From the Department of Pharmacology, University of Vermont, Burlington, Vermont 05405
Received for publication, December 26, 2000, and in revised form, February 7, 2001
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ABSTRACT |
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The transcription factor NFAT (nuclear
factor of activated T-cells) plays a central role in mediating
Ca2+-dependent gene transcription in a
variety of cell types. Sustained increases in intracellular calcium
concentration ([Ca2+]i) are
presumed to be required for NFAT dephosphorylation by the
Ca2+/calmodulin-dependent protein calcineurin and
its subsequent nuclear translocation. Here, we provide the first
identification and characterization of NFAT in native smooth muscle,
showing that NFAT4 is the predominant isoform detected by reverse
transcriptase-polymerase chain reaction and Western blot analysis. PDGF
induces NFAT4 translocation in smooth muscle, leading to an increase in
NFAT transcriptional activity. NFAT4 activation by PDGF depends on
Ca2+ entry through voltage-dependent
Ca2+ channels, because its nuclear accumulation is
prevented by the Ca2+ channel blocker nisoldipine and the
K+ channel opener pinacidil. Interestingly, elevation of
[Ca2+]i by membrane
depolarization or ionomycin treatment are not effective stimuli for
NFAT4 nuclear accumulation, indicating that Ca2+ influx is
necessary but not sufficient for NFAT4 activation. In contrast,
membrane depolarization readily activates the
Ca2+-dependent transcription factor CREB
(cAMP-responsive element-binding protein). The calcineurin blockers CsA
and FK506 also prevented the PDGF-induced NFAT4 nuclear localization.
These results indicate that both the nature of the calcium
signal and PDGF-induced modulation of nuclear import-export of NFAT are
critical for NFAT4 activation in this tissue.
Calcium ions (Ca2+) play a central role in the
physiology of all cells. In arterial smooth muscle, for example, global
changes in intracellular calcium concentration
([Ca2+]i)1 control
tonic contractions that lead to changes in arterial diameter (1, 2). In
the gut, the phasic behavior of ileal smooth muscle cells that is
essential for the normal function of this tissue is determined by
recurrent action potentials that are mediated by
voltage-dependent Ca2+ channels (VDCC). These
action potentials deliver surges of
Ca2+ to the interior of the cell in the form of repetitive
Ca2+ spikes (3). Ca2+ signal modulation in
smooth muscle takes on additional forms, including localized transient
releases of Ca2+ through ryanodine receptors in the
sarcoplasmic reticulum, known as Ca2+ sparks, as well as
propagating Ca2+ waves that traverse the length of the cell
and display distinctive frequency and amplitude properties (2, 4,
5).
It is becoming increasingly clear that
[Ca2+]i also serves an important
second messenger role in the regulation of gene expression in smooth
muscle. We have recently demonstrated that activation of the
cAMP-responsive element-binding protein (CREB) and subsequent
c-fos expression can be induced by depolarization in native
arterial smooth muscle, a process that is mediated by calmodulin-dependent kinase (CaMK) and is dependent on
Ca2+ influx through VDCC (6). The
Ca2+-sensitive transcription factor, NFAT (nuclear factor
of activated T-cells) may play a role in smooth muscle, as suggested by
results from the A7r5 aortic smooth muscle cell line (7), but this possibility has remained unexplored in native tissue.
NFAT represents a family of Ca2+-dependent
transcription factors comprising four well-characterized members,
designated NFAT1 (NFATc2/p), NFAT2 (NFATc1/c), NFAT3 (NFATc4), and
NFAT4 (NFATc3/x) (8). Although originally thought to be largely
restricted to cells of the immune system, NFAT has since been shown to
play a role in other cell types, including cardiac and skeletal
myocytes and neurons. In non-immune cells, NFAT has been shown to
regulate heart valve development, control the differentiation of
skeletal myocytes into slow- or fast-twitch fiber types, and contribute to the development of hypertrophy in cardiac and skeletal myocytes (9-13). It has also been suggested that NFAT plays a role in long term
memory in neurons (14).
Most NFAT isoforms are constitutively expressed and exist as
transcriptionally inactive, cytosolic phosphoproteins. Stimuli that
activate NFAT do so, in part, by increasing
[Ca2+]i, and NFAT activation
appears to be strictly dependent on this increase. Upon elevation of
[Ca2+]i, the
Ca2+/calmodulin-dependent phosphatase
calcineurin dephosphorylates NFAT, leading to the unmasking of nuclear
localization signals and the translocation of NFAT to the nucleus
(15-17). Calcineurin-dependent dephosphorylation of NFAT
has been shown to be inhibited by the immunosuppressant drugs
cyclosporin A (CsA) and FK506 (16). The subcellular localization of
NFAT is dynamically dependent on a balance between the cytosolic
phosphatase activity of calcineurin and the activity of incompletely
characterized nuclear kinases, which promote export of NFAT from the
nucleus. Both import and export processes can be regulated in an
isoform-selective manner (18-22).
Temporal and spatial features of the Ca2+ signal are
important determinants of NFAT activation. Stimuli that provoke a
sustained moderate elevation in global
[Ca2+]i have consistently been
shown to effectively promote NFAT nuclear translocation in
non-excitable cells. In these cell types, the Ca2+
requirement may be provided by release from intracellular stores coupled with influx of extracellular Ca2+ through
capacitative Ca2+ entry pathways (23). In hippocampal
neurons, NFAT activation is mediated by Ca2+ influx through
L-type Ca2+ channels, but not NMDA receptors, suggesting
the possibility of privileged communication between specific
Ca2+ entry pathways and NFAT activation (14). NFAT activity
can also be induced in neurons by a brief depolarizing stimulus,
indicating that the Ca2+ signaling component of NFAT
activation may display cell-type specific requirements.
Ca2+ oscillations, which can occur in both excitable and
non-excitable cells, can also increase the efficiency and specificity
of gene expression by selectively activating certain transcription
factors, including NFAT (24, 25).
Platelet-derived growth factor (PDGF) plays an important role in a
number of pathophysiological processes. It has been shown to contribute
to the healing of gastrointestinal ulcers, stimulate colonic tumor
growth by promoting angiogenesis (26, 27), and has been suggested to
play a role in the pathogenesis of idiopathic inflammatory bowel
disease (28). PDGF is also a potent smooth muscle cell mitogen that has
been shown to activate NFAT promoter-reporter constructs in cultured
smooth muscle cells (7). Although NFAT has recently received
considerable attention in the context of cardiac and skeletal muscle,
the existence of NFAT in native smooth muscle has not been
demonstrated. Likewise, the activation requirements for NFAT in this
tissue have not been described. We show here that NFAT4 is the
predominant isoform expressed in native smooth muscle. We provide
evidence that PDGF induces a nuclear translocation of NFAT4 that is
dependent on the influx of extracellular Ca2+ through
L-type VDCCs. We further show that Ca2+ influx alone is not
sufficient to induce nuclear accumulation of NFAT4 and demonstrate
differential effects of distinct Ca2+ signals on the
activation of the transcription factors NFAT4 and CREB.
Tissue Preparation--
Adult female CD-1 mice (20-25 g,
Charles River Laboratories, Canada) were euthanized by peritoneal
injection of pentobarbital solution (200 mg/kg). For isolation of ileal
strips, a segment of ileum was detached from mesenterium and the outer
longitudinal and several layers of the inner circular smooth muscle
were removed. Ileal smooth muscle sheets were cut into sections for use
in immunofluorescence experiments. For RT-PCR analysis of NFAT
expression in isolated cells, smooth muscle cells were enzymatically
dispersed as described by Gomez and Swärd (29). Briefly, the
sheets were cut into pieces and incubated for 10 min at 35 °C in 2 ml of the dispersion medium (in mM: 110 NaCl, 5 KCl, 0.16 CaCl2, 2 MgCl2, 10 HEPES, 10 NaHCO3, 0.5 KH2PO4, 0.5 NaH2PO4, 10 glucose, 0.49 EDTA, 10 taurine, pH
7.0) containing 1.5 mg ml Immunofluorescence--
Ileal sheets (1-3 mm wide, 5-8 mm
long) were treated at room temperature with various agents and times as
specified in the text and then mounted onto glass slides. After
air-drying for 5 min, sheets were fixed with 3.7% formaldehyde in
phosphate-buffered saline (PBS, pH 7.4) for 15 min, permeabilized with
0.1% Triton X-100 in PBS for 10 min and blocked for 1 h with 2%
bovine serum albumin in PBS. Primary antibody dilutions in 2% bovine
serum albumin/PBS, rabbit anti-NFAT4/c3 (Santa Cruz Biotechnologies) (1:250 dilution) and rabbit anti-P-CREB (1:250 dilution), were applied
overnight at 4 °C. Secondary antibody, Cy3-anti-rabbit IgG or
Cy5-anti-rabbit IgG (Jackson ImmunoResearch Laboratories) (1:500
dilution) was applied for 1 h at 25 °C. The fluorescent nucleic
acid dye YOYO-1 (1:10000 dilution) was used for identification of
nuclei. After washing, the sheets were mounted (Aqua Polymount mounting
medium, Polysciences) and examined at × 40 magnification using a
Bio-Rad 1000 laser scanning confocal microscope. Red fluorescence of
NFAT4/P-CREB was measured at an excitation wavelength of 550 and/or 650 nm, and emission was measured at 570 and/or 670 nm, for Cy3 and Cy5
respectively. Specificity of immune staining was confirmed by the
absence of fluorescence in sheets incubated with primary or secondary
antibodies alone. For scoring of NFAT4- and P-CREB-positive nuclei,
multiple fields for each smooth muscle strip were imaged and counted by
two independent observers under double-blind conditions. For
quantification, a cell was considered positive if co-localization
(yellow) was observed in the nucleus, whereas a cell was considered
negative if no co-localization (green) was visualized.
RT-PCR for NFAT Isoforms--
Total RNA was prepared from tissue
or isolated ileal smooth muscle cells using the Trizol-LS Reagent (Life
Technologies, Inc.) and the Micro-FastTrack 2.0 RNA Isolation Kit
(Invitrogen) respectively, according to the manufacturer's protocol.
Total RNA was DNase-treated and then reverse-transcribed using oligo-dT
primers and the Sensiscript RT Kit (Qiagen), as described by the
manufacturer. PCR reactions were performed using AmpliTaq Gold (Perkin
Elmer Life Sciences) with the following sets of primer pairs: for
NFAT1, F, 5'-ACATCCGCGTGCCCGTGAAAGT-3' and R,
5'-CTCGGGGCAGTCTGTTGTTGGATG-3'); NFAT2, F,
5'-CATGCGCCCTCTGTGGCCCTCAAA-3' and R, 5'-GGAGCCTTCTCCACGAAAATG-3');
NFAT3, F, 5'-GAAGCTACCCTCCGGTACAGAG-3' and R,
5'-GCTTCATAGCTGGCTGTAGCC-3'); NFAT4, F, 5'-CTACTGGTGGCCATCCTGTTGT-3' and R, AGCTCGTGGGCAGAGCGCTGAGAGCACTC-3'); and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH,
CLONTECH). Amplification conditions were 94 °C,
10 min; 30-45 cycles at 94 °C, 1 min; 55 °C, 1 min; 72 °C, 2 min and extension for 10 min at 72 °C. Amplified PCR products were
separated by agarose gel electrophoresis and detected by ethidium
bromide staining.
Western Blot Analysis--
Tissue samples were homogenized in
cell lysis buffer (in mM: 50 Tris, 150 NaCl, 1 EDTA, 1%
Nonidet P-40, pH 8.0) containing phenylmethylsulfonyl fluoride (1 mM), pepstatin (20 µg/ml), leupeptin (20 µg/ml), and
aprotinin (0.5 µg/ml). Protein concentrations were determined by the
Bradford dye-binding assay using bovine serum albumin as a standard.
Aliquots of tissue extracts containing equal amounts of protein were
separated by SDS-polyacrylamide gel electrophoresis on 8% gels using
the Laemmli buffering system. Proteins were transferred to Immuno-Blot
polyvinylidene difluoride membranes (Bio-Rad) and blocked by rocking
for 1 h at room temperature in blocking buffer (Tris-buffered
saline with 0.1% Tween 20 and 5% nonfat dry milk). Blots were exposed
to primary antibodies for 1 h at room temperature, multiply washed
with TBST, treated with secondary antibody (HRP-conjugated) for 45 min,
and followed by a final series of washes with TBST. Primary (rabbit
polyclonal anti-NFAT4, Santa Cruz Biotechnology) and secondary
antibodies were prepared in blocking buffer, and all treatments were
performed at room temperature.
Luciferase Reporter Assay--
Transgenic mice, uniformly
expressing a promoter construct containing three tandem copies of the
interleukin-2 promoter distal NFAT-binding domain inserted into a
minimal expression vector upstream of a luciferase construct, were used
to determine NFAT transcriptional activity. In a given experiment,
ileal strips were collected from 2 mice, pooled, and then divided into
individual treatment groups (typically 4-6 strips/group). After
treatment, samples were snap frozen in liquid nitrogen and stored at
Statistics--
Results are expressed as means ± S.E.
where applicable. All statistical analysis was performed using GraphPad
software (Prism 3.0). Statistical significance was determined using the
two-tailed unpaired Student's t test.
NFAT4 Is the Predominant Isoform Expressed in Native Smooth
Muscle--
We have used RT-PCR analysis and immunoblotting to
identify NFAT isoforms expressed in native ileal smooth muscle.
Previous results from experiments employing the rat A7r5 aortic smooth muscle cell line suggested that the NFAT1 and NFAT2 isoforms are expressed in smooth muscle (7), although expression of additional isoforms could not be ruled out in this study. Using RT-PCR analysis, we find no evidence for NFAT1 or NFAT2 expression in native ileal smooth muscle sheets (Fig. 1B)
using primer pairs that efficiently amplify NFAT1 from spleen and NFAT2
from thymus (Fig. 1A). Instead, we find that the NFAT4 and,
perhaps to a lesser extent, the NFAT3 isoforms are constitutively
expressed, as shown in Fig. 1B. We have found a similar
pattern of NFAT isoform expression in native arterial smooth muscle of
the aorta and the cerebral vasculature (30, 31). NFAT4 appears to be
the predominant isoform expressed, based on results obtained from an
RT-PCR analysis of isolated smooth muscle cells (Fig. 1C).
NFAT3 identified in RT-PCR analysis of intact ileal strips (Fig.
1B) may represent expression in co-isolated neurons of the
nerve plexus between longitudinal and circular smooth muscle layers,
which is consistent with previous observations in neurons (14).
At the protein level, NFAT4 isolated from ileal smooth muscle strips
runs as multiple high molecular mass bands on SDS gels with a single
band at ~160 kDa, which is similar to that previously reported
in Jurkat cells (32) and as additional, apparently nonspecific bands at
75-100 kDa (Fig. 1D). Differences in the mobility of the
high molecular mass band between smooth muscle and thymus (which
expresses NFAT4 at high levels) may reflect tissue differences in the
expression of known NFAT4 splice variants (33).
NFAT4 Nuclear Translocation Is Induced in Ileal Smooth Muscle by
Treatment with PDGF--
The potent smooth muscle cell mitogen PDGF
has been previously shown to induce NFAT activation in the rat A7r5
aortic smooth muscle cell line (7). To explore NFAT activation in
native smooth muscle, we treated ileal smooth muscle strips with PDGF (10 ng/ml) for 30 min and analyzed for NFAT nuclear accumulation by
immunostaining with an antibody specific for NFAT4. Sections were also
stained with the DNA binding dye YOYO-1 to identify nuclei. PDGF
induces nuclear translocation of NFAT4 in ileal smooth muscle (Fig.
2), with 47.4 ± 6.7% of the cells
in PDGF-treated strips exhibiting nuclear localization of NFAT4,
compared with 5.8 ± 1.2% of the cells in untreated control
strips. PDGF-induced nuclear accumulation is completely blocked in the
presence of the calcineurin inhibitors CsA or FK506 (Fig. 2), which
reduce the percentage of NFAT-positive nuclei to 5.7 ± 5.7 and
4.3 ± 2.0, respectively. PDGF-induced NFAT4 nuclear translocation
appears to reflect a direct action on smooth muscle rather than an
indirect action through associated neurons in the plexus layer since
stimulation with PDGF in the presence of a mixture of neurotransmitter
inhibitors that includes atropine, tetrodotoxin, phentolamine, and
propanolol (1 µM each), does not prevent NFAT4 nuclear
accumulation (60.9 ± 12.8% NFAT4-positive nuclei, Fig. 2). The
percentage of NFAT4-positive nuclei (5.4 ± 1.9%) in ileal strips
treated with the neurotransmitter inhibitory mixture alone does not
significantly differ from controls (Fig. 2).
Nuclear translocation of NFAT4 is first evident at 10-15 min after
exposure to PDGF, with a clustering of NFAT4 staining in the vicinity
of the nuclear envelope (Fig. 3). After
30 min of PDGF treatment, redistribution to the nucleus is observed.
The clustering of NFAT4 around the nucleus prior to translocation suggests the possibility that a distinct docking step is involved in
the nuclear translocation process.
PDGF Induces Transcriptional Activity in Ileal Smooth
Muscle--
To determine whether the observed NFAT4 nuclear
accumulation is associated with increased transcriptional activity,
ileal smooth muscle strips from a transgenic mouse that uniformly
expresses an NFAT luciferase promoter-reporter construct (34) were
treated with PDGF (10 ng/ml) and harvested after 6 or 24 h in
serum-free medium (37 °C). Samples were homogenized as described
under "Experimental Procedures" and assayed for protein-normalized
luciferase activity. We found that the PDGF-induced NFAT4 accumulation
is accompanied by an increase in NFAT-dependent
transcriptional activity (Fig. 4). This
transcriptional activity is transient, being evident at 6-h post-PDGF
treatment, but not after 24 h, and is effectively induced by a
brief (30 min) exposure to PDGF.
PDGF-induced NFAT4 Nuclear Translocation Is Dependent on
Voltage-dependent Ca2+
Channels--
[Ca2+]i regulates
nuclear accumulation of NFAT, and both the source of Ca2+
and the nature of the Ca2+ signal may be important in
determining the nature of the response (24, 25). In ileal strips,
treatment with the dihydropyridine inhibitor of
voltage-dependent L-type Ca2+ channels,
nisoldipine (100 nM), results in a complete abrogation of
PDGF-induced NFAT4 nuclear accumulation (Fig.
5C; 6.7 ± 5.6% NFAT4-positive nuclei). Nisoldipine is equally effective when added
before (Fig. 5C) or concurrent with PDGF exposure (data not
shown). Thus, PDGF-induced mobilization of NFAT4 is critically dependent on Ca2+ influx through VDCC. To provide
additional evidence for VDCC involvement, we examined the effects of
hyperpolarizing the cell membrane, which would decrease
Ca2+ entry by closing VDCC. Pinacidil (1 µM),
which induces membrane hyperpolarization by opening ATP-sensitive
K+ channels (35), completely blocked the PDGF-induced
nuclear translocation of NFAT4 (Fig. 5D; 6.74 ± 1.67%
NFAT4-positive nuclei).
These results indicate that functional VDCC are required for
PDGF-induced nuclear accumulation of NFAT. To determine whether direct
activation of VDCC is sufficient to induce NFAT4 nuclear accumulation,
we treated ileal smooth muscle with 60 mM K+
for up to 30 min, a treatment that has been previously shown to cause a
graded and sustained increase in
[Ca2+]i in this preparation as a
result of VDCC activation (29, 36). Surprisingly, depolarization was
incapable of inducing the nuclear accumulation of NFAT4 (Fig. 5 and
6, 10.5 ± 3.5% NFAT4-positive nuclei). Similar results were obtained with the Ca2+
ionophore ionomycin (1 µM, 30 min, 13.1 ± 2.3%
NFAT4-positive nuclei, n = 4, total cells = 930).
These data indicate that although Ca2+ influx is necessary
for PDGF-mediated NFAT4 nuclear accumulation, it is not a sufficient
stimulus alone.
Further, we tested whether PDGF was still capable of inducing NFAT4
nuclear accumulation in the presence of a depolarizing stimulus. The
PDGF-stimulated increase in NFAT4 nuclear localization was completely
blocked in ileal strips treated for 30 min with PDGF (10 ng/ml) and 60 mM K+ (Fig. 5, 8.6 ± 3.8% positive
nuclei). These results suggest that NFAT4 nuclear accumulation may
require the repetitive Ca2+-spiking characteristic of ileal
smooth muscle (3), which might be modified by PDGF, but is completely
abolished by high K+ (29, 36). In time course experiments,
neither continuous exposure (30 min) nor brief pulses of 60 mM K+ (5, 10, 15 min) were able to induce NFAT4
nuclear accumulation (Fig. 6). Even when time course experiments were
performed in the presence of the L-type Ca2+ channel
agonist Bay K 8644 to increase Ca2+ entry, depolarization
with 60 mM K+ failed to induce NFAT4 nuclear
accumulation (data not shown).
Ca2+-elevating Stimuli Have Differential Effects on
NFAT4 and CREB--
We have previously reported that depolarizing
stimuli activate the Ca2+-sensitive transcription factor
CREB in cerebral artery smooth muscle (6), increasing the fraction of
nuclei staining for P-CREB. This response is dependent on the influx of
extracellular Ca2+ through VDCC and appears to be mediated
by CaMK. In ileal smooth muscle, depolarization with 60 mM
K+ also results in a rapid and robust increase in P-CREB
(Fig. 7B). Like NFAT4, CREB is
also activated by PDGF in ileal smooth muscle, as evidenced by the
prominent nuclear P-CREB staining observed after 30 min of PDGF
stimulation (Fig. 7C). Thus, CREB is activated by both PDGF
and depolarizing stimuli, whereas depolarization-induced Ca2+ influx through VDCC alone is insufficient to stimulate
NFAT4 nuclear translocation. These results clearly indicate that these two Ca2+-sensitive transcription factors respond
differentially to a given Ca2+-elevating stimulus and
provide evidence for Ca2+ signal discrimination at the
transcription factor level.
We have found that NFAT4 is expressed in native smooth muscle and
can be activated by stimulation with the smooth muscle mitogen PDGF.
This activation is dependent on Ca2+ influx through VDCCs
and can be blocked by calcineurin inhibitors CsA and FK506.
Surprisingly, sustained increases in global
[Ca2+]i induced by treatment with
ionomycin or membrane depolarization with K+ failed to
activate NFAT4 in this tissue.
The predominant isoform of NFAT expressed in native ileal smooth muscle
is NFAT4, which we have also found abundantly expressed in aortic and
cerebral artery smooth muscle tissue (30, 31). NFAT1 and NFAT2
isoforms, which are ubiquitously expressed in cells of the immune
system and in other cell types, do not appear to be constitutively
expressed at significant levels in these tissues. This is in sharp
contrast to results reported for the A7r5 smooth muscle cell line,
which expresses both NFAT1 and NFAT2 isoforms (7), suggesting that
significant changes in the regulation of NFAT expression take place
during the process of cell line establishment. Although it is known
that NFAT4 is highly expressed in thymus cells (37) and is also
expressed in skeletal muscle (33), these data provide the first
evidence that this NFAT isoform is highly expressed in smooth muscle.
It has become axiomatic that stimuli that induce sustained increases in
[Ca2+]i are sufficient and
necessary for NFAT nuclear accumulation. The failure of
depolarization-induced elevation in
[Ca2+]i to induce NFAT4 nuclear
accumulation in native ileal smooth muscle was therefore unexpected.
Ca2+ influx is clearly necessary for PDGF-induced NFAT4
nuclear translocation in native ileal smooth muscle, as evidenced by
the ability of VDCC inhibitors and membrane hyperpolarizing agents to
completely prevent this translocation. It is possible that modulation
of the Ca2+ signal may play an important facilitating role
in PDGF-induced NFAT4 nuclear accumulation, such that the tonic
increase in [Ca2+]i induced by
depolarization with K+ may lack information that is
critical to the response of NFAT in this tissue. In ileal smooth
muscle, the presence of spontaneous action potentials generates
repetitive [Ca2+]i spiking that
may constitute an appropriately modulated Ca2+ signal. PDGF
has also been shown to give rise directly to Ca2+ waves or
oscillations (38, 39). The spontaneous phasic contractile behavior and
repetitive Ca2+ spiking is blocked in the presence of 60 mM K+, and it is likely that any complex or
modulated Ca2+ signal generated by PDGF stimulation would
be similarly abrogated in the presence of depolarizing concentrations
of K+. In either case, a modulated Ca2+ signal
would be replaced by tonic increases in
[Ca2+]i. The fact that PDGF fails
to induce NFAT4 nuclear localization in the presence of 60 mM K+ is consistent with a role for
Ca2+ signal modulation because elevated global
Ca2+ induced by depolarization would otherwise be predicted
to increase, not decrease, PDGF-induced NFAT4 nuclear translocation.
If spontaneous action potentials contribute an
appropriately modulated Ca2+ signal, these results further
suggest that nuclear export greatly exceeds Ca2+-driven
nuclear import in ileal smooth muscle; otherwise, the oscillating
Ca2+ signal provided by spontaneous phasic contractions
would be predicted to promote the constitutive nuclear localization of
NFAT. Thus, ileal smooth muscle may be unique with respect to NFAT
activation in that a sufficient Ca2+ signal may exist under
basal conditions, and the regulation of nuclear export dynamics may be
the critical factor that determines the cellular localization of
NFAT.
Accordingly, PDGF may induce NFAT4 nuclear accumulation primarily by
decreasing the basal rate of nuclear export. Nuclear export of various
NFAT isoforms is promoted by the activity of glycogen synthase kinase-3
(GSK-3), which phosphorylates conserved serines in the N terminus that
are required for nuclear export. There is evidence that PDGF inhibits
GSK-3 activity in fibroblasts, suggesting a possible mechanism by which
PDGF might promote NFAT nuclear accumulation (40). NFAT4 is unique
among NFAT isoforms in that its nuclear export can be regulated by the
activity of c-Jun N-terminal kinase (JNK; Ref. 21) and it is
conceivable that the effects of PDGF on NFAT4 may be caused by
inhibition of JNK activity. NFAT nuclear import can also be inhibited
by a number of mechanisms and PDGF might act to disinhibit these processes or directly promote NFAT4 translocation by other means. Although inhibition of nuclear export or potentiation of import represent attractive mechanisms to account for these results, the
Ca2+-independent mechanisms by which PDGF may regulate
NFAT4 localization remain to be elucidated.
Unlike NFAT4, CREB is activated by membrane depolarization with
elevated external K+ in ileal smooth muscle, suggesting
that CREB is capable of responding to global increase in
[Ca2+]i alone, whereas NFAT4 is
not. These results clearly indicate that Ca2+-sensitive
transcription factors are capable of discriminating between different
Ca2+ signals, resulting in their differential activation.
The physiological or pathological implication of differential
transcription factor activation mechanisms has not been explored in
native smooth muscle, which exhibits a rich diversity of
Ca2+ signaling modalities and a phenotypic plasticity that
clearly reflects the activity of underlying genetic regulatory mechanisms.
The targets of Ca2+-sensitive transcription factors that
may play a role in maintaining the contractile smooth muscle phenotype, or contribute to adaptive or pathological changes in smooth muscle structural or functional properties, are unknown. Smooth muscle cells
have been found to undergo rapid and profound phenotypic changes
during culture in vitro (41). In guinea pig ileal strips cultured in the presence of serum, it has been shown that force development in response to contractile stimuli is diminished and that
expression of L-type Ca2+ channels is down-regulated. The
down-regulation of VDCCs is prevented by treatment with a
dihydropyridine VDCC blocker (29) or by pretreatment with the
calcineurin inhibitor CsA (42), suggesting that the changes in VDCC
expression are Ca2+-dependent and that the
Ca2+-dependent modulation of VDCC expression
may be regulated by NFAT. A number of ion channel genes and regulatory
subunits, including those for L-type Ca2+ channels
(e.g.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 collagenase (FLUKA,
Switzerland), 1 mg ml
1 papain (Worthington Biochemical
Corporation, Lakewood, NJ), 4.5 mg ml
1 bovine serum
albumin (Sigma) and 1 mg ml
1 dithioerythiol. After
isolation, cells were allowed to settle in a chamber mounted on an
inverted microscope and collected using a microbore pipette (5-10 µm
diameter). The cells were transferred to nuclease-free microcentrifuge
tubes, flash-frozen in liquid nitrogen, and stored at
80 °C for
future use in RT-PCR.
80 °C until ready for processing. Frozen samples were pulverized and homogenized in 50-100 µl of 1× cell culture lysis reagent (Invitrogen) with the aid of a motorized pestle. An aliquot of clarified supernatant (20 µl), obtained by centrifugation at
12,000 × g for 5 min, was added to 100 µl of
luciferase substrate reagent (Invitrogen) and assayed for luciferase
activity using a Turner Designs 20/20 luminometer. Optical density
measurements were normalized to starting protein concentration
determined using a protein assay reagent (Bio-Rad) and expressed as
relative luciferase units.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
NFAT4 is the predominant isoform in ileal
smooth muscle (SM). A, RT-PCR analysis for
NFAT1, NFAT2, NFAT3, and NFAT4 mRNA showing amplification in spleen
(SPLN), heart (HRT), and thymus (THY).
RNA was extracted using TRIzol, and PCR amplification and primers were
as described under "Experimental Procedures." B, using
the same primer pairs as in A, NFAT3 and NFAT4 were
identified in ileal sheets, whereas in enzymatically isolated ileal
smooth muscle cells, only NFAT4 was amplified (C).
Asterisks indicate 600-base pair level. D,
Western blot showing expression of NFAT in ileal smooth muscle, thymus,
but not in heart. Size markers are indicated on the left.
Experiments were repeated at least twice with identical results.
View larger version (33K):
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Fig. 2.
Calcineurin mediates
PDGF-induced NFAT4 nuclear accumulation in intact smooth muscle.
Representative immunofluorescence images showing cytosolic localization
of NFAT4 in non-stimulated or control conditions (A) and
nuclear localization following PDGF treatment (10 ng/ml, B)
in ileal smooth muscle. Cells were co-stained with the DNA-binding dye
YOYO-1; yellow indicates nuclear co-localization of NFAT4
(red) and YOYO (green). Lower panel
summarizes the percentage of NFAT nuclear localization in ileal
smooth muscle strips in the control and after a 30-min treatment with
PDGF (10 ng/ml), CsA (1 µM), and FK 506 (1 µM). Ileal strips were treated with a nerve mixture
(nc) containing neurotransmitter inhibitors atropine,
tetrodotoxin, phentolamine, and propanolol (1 µM each) in
the presence or absence of PDGF (10 ng/ml). ***, p < 0.001, PDGF compared with Control, CsA, FK506, and nc alone;
whereas PDGF and PDGF + nc are not significantly different
(n, number of animals; i, analyzed images; and c,
total number of cells counted).
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Fig. 3.
Time course of PDGF-induced NFAT4
translocation. Ileal smooth muscle strips stained with anti-NFAT4
antibody following PDGF exposure (10 ng/ml) for 5, 10, 15, and 30 min.
Note clustering of NFAT4 in the proximity of the nuclear envelope
already at 10 and 15 min. Arrows indicate examples of empty
nuclei (black holes).
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Fig. 4.
PDGF exposure induces a transient increase in
NFAT4 transcriptional activity. Ileal smooth muscle strips from
NFAT-luciferase transgenic mice were pooled and divided into five
treatment groups. Strips were collected 6 or 24 h after exposure
to 10 ng/ml PDGF for 30 min (PDGF 0.5h) or continuously for
the duration of the experiment (PDGF 6h, PDGF 24h). Data are
expressed as optical density normalized to protein concentration
(n = 3, *, p < 0.05).
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Fig. 5.
Inhibition of VDCC prevents PDGF-induced
NFAT4 nuclear accumulation. Representative images of ileal smooth
muscle stained with anti-NFAT4 antibody (red) and with the
DNA-binding dye YOYO-1 (green). Yellow nuclei
reflect co-localization of NFAT4 and YOYO-1. Strips were treated for 30 min at room temperature as indicated below each field. Lower
panel summarizes the percentage of NFAT4 nuclear localization
observed in ileal strips in control conditions and after a 30-min
treatment with PDGF (10 ng/ml), nisoldipine (100 nM),
pinacidil (1 µM), and high K+ buffer (60 mM). ***, p < 0.001, PDGF
compared with control, PDGF + nisoldipine, PDGF + pinacidil, high
K+ buffer, and PDGF + high K+ buffer
(n, number of animals; i, analyzed images; and
c, total number of cells counted).
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Fig. 6.
High K+-induced depolarization is
not a sufficient stimulus for NFAT4 nuclear accumulation. Ileal
smooth muscle strips stained with anti-NFAT4 antibody following
K+ exposure (60 mM) for 5, 10, 15, and 30 min.
Note black holes indicating absence of NFAT4 nuclear
staining (arrows).
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Fig. 7.
Membrane potential depolarization and PDGF
induce increases in P-CREB in ileal smooth muscle.
Immunofluorescence images showing ileal smooth muscle strips stained
with anti-P-CREB in control conditions (A) and after a
30-min treatment with high K+ (60 mM,
B) and PDGF (10 ng/ml, C). A representative
experiment of four is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1C), ryanodine receptors (e.g. RYR2),
IP3 receptors (e.g. IP3R1), voltage-dependent
K+ channels (e.g. Kv1.5) and the
subunit of
the large conductance Ca2+-dependent
K+ channel contain NFAT response elements within their
promoter regions. These observations suggest the possibility that this Ca2+-dependent transcription factor may play a
role in regulating Ca2+ homeostasis by regulating the
expression of ion channel gene assemblages. Ultimately, the
identification of NFAT-regulated genes in native smooth muscle will be
crucial for developing an understanding of the physiological role that
this transcription factor plays in this genetically dynamic tissue.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Mercedes Rincón (University of Vermont) for helpful discussions and for providing the transgenic mice containing the NFAT-luciferase promoter-reporter construct for this work and Dr. Karen Lounsbury (University of Vermont) for helpful technical assistance. We also thank the Cell Imaging Facility for assistance in confocal microscopy.
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FOOTNOTES |
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* This work was supported by Grants HL44455, HL63722, DDK53832 (to M. T. N.) and NIH Postdoctoral Cardiovascular Training Grant HL07647-12 (to D. C. H-E.), and a Graduate Research Supplement to HL44455 (to A. S. S.) as well as by grants from the Swedish Medical Research Council, K. & A. Wallenberg, and Dr. P. Håkansson Foundation (to M. F. G.).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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 802-656-2500; Fax: 802- 656-4523; E-mail: nelson@salus.med.uvm.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M011684200
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ABBREVIATIONS |
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The abbreviations used are: [Ca2+]i, intracellular calcium concentration; VDCC, voltage-dependent calcium channels; NFAT, nuclear factor of activated T-cells; PDGF, platelet-derived growth factor; CsA, cyclosporin A; RT-PCR, reverse transcriptase-polymerase chain reaction; CREB, cAMP-responsive element-binding protein; CaMK, calmodulin-dependent kinase; GSK-3, glycogen synthase kinase-3; JNK, c-Jun N-terminal kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nc, neurotransmitter inhibitor mixture; PBS, phosphate-buffered saline; P-CREB, phosphorylated CREB.
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