPK) pathway,
cAMP metabolism, phospholipase D, and A2 in various cells
(9-14). Both molecular and pharmacological studies have shown that
1-ARs consist of three subtypes:
1A-,
1B-, and
1D-AR (15-19). The three
different
1-AR subtypes are expressed in different
tissues and various cell types. Therefore, studies on the physiological
effects mediated by
1-ARs in individual tissues are
complicated by the co-existence of multiple
1-AR
subtypes (20). Although the pharmacological properties of each subtype
are well documented (21-23), little is known regarding the
physiological role of each receptor subtype at a cellular level and in
the regulation of cell proliferation. In this study, we explored the
role of each
1-AR subtype in cell proliferation using a
heterologous expression system. Our results show that these highly
homologous receptor subtypes have distinct signal transduction coupling
properties and have differential effects on cell proliferation.
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EXPERIMENTAL PROCEDURES |
Materials--
Phentolamine hydrochloride was from Ciba
Pharmaceutical Corp. (Summit, NJ). Phenylephrine was from Sigma. Ham's
F-12 medium, Lipofectin, and G418 (Geneticin disulfate) were from
Invitrogen. Fura-2/acetoxymethyl ester (Fura-2/AM) was from Dojindo
(Kumamoto, Japan). Triton X-100 (polyoxyethylene (10) octhylphenyl
ether) was from Wako Pure Chemical Industries (Osaka, Japan). MAPK
substrate peptide and Whatman P81 phosphocellulose discs were from
Invitrogen. RpcAMP (Rp-isomer cyclic AMP) and dibutyrl cyclic AMP were
from Sigma. Anti-p27Kip1 polyclonal antibody (sc-776) was from
Santa Cruz Biotechnologies (Santa Cruz, CA). All other chemicals were
of reagent grade. The CHO-K1 cell line was obtained from American Type
Culture Collection (Rockville, MD).
Transfections and Culture Conditions--
Genes encoding the
human
1A-,
1B-, or
1D-AR were ligated
into the EcoRI site of the eukaryotic expression vector,
pSVK3, containing the neomycin-resistance gene of pMAM-neo (pSVK3neo) as described previously (23). Wild-type CHO-K1 cells were grown in
Ham's F-12 medium containing L-glutamine supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO2 in an air-ventilated
humidified incubator. Cells were passaged using trypsin. For stable
expression of human
1A-,
1B-, or
1D-AR subtype, CHO-K1 cells were seeded at a density of
2 × 104 cells in 35-mm tissue culture dishes. One day
after seeding the cells, the medium was removed, and 1 ml of serum-free
F12 medium containing 13.8 µg of Lipofectin (24) and 9.2 µg of the
recombinant expression plasmid was added to the cells. At 24 h
after DNA-lipid treatment, 1 ml of F12 medium containing 20% fetal
bovine serum was added. The cells were passaged at a low density ~72
h later. Single colonies resistant to the antibiotic G418 (600 µg/ml)
were isolated and maintained in F12 medium with 10% fetal bovine serum and 200 µg/ml G418. Three clonal CHO cell lines for each
1-AR subtype (
1A-,
1B-, or
1D-AR), which stably express the
1-AR at
similar level, were established and used for subsequent studies. Specifically, one line of CHO stable transfectants was designated as
CHO
1A, CHO
1B, and CHO
1D.
The ligand-binding characteristics of the receptors expressed in CHO
cells were determined in a preliminary series of radioligand-binding studies performed as described previously (23) using
[125I]HEAT (specific activity 2,200 Ci/mmol, PerkinElmer
Life Science) as the radioligand for
1-AR. Membrane
preparations from CHO cells stably expressing the cloned human
1-ARs showed saturable binding of
[125I]HEAT; Bmax values were
1.3 ± 0.2, 5.5 ± 0.1, and 1.1 ± 0.1 pmol/mg protein
with Kd values of 110 ± 21, 60 ± 1, and
300 ± 26 pM for the CHO
1A, CHO
1B, and CHO
1D
(n = 3 each), respectively. Competition isotherms
showed the expected pharmacology for each receptor subtype (23).
[3H]Thymidine Incorporation--
We employed the
method of Kato et al. (25). Confluent cells grown in 96-well
plates were rendered quiescent by serum deprivation for 24 h. The
cells were then incubated with 10% serum with or without 10 µM phenylephrine for 18 h and for an additional
6 h with [6-3H]thymidine (1 µCi/ml, PerkinElmer
Life Sciences). The cells were rinsed twice with ice-cold
phosphate-buffered saline, precipitated with cold 5% trichloroacetic
acid, and then washed with cold ethanol and solubilized with 0.5 N NaOH. DNA radioactivity was measured by scintillation counting.
Cell Cycle Analysis by Flow Cytometry--
Cells at 50%
confluence in 35-mm culture dishes were rendered quiescent by serum
deprivation for 24 h. The cells were then incubated with 10%
serum with or without 10 µM phenylephrine for 48 h.
Flow cytometric analysis of each CHO cell stained with propidium iodide
was used to estimate the cell cycle phase distributions using the
CycleTESTTM Plus DNA Regent Kit (BD Biosciences).
Subsequent flow-cytometric analysis was performed with a FACScan flow
cytometer (BD Biosciences). Data were analyzed using the CellFIT
software (BD Biosciences).
Measurement of Inositol 1,4,5-Trisphosphate--
Cells at 50%
confluence in 35-mm culture dishes were rendered quiescent by serum
deprivation for 24 h and treated with phenylephrine (10 µM) for 10 s at 37 °C. The medium was
subsequently removed, and cells were scraped into 1 ml of 20% (w/v)
perchloric acid on ice. After centrifugation, the supernatant was
adjusted to pH 7.0 with HEPES-KOH solution, and the sediment was
eliminated by centrifugation. Amounts of inositol 1,4,5-trisphosphate
in a sample were measured by a radioreceptor assay with the
D-myo-inositol 1,4,5-triphosphate
[3H] assay kit (TRK 1000, Amersham Biosciences).
MAPK Assay--
Cells were rendered quiescent by serum
deprivation for 24 h before stimulation with 10% serum in the
presence or absence of phenylephrine for 30 min at 37 °C. Cell
monolayers were washed in phosphate-buffered saline, pH 7.4, and lysed,
and the overall activity of ERK1/2 MAPK in the lysate was determined
using the BiotrakTM MAPK assay (Amersham Biosciences) in
accordance with the manufacturer's instructions. Aliquots of 15 µl
of cell lysates freshly obtained or stored at
80 °C corresponding
to 10 µg of protein were incubated at 30 °C with 15 µl of a
reaction mixture containing a synthetic specific peptide substrate and
[
-32P]ATP (1 µCi; 1.2 mM, PerkinElmer
Life Sciences). After 30 min, the reaction was stopped and
samples were spotted on 3-cm discs of binding paper. After several
washes in 0.75% phosphoric acid and water, the radioactivity on each
disc was then determined by scintillation counting.
Measurements of cAMP Production--
cAMP production in intact
transfected CHO cells was determined as described previously (26). Each
transfected CHO cell population was seeded in 6-well plates at a
density of 5 × 105 cells/well and cultured for 12-16
h. The cells were washed twice with phosphate-buffered saline,
incubated for 30 min in HEPES buffer, and then the medium was replaced
with the fresh HEPES buffer containing 1 mM
isobutylmethylxanthine for 10 min. The reaction was started by adding
phenylephrine (10 µM) or forskolin (10 µM).
After incubation for 10 min, the medium was aspirated and the reaction
was stopped with 6% (w/v) trichloroacetic acid. cAMP levels were
determined by radioimmunoassay (Yamasa cAMP Assay Kit, Yamasa Shoyu
Co., Chiba, Japan). Phentolamine was added 10 min prior to agonist
stimulation in HEPES buffer containing 1 mM
isobutylmethylxanthine and 1 µM propanolol.
Adenylyl Cyclase Assay--
Each transfected CHO cell population
was cultured in 15-cm dishes, suspended in lysis buffer (62.5 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 0.1 mM benzamidine, 5 µg/ml pepstatin A, and 1 µg/ml leupeptin), and homogenized using the Cell Disruption Bomb
(Parr Instrument Co., Moline, IL) at 400 p.s.i. for 30 min. The
homogenate was centrifuged at 35,000 × g for 20 min at
4 °C, and the pellet was resuspended in 10 mM Tris-HCl,
pH 7.4, containing 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 5 µg/ml pepstatin A, and 1 µg/ml leupeptin and then used as membrane
preparations. The standard assay mixture contained 20 µg of membrane
proteins in 100 µl of 50 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl2, 1 mM isobutylmethylxanthine, 0.5 mM ATP, 1 mM dithiothreitol, 1 µM guanosine
5'-3-O-(thio)triphosphate or guanosine
5'-O-(3-thiotriphosphate), and 1 µM propanolol.
Reactions were started by the addition of the agonists, carried out for
15 min at 37 °C, and terminated by boiling at 90 s. The cAMP
levels were determined using a cAMP radioimmunoassay kit (Yamasa Shoyu
Co.).
Immnoblotting with Anti-p27Kip1--
Following the
experimental treatments, cells were washed with phosphate-buffered
saline and lysed with 1% Nonidet P-40 in a buffer consisting of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2,
10% glycerol, 0.4 mM sodium orthovanadate, 10 mM EDTA, and protease inhibitors (1 mM
phenylmethylsulfonyl fluoride and 2.5 µg/ml each of leupeptin and
aprotinin). After 30 min on ice, the cell extracts were clarified by
centrifugation for 10 min at 14,500 × g at 4 °C.
The p27Kip1 quantitation was adjusted for protein load, and
equal amount of the lysates (10 µg of protein) were mixed with 1×
Laemmli sample buffer, boiled for 5 min, and resolved by
SDS-polyacrylamide gel electrophoresis on a 12% gel. The proteins were
electrophoretically transferred to polyvinylidine fluoride membranes as
recommended by the manufacturer. The blots were first incubated with
blocking buffer (l% bovine serum albumin in 10 mM
Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) and
probed with rabbit polyclonal anti-p27Kip1 antibody (sc-776).
The immune complexes were detected using the ECL reagents from Amersham
Biosciences and quantitated by densitometry using NIH Image software.
The protein concentration was measured using the bicinchoninic acid
protein assay kit (Pierce, Rockford, IL).
Statistics--
Values are expressed as the mean ± S.E.
Two-way analysis of variance with 95 or 99% confidence limits followed
by a Student's t test on individual sets of data was
performed with the analytical software StatView 4.0 (SAS Institute
Inc., Cary, NC).
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RESULTS |
Activation of
1A-AR or
1B-AR but Not
1D-AR Inhibits Serum-stimulated Cell
Proliferation--
The cell proliferation of CHO cells stably
expressing each
1-AR subtype (CHO
1A, CHO
1B, and
CHO
1D) was measured by [3H]thymidine incorporation
after serum deprivation. Cells were then exposed to serum in either the
presence or the absence of
1-AR stimulation. The
addition of 10% serum to serum-deprived cells markedly stimulated DNA
synthesis in all CHO cell lines expressing any of the
1-AR subtypes. The activation of
1-AR by
phenylephrine (10 µM) inhibited the serum-promoted
[3H]thymidine incorporation in CHO
1A and CHO
1B
cells, whereas such an inhibitory effect was not observed when treating
CHO
1D cells with phenylephrine (Fig.
1). [3H]Thymidine
incorporation experiments were also performed for additional two clonal
CHO stable transfectants for each
1-AR (
1A-,
1B-, or
1D-AR), and
we observed similar subtype-specific anti-proliferative effects (data
not shown). Thus, the results clearly showed
1-AR
subtype-dependent differential effects on the cell
proliferation.

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Fig. 1.
Effect of
1-AR subtype activation on
serum-stimulated cell proliferation.
[3H]Thymidine incorporation was measured in each
CHO cell line stably expressing 1A-, 1B-,
or 1D-AR cDNA following growth in serum-free medium
(Basal) or in serum-enriched medium alone (Serum)
or serum with phenylephrine (Serum/PE) or serum with
phenylephrine/phentolamine (Serum/PE/Phent). The results are
expressed as the mean ± S.E. of four different experiments. **,
p < 0.01 versus serum alone.
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Activation of
1A- or
1B-AR but not
1D-AR Inhibits Serum-promoted Cell Cycle
Progression--
To explore the mechanism underlying the
1-AR subtype-dependent differential effects
on DNA synthesis and morphological alterations, the effects of
1-AR activation on the cell cycle progression were
examined by flow cytometric analysis in each of the CHO cell lines.
Three distinct cell populations corresponding to the G1, S,
and G2/M phases of the cell cycle were readily observed in serum-deprived cells. In addition, there were no significant
differences among the cell cycle profiles of CHO
1A, CHO
1B, and
CHO
1D cells (Fig. 2). Treatment with
serum resulted in a 2- to 3-fold increase in the cell population in S
phase with a concomitant reduction in the cell population in
G1 in all CHO cell groups, indicating entry of a
significant number of cells into S phase. The addition of 10 µM phenylephrine to serum, however, markedly and
reproducibly blocked the cell cycle progression in CHO
1A and
CHO
1B. In contrast, the addition of 10 µM
phenylephrine to CHO
1D cells failed to block the serum-induced
proliferation (Fig. 2). These results indicate that
1-AR
activation has an inhibitory effect on serum-promoted cell cycle
progression in CHO
1A and CHO
1B cells, whereas stimulation of
CHO
1D may have been mildly growth promoting. The
phenylephrine-stimulated cell cycle blockade observed in CHO
1A and
CHO
1B was reversed by treatment with the
1-AR
antagonist, phentolamine, at 10 µM (Fig. 2).

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Fig. 2.
Effect of
1-AR subtype activation on cell cycle
progression as analyzed by flow cytometry. Cells rendered
quiescent by serum deprivation for 24 h were treated with 10%
serum in the presence or absence of 10 µM phenylephrine
for 48 h. Furthermore, the effect of direct stimulation of Gs
protein by cholera toxin on cell proliferation was examined in each
1-AR-expressing CHO cell (bottom). Cells were
then collected by trypsinization, fixed, and stained with propidium
iodide. Subsequent flow-cytometric analysis was performed with a
FACScan flow cytometer. Data were analyzed using the CellFIT software.
Histograms show relative DNA content (x axis) and relative
cell number (y axis). Flow-cytometric analysis was performed
on six independent pools of CHO cells in each treatment group, and the
results of the cell cycle study are presented as percent of cells in
the indicated cell cycle stage. PE, phenylephrine;
Phent, phentolamine; CTX, cholera toxin.
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1-AR Subtypes Similarly Stimulate Inositol
1,4,5-Trisphosphate Production and Serum-stimulated MAPK Activation but
Differentially Induce cAMP Production--
We postulated that
participation of different intracellular signaling transduction
pathways might explain the differential regulation of cell
proliferation by
1-AR subtype activation. To evaluate
this possibility, we first examined the production of inositol
1,4,5-trisphosphate after
1-AR activation.
1-AR-activated production of inositol
1,4,5-trisphosphate was observed in all
1-AR-expressing
CHO cells (Fig. 3A) but not in
untransfected CHO-K1 cells (data not shown). When MAPK activity (ERK
isoforms) was measured,
1-AR activation had no
discernible effect on serum-stimulated MAPK activity in all CHO cells
stably expressing each
1-AR subtype (Fig.
3B). These results indicated that these early events caused after receptor stimulation were not different upon activation of
different
1-AR subtypes.

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Fig. 3.
Effect of
1-AR subtype activation on
intracellular signal transduction pathways. A, effect
of 1-AR subtype activation on inositol
1,4,5-triphosphate production. B, effect of
1-AR subtype activation on the serum-promoted MAPK
activation. C, effect of 1-AR subtype
activation on intracellular cAMP production. D, effect of
1-AR subtype activation on adenylyl cyclase activity in
CHO cells stably expressing each 1-AR subtype. In all
experiments, phentolamine (10 µM) treatment was started
30 min prior to the phenylephrine stimulation. Values are the mean ± S.E. of at least three different experiments, each performed in
duplicate. **, p < 0.01 versus basal.
IP3, inositol 1,4,5-triphosphate; PE,
phenylephrine; Phent, phentolamine; FSK,
forskolin.
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We next decided to examine the ability of
1-AR
activation to induce production of cAMP in CHO
1A, CHO
1B, and
CHO
1D cells. As shown in Fig. 3C,
1-AR
stimulation resulted in marked cAMP production in CHO
1A and CHO
1B
but not in CHO
1D. This was also found to be true for additional two
clonal CHO stable transfectants for each
1-AR
(
1A-,
1B-, or
1D-AR) (data
not shown). On the other hand, an adenylyl cyclase activator, forskolin
(5 µM), elicited an ~10- to 25-fold increase in cAMP
production above basal levels in all
1-AR-expressing CHO
cells (Fig. 3C) as well as in untransfected CHO-K1 cells
(data not shown). Furthermore, we examined the effects of
1-AR activation on the adenylyl cyclase activity of
membranes from each transfected CHO cell line.
1-AR
activation increased the adenylyl cyclase activity in the CHO
1A and
CHO
1B membranes but not in the CHO
1D (Fig. 3D). The
GTP analog, GTP
S (10 µM), elicited an ~2- to 4-fold
increase in adenylyl cyclase activity above basal levels in all
CHO
1A, CHO
1B, and CHO
1D cells.
1-AR-mediated cAMP production and adenylyl cyclase activation in CHO
1A and CHO
1B were abolished by the
1-AR antagonist,
phentolamine (10 µM). The
1-AR-induced
cAMP response was not observed in wild-type CHO-K1 cells (data not
shown). Hence, the above data show that stimulation of
1A- and
1B-AR but not
1D-AR promotes
cAMP synthesis in part by activating adenylate cyclase.
cAMP Suppresses Cell Proliferation in Transfected CHO
Cells--
We next examined the correlation between cAMP signaling
pathway and
1-AR-mediated anti-proliferative effects as
observed in CHO
1A and CHO
1B. cAMP-inducing agents, cholera toxin,
forskolin, and dibutyrl cyclic AMP, all inhibited serum-promoted
[3H]thymidine incorporation to 30~40% of control
levels in all three transfected CHO cells (Fig.
4), suggesting that cAMP signaling is an
important anti-mitogenic signal in CHO cells. On the other hand,
treatment with RpcAMP, an antagonist of cAMP, significantly blocked
1-AR-mediated inhibitory effects on cell proliferation in CHO
1A and CHO
1B (Fig. 4). A series of experiments using cAMP modulators were also performed for additional two clonal CHO stable transfectants for each
1-AR (
1A-,
1B-, or
1D-AR), and we observed basically
similar subtype-dependent behavior (data not shown).

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Fig. 4.
Effect of various agents that modulate cAMP
pathway on serum-stimulated cell proliferation.
[3H]Thymidine incorporation was measured in each CHO cell
line stably expressing 1A-, 1B-, or
1D-AR cDNA. The results are expressed as the
mean ± S.E. of four different experiments. ++, p < 0.01 versus serum free. **p < 0.01 versus serum alone. PE, phenylephrine;
Phent, phentolamine; CTX, cholera toxin;
FSK, forskolin.
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Enhanced cAMP Production Inhibits the Serum-promoted Cell
Cycle--
We next examined whether
1-AR-mediated cAMP
production may have an inhibitory effect on the cell cycle progression
of CHO
1A and CHO
1B cells. The effect of direct stimulation of Gs
protein by cholera toxin on cell proliferation was examined in each
1-AR-expressing CHO cell. As shown in Fig. 2,
bottom, the addition of 10 µM cholera toxin
resulted in a decreased cell population in S phase in all CHO cell
lines. This change in cell cycle profile was similar to that observed
in the CHO
1A and CHO
1B cells. Thus, the data indicate that the
differential effect of
1-AR subtype stimulation on cell
proliferation may be attributed to the differential activation of the
cAMP-mediated signaling pathway by each
1-AR subtype.
cAMP-mediated Cell Cycle Blockade Caused by
1A-AR or
1B-AR but Not by
1D-AR Is Associated with
an Induction of the Cyclin-dependent Kinase Inhibitor
p27Kip1--
p27Kip1 is a
cyclin-dependent kinase inhibitor that negatively regulates
cell cycle progression and enforces cell cycle arrest in a
cAMP-dependent manner (25). Hence, we further examined whether changes in p27Kip1 expression levels could account for
the differential effect of
1-AR subtype stimulation on
cell cycle progression. As shown in Fig.
5, immunoblot with a specific
anti-p27Kip1 antibody revealed that serum treatment caused a
significant reduction in the protein expression level of
p27Kip1 with similar kinetics in all
1-AR-expressing CHO cell lines. On the other hand, the
activation of
1-AR by phenylephrine blocked this
serum-induced reduction of p27Kip1 expression only in the
CHO
1A and CHO
1B cells but not in the CHO
1D cells (Fig. 5).
These phenylephrine-induced changes in the expression of
p27Kip1 in CHO
1A and CHO
1B were blocked by pretreatment
with phentolamine (data not shown). Corresponding with the effects on
serum-induced cell proliferation, we observed that treatment with
cholera toxin and forskolin inhibited the serum-induced reduction in
p27Kip1 expression in all three CHO cell lines as observed when
1A/
1B-AR are activated (Fig.
6). Also, these phenylephrine-induced
changes in the expression of p27Kip1 in CHO
1A and CHO
1B
were blocked by RpcAMP (Fig. 6).

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Fig. 5.
Effect of
1-AR subtype activation on
serum-induced decrease in the level of the cyclin-dependent
kinase inhibitor p27Kip1. Serum-starved cells were
stimulated with 10% serum in the presence or absence of phenylephrine
or cholera toxin. Cells were then harvested at the indicated times, and
total cell lysates were electrophoresed and immunoblotted with
anti-p27Kip1 antibody. Both the autoradiograph (A)
and quantitative data obtained by densitometry using NIH Image software
(B) are shown. PE, phenylephrine.
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Fig. 6.
Effect of various agents that modulate cAMP
pathway on the level of the cyclin-dependent kinase
inhibitor p27Kip1. Serum-starved cells were stimulated
with 10% serum in the presence or absence of phenylephrine. Cells were
then harvested 48 h after serum addition, and total cell lysates
were electrophoresed and immunoblotted with anti-p27Kip1
antibody. Both the autoradiograph (A) and quantitative data
obtained by densitometry using NIH Image software (B) are
shown. In all experiments, phentolamine (10 µM) treatment
was started 30 min prior to the phenylephrine stimulation. The results
are expressed as the mean ± S.E. of three different experiments.
*, p < 0.05; **, p < 0.01 versus serum alone (lane 2). Lane 1,
basal; lane 2, serum; lane 3, serum with
phenylephrine; lane 4, serum with
phenylephrine/phentolamine; lane 5, serum with
phenylephrine/RpcAMP; lane 6, serum with cholera toxin; and
lane 7, serum with forskolin.
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|
 |
DISCUSSION |
In this study, we explored the roles of each
1-AR
subtype in cell proliferation using a heterologous expression system.
The activation of
1A- or
1B-AR but not
1D-AR inhibited serum-promoted cell proliferation as
evidenced by changes in the cell cycle profiles. In view of the
differential expression profiles obtained between
1A/
1B-AR- and
1D-AR-activated cells, we examined the molecular mechanism for the
1-AR subtype-dependent
differential effect on cell proliferation. We found that all of the
1-AR subtypes share induction of inositol
1,4,5-trisphosphate production and activation of MAPK pathways (ERK
isoforms), but only
1A- and
1B-ARs are
coupled to the cAMP signaling pathway. Furthermore, we observed that
1A- or
1B-AR but not
1D-AR
can block the serum-induced depletion of the cell
cycle-dependent kinase inhibitor p27Kip1, which is
known to mediate cAMP-induced G1 phase arrest. Taken together, our present data show that the
1A- and
1B-ARs but not
1D-AR are coupled to cAMP
synthesis and block serum-promoted depletion of p27Kip1, and
thereby inhibit serum-promoted cell proliferation.
1-AR stimulation generally promotes cell growth in
various cell types. However, recent studies have reported an
anti-proliferative effect of
1-AR activation in certain
cell types (27-29). In the process of hepatic regeneration after
hepatectomy,
1-AR stimulation is thought to act as
anti-proliferative signal in hepatic cells (28, 29). Also, it was
reported that the activation of
1B-AR in HepG2 human
hepatoma cells stably transfected with a rat
1B-AR cDNA inhibited cell growth via a p42MAPK and
p21WAF1/CIP1-dependent
mechanism (28). This study confirms an inhibitory effect of
1-AR activation on the serum-promoted cell proliferation in a heterologous expression system and shows that the inhibitory effect is subtype-dependent and mediated through cAMP
production. In agreement with our findings, Keffel et al.
(30) recently has reported that
1-AR subtypes are
differentially coupled to growth promotion and growth inhibition in CHO
cells (30). However, Keffel et al. (30) concluded that the
opposite effects of
1-AR subtypes on cellular growth are
because of quantitative differences in the ERK isoforms in the MAPK
signal transduction pathway. Although we confirmed the
subtype-dependent differential extent of MAPK activation
that is previously reported by Keffel et al. (30) in the
serum-free conditions (data not shown), we also found that serum
activates the MAPK pathway more dramatically than
1-AR activation. In fact, as shown in Fig. 3B of this study,
1-AR activation had a small additive stimulatory effect
on the serum-promoted MAPK activation in all subtypes. Therefore, we
consider that the anti-proliferative effect observed upon activation of
1A/
1B-AR but not of
1D-AR
may not be solely explained by the quantitative difference in MAPK activation.
1-ARs have been reported to have a stimulating effect on
adenylyl cyclase activity in certain native tissues. In fact, the activation of
1-AR increases cAMP production in tissue
sections of rat brain or rat liver (9, 10). However, the molecular mechanism and physiological significance of the
1-AR-induced cAMP production have remained uncertain.
This report proposes for the first time that
1-ARs
activate the cAMP cascade in a subtype-dependent manner and
that this differential activation is closely associated with the
anti-proliferative effect of the
1-AR subtype. We
previously examined the mechanism of
1-AR-mediated cAMP
synthesis using
1B-AR as a model and found that
1B-AR-mediated cAMP synthesis is because of a direct
coupling of
1B-AR to the Gs protein (26). However, in
other studies, the mechanism of
1-AR-mediated induction
of cAMP synthesis was shown to be secondary to its phosphatidylinositol
hydrolysis/protein kinase C activation (12, 31, 32). Hence,
1-AR-stimulated cAMP production is now considered to
involve multiple mechanisms including direct activation of Gs/adenylyl
cyclase and a secondary effect through activation of
phosphatidylinositol hydrolysis. However, our data showing that all
subtypes are efficiently coupled to phosphatidylinositol hydrolysis but
that only
1A- and
1B-AR promote cAMP
production provide support for a mechanism by which
1-AR-mediated cAMP synthesis is the result of direct
coupling to a Gs protein.
This study shows that activation of
1-ARs inhibits the
serum-promoted cellular growth in a cAMP-dependent manner.
Several studies (34, 35) have reported that cAMP inhibits growth
factor-induced mitogenesis by antagonizing the ERK/MAPK pathway at the
Raf-1 level. In this study, however, we did not obtain any evidence in
support of such a mechanism. Cell cycle blockade is recognized as
another potential mechanism for cAMP/protein kinase A-mediated inhibition of cell proliferation in certain cells (36-38). In
mammalian cells, the cell division cycle is controlled by the formation and activation of protein kinase complexes consisting of cyclins and
cyclin-dependent kinases (39-41). The activity of
cyclin-cyclin-dependent kinase complexes controls the cell
cycle checkpoints in a coordinated way. By inhibiting the kinase
activity of the cyclin-dependent kinases,
cyclin-dependent kinase inhibitor proteins interfere with
phosphorylation events critical for cell cycle transition (33, 41).
Recently, it was reported that cAMP blocks colony-stimulating factor-induced mitogenesis in macrophages by inhibiting the cyclin D1-cyclin-dependent kinase 4 activity via increasing the
expression of p27Kip1 (25). Our results show that serum
stimulation is capable of activating the G1/S transition
with a concomitant decrease in the expression level of p27Kip1
and that these events are inhibited by
1-AR
subtype-dependent activation of cAMP. Therefore, it is
possible that serum-promoted cell growth may be mediated by a decrease
in the steady-state level of p27Kip1 and that the
subtype-dependent anti-proliferative effect could be caused
by a cAMP-dependent inhibition of serum-promoted
down-regulation of p27Kip1. This study provides evidence for
the first time for regulation of cell proliferation through a
cAMP/p27Kip1-related mechanism rather than through an
ERK/MAPK-related mechanism.
In conclusion, we hereby report that a gene family of highly related
1-ARs exert subtype-dependent differential
effects on cell proliferation, although
1-ARs are
generally viewed to promote cell proliferation. Moreover, we found that
the subtype-dependent differential effects are because of
the different signal coupling associated with each subtype. Thus,
activation of
1A- and
1B-ARs but not
1D-AR, exerts anti-proliferative effects via the
cAMP/p27Kip1 pathway. The present study was performed by using
an
1-AR overexpression system where receptor couplings
are sometimes promiscuous. Hence, direct extrapolation of the present
findings to a natural system requires caution. Furthermore, in
vivo cells naturally co-express varying levels of multiple or all
subtypes. The physiological output of adrenergic stimulation in
vivo system may consist of complex cellular responses, which
involve subtype-dependent differential effects as observed
in this study.