Am J Physiol Cell Physiol 287: C1192–C1201, 2004.
First published June 30, 2004; doi:10.1152/ajpcell.00158.2004.
ATP-induced mitogenesis is mediated by cyclic AMP response
element-binding protein-enhanced TRPC4 expression and
activity in human pulmonary artery smooth muscle cells
Shen Zhang, Carmelle V. Remillard, Ivana Fantozzi, and Jason X.-J. Yuan
Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine,
University of California, San Diego, La Jolla, California 92093-0725
Submitted 24 March 2004; accepted in final form 15 June 2004
capacitative Ca2⫹ entry; proliferation; vascular smooth muscle
ceptors (including at least 6 subtypes: P2X, P2Y, P2T, P2U, P2Z,
and P2D), which are linked to phospholipases and adenylate
cyclase (7, 65). Activation of P2 purinoceptors by ATP and
UTP (24) in pulmonary artery smooth muscle cells (PASMC)
causes pulmonary vasoconstriction and in fibroblasts promotes
the progression of pulmonary vascular remodeling by stimulating fibroblast and SMC proliferation (19, 56).
While increased pulmonary vasoconstriction can underlie
the increased pulmonary vascular resistance (PVR) in patients
with pulmonary arterial hypertension, pulmonary vascular remodeling due to increased proliferation of PASMC in the
tunica media, resulting in medial hypertrophy, also contributes
greatly to the increased PVR. Increased extracellular or interstitial ATP level has been found to be involved in the progression of pulmonary hypertension (38, 56) as well as in the
regulation of cell cycle progression and cell proliferation (19,
36, 56), all of which depend on elevations in cytosolic free
Ca2⫹ concentration ([Ca2⫹]i). Investigators at our laboratory
previously showed that store-operated Ca2⫹ (SOC) influx
through canonical transient receptor potential (TRPC) channels
may underlie part of the agonist-mediated Ca2⫹ response via
capacitative Ca2⫹ entry (CCE) in PASMC (21, 61, 72). In all
of these studies, enhanced CCE influenced either vasoconstriction or proliferation. Along with TRPC1 and TRPC6, TRPC4
is a TRPC isoform that has been implicated in forming receptor-operated Ca2⫹ (ROC) and/or SOC channels in many cell
types (2, 9, 17, 30, 39, 51, 60, 62, 66, 72).
In this study, we hypothesized that extracellular ATP induces human PASMC proliferation via upregulation of TRPC4
expression and increase in CCE. Furthermore, we proposed
that ATP-induced phosphorylation of the cyclic AMP response
element-binding protein (CREB), a critical transcription factor
in the regulation of cell survival (14, 49, 64), is involved in the
increased transcription of TRPC4 induced by ATP.
NUCLEOTIDES SUCH AS ATP, ADP, AND UTP act as extracellular
messengers and play an important role in regulating vascular
tone and cell proliferation and differentiation (12, 18, 25, 41,
56). Intracellular ATP is an essential molecular energy source,
while extracellular ATP, which can be released from vascular
endothelial cells (44, 71), damaged vessel walls (4), hypoxic
myocardium (15), and aggregating platelets (29), serves as a
potent vasoconstrictor (20, 24) and smooth muscle and endothelial cell mitogen (7, 19). ATP also can be released from
perivascular nerves as an excitatory neurotransmitter (6). The
contractile and mitogenic effects of extracellular ATP on
vascular smooth muscle cells (VSMC) are mediated by activation of a family of G protein-coupled receptors, P2 purino-
Cell preparation and culture. Human PASMC from healthy individuals were purchased from Cambrex and used at passages 4 – 6.
PASMC were cultured as described previously (61). Briefly, PASMC
were plated onto coverslips or petri dishes and incubated in a humidified atmosphere of 5% CO2 in air at 37°C in the smooth muscle
growth medium (SMGM; Cambrex). The SMGM was composed of
smooth muscle basal medium (SMBM) supplemented with 5% fetal
bovine serum (FBS), 0.5 ng/ml human epidermal growth factor, 2
Address for reprint requests and other correspondence: J. X.-J. Yuan,
Division of Pulmonary and Critical Care Medicine, Dept. of Medicine, #0725,
Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0725
(E-mail: [email protected]).
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.
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MATERIALS AND METHODS
0363-6143/04 $5.00 Copyright © 2004 the American Physiological Society
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Zhang, Shen, Carmelle V. Remillard, Ivana Fantozzi, and
Jason X.-J. Yuan. ATP-induced mitogenesis is mediated by cyclic
AMP response element-binding protein-enhanced TRPC4 expression
and activity in human pulmonary artery smooth muscle cells. Am J
Physiol Cell Physiol 287: C1192–C1201, 2004. First published June
30, 2004; doi:10.1152/ajpcell.00158.2004.—Extracellular ATP and
intracellular cyclic AMP response element-binding protein (CREB, a
transcription factor) promote cell proliferation in many cell types. The
canonical transient receptor potential (TRPC) channels, which putatively participate in forming store- and receptor-operated Ca2⫹ channels, have been implicated in the pulmonary vascular remodeling
processes. A link between extracellular ATP, CREB activation, and
TRPC4 channel expression and activity has not been shown in human
pulmonary artery smooth muscle cells (PASMC). Long-term (24 – 48
h) treatment of human PASMC with a low dose (100 ␮M) of ATP,
which did not trigger a transient rise in free cytosolic Ca2⫹ concentration ([Ca2⫹]i) when applied acutely to the cells, caused marked
increases in CREB phosphorylation and TRPC4 protein expression.
The time course indicated that the ATP-mediated CREB phosphorylation preceded TRPC4 upregulation, whereas transfection of a nonphosphorylatable CREB mutant abolished ATP-mediated TRPC4 expression. Furthermore, treatment of human PASMC with ATP also
enhanced the amplitude of capacitative Ca2⫹ entry (CCE) induced by
passive store depletion, whereas the small interfering RNA specifically targeting TRPC4 attenuated ATP-mediated increases in TRPC4
expression and CCE amplitude and inhibited ATP-induced PASMC
proliferation. These data suggest that low-dose ATP exerts part of its
mitogenic effect in human PASMC via CREB-mediated upregulation
of TRPC4 channel expression and activity and the subsequent increase in CCE and [Ca2⫹]i.
TRPC4 IS INVOLVED IN ATP-INDUCED CELL PROLIFERATION
AJP-Cell Physiol • VOL
nescence detection system (Amersham). In some experiments, the
protein kinase inhibitors N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and KT-5823 (KT)
(Sigma) were incubated along with the cells for 48 h before protein
extraction and Western blot analysis. Band intensity is reported in
arbitrary units (a.u.), which are a measure of band intensity relative to
␣-actin controls. The time courses of ATP-mediated CREB phosphorylation and TRPC4 upregulation were best fitted using the SigmaPlot
program.
Synthesis and transfection of siRNA. The 21-nucleotide siRNA
sequence targeting TRPC4 (sense: 5⬘-ACUCUUGGUUCAGAAAGGATT-3⬘; antisense: 5⬘-UCCUUUCUGAACCAAGAGUTT-3⬘) and
a scrambled TRPC4 sequence (sense: 5⬘-GUGUCGUAAGUUCAUCCGATT-3⬘; antisense: 5⬘-UCGGAUGAACUUACGACACTT-3⬘)
were synthesized, and these were purchased from Sequitur. The
human PASMC grown in SMGM to ⬃80% confluence were transfected with either siRNA (20 nM) using the Gene Porter 2 transfection
reagent kit (Gene Therapy Systems). After transfection, cells were
incubated at 37°C in serum-free culture medium (SMBM). Fifteen
hours after transfection, fresh growth medium was added and the cells
were left to recover at 37°C for 8 h before protein extraction. The
efficiency of siRNA transfection was determined using fluorescencelabeled siRNA; fluorescence was visible only in siRNA-transfected
cells.
Measurement of [Ca2⫹]i. PASMC were loaded with the membrane-permeable acetoxymethyl ester form of fura 2 (fura 2-AM; 3
␮M) for 30 min in the dark at room temperature (22–24°C). The fura
2-AM-loaded cells were then superfused with a standard bath solution
(see below for composition) for 20 min at 34°C to wash away
extracellular dye and to permit intracellular cleavage of fura 2-AM to
active fura 2 by esterases. The standard bath solution contained (in
mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10
glucose (pH 7.4 with 5 mol/l NaOH). Fura 2 fluorescence from the
cells and background fluorescence were collected at 32°C using Nikon
UV-Fluor objectives. The fluorescence signals emitted from the cells
were monitored continuously using an intracellular imaging fluorescence microscopy system and recorded on a personal computer for
later analysis. [Ca2⫹]i was calculated from fura 2 fluorescence emission excited at 340 and 380 nm (F340/F380) using the ratio method (21)
based on the following equation:
关Ca 2⫹兴i ⫽ Kd ⫻ 共Sf2/Sb2兲 ⫻ 共R ⫺ Rmin兲/共Rmax ⫺ R兲,
where Kd (225 nM) is the dissociation constant for Ca2⫹; Sf2 and Sb2
are emission fluorescence values at 380-nm excitation in the presence
of EGTA and Triton X-100, respectively; R is the measured fluorescence ratio; and Rmin and Rmax are minimal and maximal ratios,
respectively. In all experiments, multiple cells were imaged in a single
field, and one arbitrarily chosen peripheral cytosolic area from each
cell was spatially averaged.
Transfection of cells with the recombinant adenovirus. A recombinant adenoviral vector containing a CREB mutant (AdCREBM1) in
which the phosphorylation site at 133Ser was mutated to an alanine
was kindly provided by Dr. Anthony J. Zeleznik (University of
Pittsburgh, Pittsburgh, PA) (55). Human PASMC were transfected
using the AdCREBM1 vector as described by Tokunou et al. (63).
Briefly, confluent PASMC were washed twice with PBS and then
incubated with AdCREBM1 (added [AdCREBM1] is 10-fold the
number of cells in each culture dish) at room temperature in PBS.
After 2 h of incubation, cells were washed three times with PBS and
cultured in 0.5% FBS-SMBM for days before being used for experiments.
Statistical analysis. Data are expressed as means ⫾ SE. Statistical
analysis was performed using unpaired Student’s t-tests or ANOVA
as indicated. P ⬍ 0.05 was considered significant.
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ng/ml human fibroblast growth factor, and 5 ␮g/ml insulin. Cells were
subcultured or plated onto 25-mm coverslips using trypsin-EDTA
buffer when 70 –90% confluence was achieved. The morphology of
the cells was examined using an inverted phase-contrast microscope
attached to a digital camera.
Rat PASMC were used in some experiments to compare the
response to ATP with human PASMC. Rat PASMC were prepared
from the left and right branches of the main pulmonary artery and the
intrapulmonary arteries of male Sprague-Dawley rats (150 –200 g).
Briefly, the isolated pulmonary arteries were incubated for 20 min in
Hanks’ balanced salt solution containing 1.5 mg/ml collagenase
(Worthington Biochemical). Adventitia and endothelium were carefully removed after the incubation. The remaining smooth muscle was
then digested with 1.5 mg/ml collagenase and 0.5 mg/ml elastase
(Sigma) at 37°C. Approximately 45–50 min later, PASMC were
sedimented by centrifugation, resuspended in fresh medium, and
placed onto petri dishes or coverslips. The cells were cultured in
high-glucose (4.5 g/l) DMEM supplemented with 10% FBS, 2 mM
glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (BioFluids) and incubated in 5% CO2 at 37°C in a humidified atmosphere.
DNA synthesis determination and cell counting. [3H]Thymidine
incorporation was determined to evaluate DNA synthesis and cell
proliferation. Briefly, human PASMC were seeded in 24-well microplates at a density of ⬃2 ⫻ 104 cells/well, cultured in SMGM for 48 h,
and growth arrested in SMBM for 24 h. Treated cells were then
incubated in 0.2% FBS-SMGM with 1 ␮Ci of [3H]thymidine added to
the cells for at least 16 h. Incorporation of radioactivity into trichloroacetic acid-insoluble material was measured using a liquid scintillation counter. For ATP experiments, the growth-arrested cells were
treated with 100 ␮M ATP for 24 h before [3H]thymidine incorporation was measured. For small interfering RNA (siRNA) experiments,
cells were transfected with control siRNA with scrambled sequence
(si-Cont) and TRPC4-siRNA sequences for 0, 24, 48, or 72 h in the
continuous presence of 100 ␮M ATP before [3H]thymidine uptake
measurements. The dose-response curve of [3H]thymidine uptake in
response to ATP treatment was best fitted using the SigmaPlot
program.
For cell-counting experiments, previously growth-arrested (with
24-h SMBM) human PASMC were cultured in 0.2% FBS-SMGM
with ATP for 48 h. Cell viability after 48 h was determined using
0.45% trypan blue (Sigma). Cell number was determined using a
hemocytometer. The cell counts in the four 1-mm2 corner squares of
the hemocytometer were averaged to calculate the total number of
PASMC (1 ⫻ 104/ml) in the cell suspension.
Western blot analysis. Human PASMC were gently washed twice
in cold phosphate-buffered saline (PBS), scraped into lysis buffer [1%
Nonidet P-4 (Amaresco), 0.5% sodium deoxycholate, 0.1% SDS, 100
␮g/ml phenylmethylsulfonyl fluoride, and 30 ␮l/l aprotinin], and
incubated on ice for 30 min. Cell lysates were then sonicated and
centrifuged at 12,000 rpm for 10 min, and the insoluble fraction was
discarded. In some experiments, cell lysates were treated with the
peptide N-glycosidase F (20 U; New England Biolabs) overnight at
4°C. The protein concentration in the supernatant was determined
using a bicinchoninic acid protein assay with bovine serum albumin
serving as a standard. Ten- to 25-␮l aliquots of protein were mixed
and boiled in SDS-PAGE sample buffer for 5 min. The protein
samples separated on 10% SDS-PAGE were then transferred to
nitrocellulose membranes by electroblotting in a MINI Trans-Blot cell
transfer apparatus (Bio-Rad Laboratories) according to the manufacturer’s instruction. After incubation overnight at 4°C in a blocking
buffer (0.1% Tween 20 in PBS) containing 5% nonfat dry milk
powder, the membranes were incubated with polyclonal antibodies
against TRPC4 (Alomone Labs) and ␣-actin (Sigma). Finally, the
membranes were washed and incubated with anti-rabbit or anti-mouse
horseradish peroxidase-conjugated IgG for 90 min at room temperature. The bound antibody was detected with an enhanced chemilumi-
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TRPC4 IS INVOLVED IN ATP-INDUCED CELL PROLIFERATION
RESULTS
Fig. 1. Low doses of ATP stimulate cell proliferation without inducing Ca2⫹
transients in pulmonary artery smooth muscle cells (PASMC). A: cytosolic free
Ca2⫹ concentration ([Ca2⫹]i) measured in representative human (hPASMC) or
rat PASMC (rPASMC; inset) superfused with solutions including 0.1, 1, or 5
mM ATP. B: summarized data (means ⫾ SE) showing [Ca2⫹]i measurements
from hPASMC (n ⫽ 36) or rPASMC (inset; n ⫽ 68) exposed to cumulative
doses of extracellular ATP as in A. C: [3H]thymidine uptake in hPASMC
before (0 ATP) and after 24-h treatment with 1, 10, or 100 ␮M ATP (n ⫽ 8).
Inset: number of hPASMC counted before or after treatment with 100 ␮M
ATP for 24 h (n ⫽ 8). *P ⬍ 0.05 vs. 0 ATP or control. **P ⬍ 0.01 vs. 0 ATP
or control (Cont). ***P ⬍ 0.001 vs. 0 ATP or control.
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Extracellular ATP causes cell proliferation in a dose-dependent manner. In human PASMC, extracellular application of
ATP acutely induced [Ca2⫹]i increases in a dose-dependent
manner. Low doses (0.1–1 mM) of ATP did not affect resting
[Ca2⫹]i (⬃180 nM in this cell, average 105 ⫾ 6 nM; n ⫽ 32),
while a high dose (5 mM) of ATP caused a significant, albeit
transient, increase in [Ca2⫹]i (Fig. 1, A and B) from a resting
value of 147 ⫾ 2 nM to a peak value of 1,030 ⫾ 13 nM (n ⫽
36; peak value ⬃1,230 nM in this cell). In contrast to human
PASMC, rat PASMC exhibited much higher sensitivity to
ATP. Extracellular application of 100 ␮M and 1 mM ATP
significantly increased [Ca2⫹]i (Fig. 1, A and B, insets). These
results indicate that in human PASMC, but not in rat PASMC,
100 ␮M ATP is not sufficient to cause a transient increase in
global [Ca2⫹]i, which serves as an important mechanism or
signal transduction pathway for inducing PASMC contraction
(24). Potential underlying causes of this species-specific difference in the [Ca2⫹]i response after ATP treatment are discussed below.
By measuring [3H]thymidine uptake in human PASMC, we
confirmed the proliferative effect of low doses of ATP. After
24-h treatment with ATP, [3H]thymidine incorporation had
increased 1.2-fold (P ⬍ 0.05), 1.4-fold (P ⬍ 0.001), and
1.6-fold (P ⬍ 0.001) from control (day 0) levels in the presence
of 1, 10, and 100 ␮M ATP, respectively (Fig. 1C). The number
of cells (Fig. 1C, inset) also increased in the presence of 100
␮M ATP (1.7-fold; P ⬍ 0.01) under similar conditions. These
data show that, despite its lack of an acute effect on [Ca2]i
levels, long-term treatment of human PASMC with 100 ␮M
ATP is sufficient to induce PASMC proliferation. We therefore
focused on this low dose of ATP (100 ␮M) and attempted to
elucidate the mechanism by which it causes cell proliferation.
ATP upregulates protein expression of TRPC4 channels and
enhances CCE in PASMC. Having previously established that
TRPC channels and CCE are important in vascular smooth
muscle cell proliferation (13, 61, 72), we first verified whether
ATP could also affect CCE by regulating TRPC4 channel
expression. Western blot analysis (Fig. 2A) showed that 100
␮M ATP significantly increased TRPC4 expression over the
span of 48 h, from 88 ⫾ 2 a.u. at time 0 (just before ATP
exposure) to 118 ⫾ 1 and 136 ⫾ 1 a.u. after 24 or 48 h of
treatment, respectively (P ⬍ 0.001 vs. ATP 0 h). Because of its
putative role in underlying SOC and/or ROC formation, enhanced TRPC4 protein expression may augment Ca2⫹ entry
through SOC and/or ROC (13).
In addition to the augmenting effect on TRPC4 expression,
we then determined the effect of low-dose ATP on SOCmediated CCE. As predicted, the ATP-mediated upregulation
of TRPC4 was accompanied by a marked enhancement of
cyclopiazonic acid (CPA; 10 ␮M)-induced CCE in PASMC,
from 381 ⫾ 16 to 535 ⫾ 29 nM [Ca2⫹]i (P ⬍ 0.001 vs. control)
(Fig. 2, Ba and Bc). In addition to the increased amplitude of
CCE, the [Ca2⫹]i in the sarcoplasmic reticulum ([Ca2⫹]SR) was
also increased in PASMC treated with 100 ␮M ATP for 48 h
([Ca2⫹]i rise due to Ca2⫹ mobilization was increased from
121 ⫾ 4 to 164 ⫾ 9 nM), while the change in resting [Ca2⫹]i
was not significant (Fig. 2, Ba and Bb).
We also considered the possibility that [Ca2⫹]i might be
increased because of an alternate mechanism after CPA application in the presence of ATP and reintroduced Ca2⫹. More
specifically, the combination of store depletion and loss of
extracellular Ca2⫹ may elevate intracellular Na⫹ levels via
TRPC4 and result in diminished Na⫹/Ca2⫹ exchange activity,
as was suggested previously regarding TRPC3 channels in
carbachol-stimulated human embryonic kidney (HEK-293)
cells (53). Reintroduction of extracellular Ca2⫹ could cause the
exchanger to operate in a reverse mode, thereby causing Ca2⫹
influx rather than its “normal” Ca2⫹ extrusion. ATP-treated
human PASMC exposed to 10 ␮M KB-R9743, a Na⫹/Ca2⫹
exchange inhibitor, still exhibited a significant increase in both
Ca2⫹ release [128 ⫾ 7 nM control (n ⫽ 53), 246 ⫾ 25 nM
ATP (n ⫽ 66); P ⬍ 0.001] and CCE levels (145 ⫾ 10 nM
control, 291 ⫾ 37 nM ATP; P ⬍ 0.001), with no change in
resting Ca2⫹ levels (108 ⫾ 4 nM control, 109 ⫾ 7 nM ATP)
(Fig. 2C). These data indicate that ATP-induced CCE is me-
TRPC4 IS INVOLVED IN ATP-INDUCED CELL PROLIFERATION
diated primarily by SOC channels with little involvement of
the reverse-mode Na⫹/Ca2⫹ exchanger.
Downregulation of TRPC4 mRNA using siRNA inhibits CCE
and cell proliferation in PASMC. To confirm that the low-dose
ATP-induced TRPC4 gene upregulation (Fig. 2A) is responsible for enhanced CCE (Fig. 2B), a critical question to address
is whether TRPC4 is involved in CCE in human PASMC. The
molecular identity of SOC responsible for CCE remains un-
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Fig. 2. ATP upregulates canonical transient receptor potential TRPC4 expression and enhances capacitative Ca2⫹ entry (CCE) in human PASMC. A:
Western blot analysis (top) showing the time-dependent increase in TRPC4
protein expression induced by 100 ␮M ATP over a 48-h period; bottom:
summary of the time course of ATP-induced TRPC4 expression (n ⫽ 3
experiments). ***P ⬍ 0.001 vs. ATP 0 h. B: CCE induced by cyclopiazonic
acid (CPA; 10 ␮M) shown as a function of time in control cells and cells
treated with 100 ␮M ATP for 48 h (ATP) (a). Summarized data show resting
[Ca2⫹]i (b) as well as increases in [Ca2⫹]i via Ca2⫹ release and CCE (c) in cells
treated with (ATP) and without (control) ATP for 48 h (n ⫽ 32 cells for each
group). C: time course of CPA-induced CCE in human PASMC before (n ⫽
66) or after (n ⫽ 53) treatment with 100 ␮M ATP in the presence of 10 ␮M
KB-R9743, a Na⫹/Ca2⫹ exchanger blocker (a). Summarized data show resting
[Ca2⫹]i (b) as well as increases in [Ca2⫹]i via Ca2⫹ release and CCE (c) in cells
treated with (ATP ⫹ KB-R9743; n ⫽ 53) and without (control ⫹ KB-R9743;
n ⫽ 66) ATP for 48 h. **P ⬍ 0.01 vs. control. ***P ⬍ 0.001 vs. control. 0Ca,
Ca2⫹-free solution.
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clear. TRPC4 is a TRPC isoform that can form heterotetramers
with TRPC1 and TRPC5 and is involved in forming native
SOC in many cell types (39, 58). Therefore, we tested whether
TRPC4 in human PASMC is involved in forming Ca2⫹ channels that are activated by passive store depletion using CPA.
Using a previously identified siRNA specifically targeting
TRPC4 (si-TRPC4) (13) (Fig. 3A), we were able to inhibit
ATP-mediated upregulation of TRPC4 protein expression by
77% (Fig. 3B). The control siRNA with scrambled sequence
(si-Cont) had no effect on TRPC4 expression. Furthermore, in
PASMC treated with 100 ␮M ATP for 48 h, inhibition of
TRPC4 expression with si-TRPC4 markedly attenuated the
CPA-mediated CCE (Fig. 3C), bringing the amplitude of CCE
down to 137 ⫾ 7 nM from a control (i.e., si-Cont) value of
447 ⫾ 17 nM. These data suggest that TRPC4 is involved, at
least in human PASMC, in forming heterotetrameric store
depletion-activated SOC channels. In parallel, si-TRPC4
caused a significant decrease in ATP-induced PASMC proliferation (Fig. 3D) over a 72-h period compared with cells
treated with si-Cont. These results suggest that ATP-mediated
upregulation of TRPC4 protein expression contributes to the
enhancement of CCE and proliferation in human PASMC.
ATP-induced CREB activation upregulates TRPC4 expression in human PASMC. CREB has been identified as an
important signal transduction element in agonist-mediated
VSMC proliferation (32, 63). Consistent with its mitogenic
effect on human PASMC, low-dose ATP (100 ␮M) significantly increased phosphorylation of CREB (Fig. 4A). Blockade
of P2 receptors with suramin (100 ␮M) markedly attenuated
ATP-mediated CREB phosphorylation (Fig. 4A), suggesting
that ATP-mediated CREB phosphorylation is due to activation
of a suramin-sensitive P2X or P2Y receptor.
ATP induced CREB phosphorylation in a time-dependent
manner. Short-term (1–2 h) treatment of quiescent human
PASMC with 100 ␮M ATP had little effect on the level of
phosphorylated CREB (pCREB) (Fig. 4Ba). However, pCREB
levels were significantly elevated after 4 h of treatment; the
increase in pCREB levels maximized at 8 h and persisted after
24 h of treatment (Fig. 4Ba). Compared with the time course of
the ATP-mediated effect on TRPC4 protein expression, these
results indicate that ATP-induced CREB phosphorylation precedes TRPC4 upregulation (Fig. 4Bb) and that phosphorylated
CREB may be involved in enhancing TRPC4 gene expression.
Indeed, transfection of a nonphosphorylatable CREB mutant
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TRPC4 IS INVOLVED IN ATP-INDUCED CELL PROLIFERATION
(AdCREB-M1) using an adenoviral vector to human PASMC
precluded ATP-mediated increase in TRPC4 expression (Fig.
4C). This strongly suggests that CREB activation is required
for ATP-induced TRPC4 expression to occur.
Upregulated TRPC4 expression by ATP involves protein
kinase activation. As shown in Fig. 4A, the ATP-mediated
increase in CREB phosphorylation was due mainly to activa-
DISCUSSION
Pulmonary vascular remodeling due to excessive smooth
muscle and endothelial proliferation is a major cause of elevated PVR and pulmonary arterial pressure (PAP) in patients
with pulmonary hypertension (57). Upregulation of TRPC
channels is an important mediator of vascular smooth muscle
and endothelial proliferation (13, 21, 61, 72). A potent mitogen, extracellular ATP, also plays an important role in the
development of pulmonary vascular wall remodeling (19). Our
results suggest that the increased PASMC proliferation induced
by low-dose ATP is due, at least in part, to increased TRPC4
channel expression and activity via P2 receptor stimulation and
increased CREB phosphorylation. Although the data do not
fully establish a causal relationship between CCE via TRPC4encoded SOC and PASMC proliferation, the data do provide
evidence that TRPC4 and SOC are involved in PASMC pro-
Fig. 3. TRPC4-specific small interfering RNA (siRNA-TRPC4) inhibits ATPinduced increases in TRPC4 expression and CCE and attenuated ATP-induced
PASMC proliferation. A: phase-contrast and FITC-green fluorescent protein
(GFP) fluorescence images showing the incorporation of siRNA specifically
targeting TRPC4 (si-TRPC4) into human PASMC. Cells that were transfected
using siRNA are green, as shown in overlay images. B: Western blot analysis
showing the effect of control siRNA with scrambled sequence (si-Cont) and
si-TRPC4 on TRPC4 expression in human PASMC treated with 100 ␮M ATP
for 48 h (n ⫽ 3 experiments). **P ⬍ 0.01 vs. si-Cont. C: representative record
(left) showing CPA-induced changes in [Ca2⫹]i in human PASMC after 48-h
treatment with 100 ␮M ATP in the presence of si-Cont (gray trace) or
si-TRPC4 (black trace). Bar graphs show summarized data (n ⫽ 30 –31 cells)
for resting [Ca2⫹]i (middle) and increases in [Ca2⫹]i due to CPA-mediated
Ca2⫹ release and CCE in cells treated with 100 ␮M ATP for 48 h in the
presence of si-Cont (gray bars) or si-TRPC4 (black bars). ***P ⬍ 0.001 vs.
si-Cont. D: PASMC proliferation, quantified as [3H]thymidine uptake, is
plotted as a function of time of ATP (100 ␮M) treatment in cells treated with
si-Cont (gray circles; n ⫽ 7–9 experiments) or si-TRPC4 (black circles; n ⫽
6 –7 experiments). **P ⬍ 0.01 vs. day 0. The time course curves for si-Contand si-TRPC4-treated cells are significantly different (P ⬍ 0.01).
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tion of suramin-sensitive P2 receptors. At this stage, we cannot
ascertain whether CREB phosphorylation occurs because of
stimulation of P2x or P2y receptors. Suramin can block both P2x
(42) receptors, which form ligand-gated channels, and P2y
receptors (65), which belong to the superfamily of G proteincoupled receptors (GPCR) (7, 65). GPCR are coupled to many
signaling pathways, including those involved in protein kinasemediated phosphorylation. CREB phosphorylation at 133Ser by
protein kinase A (PKA) in response to cAMP and other kinases
is sufficient to induce the transcription and expression of
several genes. PKA in particular acts as a central processing
hub not only in mediating but also in transmitting the effects of
cAMP to different effector proteins, such as PKC, PKB, and
mitogen-activated protein kinase (MAPK) (7, 50). Protein
kinases may therefore be involved in the signal transduction
pathway that occurs between plasmalemmal receptor binding
by ATP and CREB phosphorylation before TRPC4 gene transcription.
We verified that PKA and PKG might be involved in the
enhanced TRPC4 transcription and expression induced by 100
␮M ATP. Inhibition of PKA or PKG with 10 ␮M H-89 or 2
␮M KT-5823 (48-h treatments) significantly attenuated ATPinduced TRPC4 upregulation (Fig. 5). Taken together, these
results suggest that ATP-mediated TRPC4 upregulation involves PKA- or PKG-induced phosphorylation of CREB.
TRPC4 IS INVOLVED IN ATP-INDUCED CELL PROLIFERATION
C1197
Fig. 4. ATP upregulates TRPC4 expression by inducing cAMP response
element-binding protein (CREB) phosphorylation. A: Western blot analysis
(left) of phosphorylated CREB (pCREB) in human PASMC treated with (ATP)
or without (Cont) 100 ␮M ATP for 8 and 12 h in the absence (⫺) or presence
(⫹) of 100 ␮M suramin. Summarized data averaged from 3 experiments are
shown at right. ***P ⬍ 0.001 vs. Cont (open bar). B: time course of
ATP-induced pCREB expression. Western blot analysis of pCREB and ␣-actin
levels (a) in cells treated with (ATP) and without (Cont) 100 ␮M ATP for 1,
2, 4, 8, 12, 24, and 48 h. Summarized data (a, bottom) from 3 experiments
indicate a transient increase in pCREB levels between 4 and 24 h of ATP
treatment. The time courses of ATP-mediated increases in pCREB (closed
circles) and TRPC4 (open circles; same data shown in Fig. 2A) are compared
in b. C: ATP-induced TRPC4 upregulation is abolished by a nonphosphorylatable CREB mutant. Western blot analysis of TRPC4 and ␣-actin levels (left)
in human PASMC infected with an empty vector (C) or with an adenoviral
vector containing the nonphosphorylatable CREB-M1 mutant (Ad-C) before
(Cont) and after (ATP) 48 h of treatment with 100 ␮M ATP. Summarized data
from 4 experiments are shown at right. *P ⬍ 0.05 vs. cells not treated with
ATP. **P ⬍ 0.01 vs. empty vector-infected cells treated with ATP.
AJP-Cell Physiol • VOL
liferation and that ATP-mediated upregulation of TRPC4 and
increase in CCE amplitude may be one of the mechanisms
involved in the mitogenic effect of ATP.
Effect of ATP on [Ca2⫹]i and CREB phosphorylation. In
human PASMC, acute application of low doses (0.1–1 mM) of
ATP did not evoke transient increases in [Ca2⫹]i (Fig. 1), while
high-dose ATP caused a large, transient increase in [Ca2⫹]i.
The former observation contrasts with reports showing that
ATP at concentrations as low as 100 ␮M can cause cyclical
Ca2⫹ oscillations in rat PASMC (present study), human coronary artery SMC, murine colonic SMC, and vascular endothelial cells (28, 31) mainly because of Ca2⫹ release from inositol1,4,5-trisphosphate (IP3)-sensitive sarcoplasmic reticulum
(SR) stores. Therefore, the difference in Ca2⫹ reactivity to
low-dose ATP we observed may be attributed to the different
expression levels of sarcolemmal purinoceptors and the different sensitivities of IP3 receptors in the SR membrane in human
PASMC relative to the other preparations.
In spite of the inability to induce transient increases in
[Ca2⫹]i, long-term treatment with the low dose of ATP markedly upregulated TRPC4 expression (Fig. 2A), enhanced CCE
amplitude (Fig. 2B), and increased proliferation (Fig. 1C) in
human PASMC. Furthermore, ATP-mediated CREB phosphorylation preceded the ATP-induced increases in TRPC4
expression (Fig. 4Bb). Inhibition of CREB phosphorylation by
overexpressing a nonphosphorylatable CREB mutant in human
PASMC abolished ATP-mediated upregulation of TRPC4 and
significantly inhibited ATP-mediated cell proliferation (data
not shown). Taken together, these results suggest that treatment
of human PASMC with ATP, at concentrations insufficient to
cause an acute increase in [Ca2⫹]i, can phosphorylate CREB,
upregulate TRPC4 expression, enhance CCE, and stimulate
proliferation (Fig. 6).
Activation or phosphorylation of CREB is often triggered by
a transient increase in [Ca2⫹]i in many cell types (8, 23, 67).
However, our results demonstrate that the ATP-induced increase in pCREB levels did not coincide with transient increases in resting [Ca2⫹]i at that time. This suggests that
low-dose ATP-induced CREB phosphorylation may initially
occur via a Ca2⫹-independent mechanism, contrary to the
mechanism with a high dose of ATP, which activates CREB by
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Fig. 5. Inhibition of protein kinase A (PKA) and PKG abolishes ATP-induced
TRPC4 upregulation. A: Western blot analysis of TRPC4 and ␣-actin levels in
human PASMC treated with 100 ␮M ATP for 48 h in the absence (Cont) and
presence of 10 ␮M N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89; a PKA inhibitor) or 2 ␮M KT-5823 (KT, a
PKG inhibitor). B: summarized data from 3 experiments showing the inhibitory effect of H-89 and KT-5823 on ATP-mediated TRPC4 expression.
***P ⬍ 0.001 vs. ATP control (open bar).
C1198
TRPC4 IS INVOLVED IN ATP-INDUCED CELL PROLIFERATION
inducing a transient increase in [Ca2⫹]i via Ca2⫹ release and
influx (8, 67). On the other hand, the change in [Ca2⫹]i induced
by low-dose ATP may occur at a more local level that does not
influence global Ca2⫹ levels. Lipp et al. (34) described the role
of IP3 receptor-mediated Ca2⫹ puffs in the regulation of
perinuclear Ca2⫹, which may then regulate key nuclear functions such as gene transcription. A similar case may also be
made for ryanodine receptor-mediated Ca2⫹ sparks, which
have been described in rat PASMC (47) and may be involved
in Ca2⫹ cross signaling between IP3 and ryanodine receptors
(73). Therefore, without more conclusive evidence, we cannot
say with certainty that ATP-induced CREB activation occurs
via a Ca2⫹-independent mechanism.
Mechanisms involved in ATP-mediated CREB phosphorylation and TRPC4 upregulation. Thrombin-induced aortic SMC
proliferation and hypertrophy are mediated by CREB-dependent gene transcription of the c-fos gene (63). Inhibition
of CREB phosphorylation attenuates neointimal proliferAJP-Cell Physiol • VOL
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Fig. 6. Schematic diagram depicting the potential mechanism by which
ATP promotes PASMC proliferation and, potentially, pulmonary hypertension. Low-level extracellular ATP activates a P2 receptor (P2x-R/P2Y-R) on
the plasma membrane of PASMC. G protein-coupled P2y receptors activate
PKA and PKG to stimulate phosphorylation and activation of CREB
(pCREB). pCREB stimulates TRPC4 expression, enhances capacitative
Ca2⫹ entry (CCE) by increasing store-operated Ca2⫹ (SOC) channel
activity, and raises [Ca2⫹]i. Increased TRPC4 may also enhance Ca2⫹ entry
through receptor-operated Ca2⫹ (ROC) channels. High-level extracellular
ATP, in addition to activating PKA/PKG, activates PKC via diacylglycerol
(DAG) and stimulates Ca2⫹ release by producing inositol-1,4,5-trisphosphate (IP3). The PKC-pCREB-mediated upregulation of TRPC4 and/or
other TRPC isoforms (e.g., TRPC3) and store depletion-mediated activation of SOC concurrently enhance CCE and increase [Ca2⫹]i. The Ca2⫹mediated positive feedback effect on activating pCREB through Ca2⫹/
calmodulin-dependent kinases (CaMK) II and IV further enhances the
augmenting effect of ATP on TRPC4 and [Ca2⫹]i. The increased [Ca2⫹]i
propels the quiescent cells into the cell cycle, and they undergo mitosis,
promoting PASMC proliferation. Excessive PASMC growth may eventually cause pulmonary vascular medial hypertrophy and contribute to the
development of pulmonary hypertension.
ation in rat carotid artery by downregulating Bcl-2, an
anti-apoptotic protein (64). Therefore, CREB is a transcription factor that plays an important role in the vascular
remodeling process.
Various kinases are able to phosphorylate CREB, including
PKA (22), PKG, PKC (69), Ca2⫹/calmodulin-dependent protein kinases (CaM) (8, 59), Akt/protein kinase B (45), MAPK
(3), and Ras-dependent protein kinase (52). The phosphorylation of CREB on 133Ser enables CREB to bind to the cAMP
response element (CRE) and to modulate transcription of the
genes (e.g., bcl-2, c-fos) whose promoters contain a CRE
binding sequence. In the present study, we have demonstrated
that ATP-induced TRPC4 upregulation was attenuated when
PKA and PKG were inhibited (Fig. 5), indicating that CREB
was activated or phosphorylated by PKA/PKG-dependent
pathways when human PASMC were treated with low-dose
ATP for 4 –24 h. The initial PKA/PKG-mediated CREB phosphorylation appeared not to be dependent on transient increases
in [Ca2⫹]i, because acute application of 100 ␮M ATP had little
effect on [Ca2⫹]i in human PASMC (Fig. 1A).
The phosphorylated CREB, by binding to the CRE, can
upregulate many proteins and factors that regulate cell
proliferation and survival (3, 32, 48, 49, 63). Although our
observations provide evidence that ATP-mediated upregulation of TRPC4 is related to phosphorylation of CREB, it is
still unclear how CREB modulates TRPC4 gene transcription. In other words, we still do not know whether ATPmediated TRPC4 upregulation is due directly to binding of
CREB to CRE in the TRPC4 gene promoter or indirectly to
a CREB-induced intermediate (e.g., c-Fos or c-Jun) that
subsequently upregulates or facilitates TRPC4 gene transcription.
In contrast to our finding that CREB phosphorylation preceded TRPC4 upregulation in human PASMC treated chronically with ATP, a recent study (46) suggested that enhanced
SOC entry (but not Ca2⫹ entry through L-type voltage-dependent Ca2⫹ channels) led to CREB phosphorylation and c-fos
transcription in rat and human cerebral arteries and rat aorta. In
light of these new findings, it is apparent that upregulation of
TRPC4 can initially be caused by Ca2⫹-dependent and -independent mechanisms. The subsequent enhancement of CCE
and rises in [Ca2⫹]i may further upregulate TRPC4 gene
expression, forming a positive feedback mechanism to ensure
the increased [Ca2⫹]i required for cell cycle progression and
PASMC proliferation. These observations also suggest that the
physiological role of TRPC4 in the regulation of vascular
contractility and VSMC proliferation is complex.
Role of TRPC channels and CCE in PASMC proliferation.
Voltage-independent SOC channels are sensitive to changes in
SR Ca2⫹ content and are activated upon SR depletion. TRPC
gene products have been suggested as potential underlying
components of native SOC in the cardiovascular system (16,
39, 43). TRPC1, TRPC4, and TRPC6 figure prominently as
components of native SOC in pulmonary VSMC and endothelial cells and are involved in regulating vascular tone, microvascular permeability, and cell proliferation (5, 13, 17, 21, 33,
40, 61, 62, 68, 70, 72).
In the present study, we have shown that ATP significantly
upregulated TRPC4 gene expression (Fig. 2A). TRPC3 was
upregulated only at high ATP concentrations, while TRPC6
TRPC4 IS INVOLVED IN ATP-INDUCED CELL PROLIFERATION
AJP-Cell Physiol • VOL
ACKNOWLEDGMENTS
We kindly thank Dr. P. A. Insel for constructive discussion about the study
design and Ann Nicholson for technical assistance.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-66012, HL-54043, HL-64945, HL-69758, and HL-66941.
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expression was unaffected by ATP treatments (data not
shown). In parallel with the increased expression of TRPC4,
CPA-induced CCE was also upregulated in PASMC treated
with ATP (Fig. 2B). Both the ATP-induced TRPC4 upregulation and CCE enhancement were abolished by TRPC4-targeted
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A rise in [Ca2⫹]i is essential for cell proliferation (1, 35);
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Physiological and pathophysiological implications. In light
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extracellular ATP concentration may play a significant role in
PASMC proliferation. Our findings may be relevant in unraveling the pathophysiological basis of severe pulmonary hypertension.
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