From the Department of Pharmacology, Expression of the antigen-regulated, cyclosporin
A-sensitive nuclear factor of activated T cells (NFAT) is not
restricted to lymphoid cells, as thought initially, but the
physiological inducers of NFAT-mediated transcription in non-lymphoid
cells are unknown. Here, cultured vascular smooth muscle cells (VSMC) are shown to express two isoforms of the NFAT family endogenously, which are localized differentially in cells under resting conditions. Using a retroviral NFAT-specific luciferase reporter, we show that VSMC
support previously unrecognized complexities in NFAT-mediated transcription, including evidence for negative regulation by
Ca2+ signaling and positive regulation through
co-activation of adenylyl cyclase and Ca2+ mobilization.
The VSMC mitogen platelet derived growth factor-BB (PDGF-BB) induces
NFAT-mediated transcription in VSMC. Thrombin and angiotensin II, which
activate G Transcription mediated by the nuclear factor of activated T
lymphocytes (NFAT)1 is
regulated tightly in response to elevations of both intracellular calcium ion (Ca2+) and diacylglycerol second messengers
following activation of phospholipase C (PLC) (1). Increased
intracellular Ca2+ stimulates
calcineurin-dependent dephosphorylation of cytoplasmic NFAT, leading to its nuclear translocation (2-5). Macrolide
immunosuppressive agents, such as cyclosporin A (CsA) and FK506 (6),
block this step. In the nucleus, NFAT forms a heteromeric
transcriptional co-activator complex with AP1 proto-oncogene partners
that are co-induced downstream of receptor activation (3). This
multimeric NFAT complex participates in the transactivation of several
cytokine genes by interactions with purine-rich genomic NFAT-responsive enhancer elements (NFRE) (7).
The first evidence for the existence of NFAT was derived from analysis
of antigen-responsive enhancer elements in the cytokine IL-2 gene
promoter in lymphocytes (8). A growing body of data now suggests that
NFAT is expressed more widely but the picture is far from complete. To
date, NFAT expression or function has been described in several types
of non-lymphoid cells, including mast (9), endothelial (10), and
neuronal cells (11). Each of the mRNAs for the known NFAT isoforms
are expressed in distinct tissue-specific patterns (12). However, the
precise non-lymphoid cell types that express NFAT isoforms in
vivo are largely unknown. For instance, it remains unclear whether
NFAT is expressed to some degree in all cells, or if its expression in
non-lymphoid tissues is restricted to certain subpopulations of cells
within tissues.
The presentation of foreign antigen on lymphocytes provides the best
understood physiological inducer of NFAT-mediated transcription. The
existence of NFAT in non-immune cells suggests that it can be directed
to regulate gene expression by other physiologic stimuli. Physiological
agonists for receptors that activate PLC signaling, such as hormones,
neurotransmitters, and growth factors, provide obvious candidates for
this function (11, 13). The extent to which such stimuli can trigger
NFAT-mediated transcription in non-lymphoid cells that express
endogenous NFAT isoforms needs to be determined. Additionally, it is
not known if NFAT transactivation properties in non-lymphoid cells
differs from that in the better understood lymphocyte context.
Vascular smooth muscle cells (VSMC) respond to a diverse group of
extracellular signals to progress through phenotypic modulation, a
process that occurs in the development and progression of
cardiovascular diseases such as hypertension and atherosclerosis (14).
Changes in gene expression patterns underlie this transition, which
serves to regulate processes involved in extracellular matrix
remodeling, VSMC growth, and migration. Cultured VSMC serve as a useful
system to understand the molecular and cellular consequences of VSMC activation, and are helpful in elucidating the genes, gene expression control pathways, and the extracellular signals associated with VSMC
activation. We speculated that NFAT might be expressed in cultured VSMC
and may participate in facilitating the VSMC activated state in
response to mitogenic signals. The experiments described herein
represent the first demonstration that NFAT is expressed in VSMC and
provide evidence that PDGF-BB, which activates a tyrosine kinase-linked
receptor, can serve as a physiological inducer of NFAT-mediated
transcription in VSMC. This response is synergized by co-stimulation of
VSMC with thrombin and angiotensin II, which activate PLC Materials and Cell Lines--
The following plasmids used in
this study were received as gifts: the retroviral plasmid pLNCX, from
A. D. Miller (Seattle, WA); plasmids pSH210 and pSH250, encoding
human NFATc1 and NFATc2, from G. Crabtree (Stanford, CA); and plasmid
pNFAT/luc, from R. Bram (Memphis, TN). A mouse monoclonal antibody
(IgG1, clone 7A6) against NFATc1 was purchased from Affinity
Bioreagents, Inc. (Golden, CO). The monoclonal antibody (GH8-G1-G9)
against NFATc2 was a gift from G. Crabtree. Mouse IgG1 in clarified
ascites was purchased from Sigma. Secondary antibodies were bought from
Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). The
tyramide-green immunochemical reporter was purchased from NEN Life
Science Products). Except where noted all cell culture media,
antibiotics and molecular biology supplies were purchased from Life
Technologies, Inc. Fetal bovine and calf serum were obtained from
Atlanta Biologicals (Norcross, GA). Luciferin and additional salts and
buffers used were purchased from Sigma. The retroviral plasmids used in
this study were subcloned and maintained in Escherichia coli
Top10F' (Invitrogen, Inc., Carlsbad, CA) in 100 µg/ml ampicillin plus
12.5 µg/ml tetracycline. The VSMC used in this study were derived
from dissociated rat thoracic aorta (passages 10 to 25) and are
maintained as a continuous primary cell line in DMEM, 4.5 µg/ml
glucose, 10% heat-inactivated calf serum with 100 units/ml each
penicillin and streptomycin (15). Amphotropic (ATCC CRL-11554)
retroviral producer cells were obtained from the American Type and
Culture Collection (Rockville, MD). Angiotensin II was purchased from
Sigma, purified human thrombin was provided generously by S. Krishnaswamy (Dept. of Medicine, Emory University, Atlanta, GA), and
PDGF-BB was purchased from Calbiochem, Inc. (San Diego, CA).
Retroviral Reporter Plasmids--
pTJM9 was created by placing a
new multicloning site just distal of the cytomegalovirus promoter in
HindIII- and ClaI-digested pLNCX (16) using the
oligonucleotides 5'-AGCTTCCATTGTGCTGGTCGACGCGTCCAGCACAATGGAT and
5'-CGATCCATTGTGCTGGACGCGTCGACCAGCACAATGGA. The cytomegalovirus promoter in pTJM9 was removed by digestion with BamHI and
HindIII, and the plasmid was closed by blunt-end ligation
after Klenow treatment to create pTJM12. A luciferase coding sequence
(17) with upstream remnant was excised with PstI and
SstI from the vector poLuc (18). After Klenow treatment, the
fragment ends were ligated with non-cohesive BstXI adapters
(5'-CTGGCGCG and 5'-CGCGCCAGCACA) and subsequently cloned into pTJM12
digested with BstXI. The resulting construct, pKA8, can
accept promoters in the unique BamHI and HindIII
(5' to 3') sites just proximal of the luciferase 5' end, and
transcribes luciferase off of the opposite strand as that for the viral
5'-LTR promoter. Plasmid pKA7 contains a ~250-base pair
BamHI-HindIII fragment with an NFAT enhancer
triplex linked to the minimal IL-2 promoter fragment excised from
pNFAT/luc (19), which was cloned into the same sites in pKA8. Plasmid
pKA9 contains a ~130-base pair BamHI-HindIII fragment for the minimal IL-2 promoter, without the NFAT enhancer, amplified from pNFAT/luc by PCR using the primers
5'-CTCGGATCCTATGTAAAACATTTTGACACC and 5'-GGCAAGCTTAGGAGTTGAGGTTACTGTGA.
To create a reporter plasmid bearing a mutation in the NFAT binding
site (mutations shown in bold letters), the oligonucleotides F87
(5'-GATCAGGACTGATCACTGTTTCATACAGAAGGC) and F88
(5'-GATCGCCTTCTGTATGAAACAGTGATCAGTCCT) were concatenized by
brief ligation before adding BamHI-digested pKA9 to the
ligation reaction. pKA10 represents a clone derived from this procedure that was identified by sequencing as having an enhancer region identical that in pKA7 except for conversion of the NFAT binding site
from GGAAAA to CTGATC.
Retroviral Production and Infection--
Retroviruses were
prepared by transient transfection of helper virus-free amphotropic
producer cells (20). Bing-CAK8 cells (ATCC CRL-11554) were grown from
original expanded stocks without selective antibiotics for less than 30 days in growth medium (GM) consisting of DMEM, 4.5 µg/ml glucose,
10% fetal bovine serum, and a mixture of 100 units/ml penicillin and
100 units/ml streptomycin in a humidified 5% CO2
atmosphere at 37 °C. To produce infectious retroviral supernatants,
the cells at 50-80% confluence in 10-cm diameter dishes were
transfected with the retroviral plasmids using calcium phosphate and 25 µM chloroquine for 6-12 h before re-feeding with 25 ml
of GM. Twenty-four hours after initiating the transfection, the GM was
aspirated and replaced with 9 ml of fresh GM before placing the dishes
in a humidified 5% CO2 atmosphere at 32 °C. Previous
work has shown this lower temperature enhances retroviral titers (21).
The supernatant containing retroviral particles was harvested after
24 h, and twice more at 12-18 h intervals thereafter replenishing
with 9 ml of GM each time. Each collected supernatant (9 ml) was
filtered through sterile 0.45-µm syringe-tip cellulose acetate disk,
aliquoted, snap-frozen in liquid nitrogen and stored at -80 °C. VSMC
(~20% confluent) grown on 6 × 35-mm multiwell plates were
infected with retroviruses by adding thawed retroviral supernatant (2 ml) containing 8 µg/ml Polybrene to each well and spinning the cells
at 2,500 rpm for 30 min at 32 °C in a Beckman model GS-6R
refrigerated centrifuge in a swinging bucket rotor before placing in a
5% CO2 incubator at 37 °C. This infection protocol was
repeated twice more at 8-12 h intervals. Forty-eight hours following
the last infection, the cells were treated with 400 µg/ml G418 in
normal growth VSMC medium (Life Technologies, Inc.). Within 7 days of
selection, the surviving cells from each well were combined, expanded,
and used subsequently for further study without subcloning and
maintained in the absence of G418. Because cell death during selection
of retrovirus-infected cultures was minimal compared with that in
retrovirus-naïve cells treated in parallel with 400 µg/ml
G418, transduction efficiency was estimated in all cases to be nearly
quantitative.
Cell Treatments and Luciferase Assays--
Recombinant VSMC
selected for reporter retroviruses were grown to confluence in 24-well
plates for 3 days in growth medium lacking G418 and then for an
additional 24 h in medium lacking serum before stimulation with
drugs and agonists. The drugs were prepared as 10-fold concentrated
stocks in phosphate-buffered saline, pH 7.4 (PBS), and added as
aliquots directly to the cells to begin stimulation. These were
performed in a humidified 5% CO2 atmosphere at 37 °C
for the indicated time before the cells were lysed and extracts were
prepared for luciferase determinations, as described previously
(22).
Western Blot Analysis--
To prepare control cell extracts
expressing defined NFAT isoforms, COS-7 cells were transfected by the
DEAE-dextran/chloroquine approach with the vector pSH107c, encoding
human NFATc1; or the vector pSH210, encoding human NFATc2 (23); or
pCDM8 as a sham control. Confluent VSMC or transfected COS cells were
rinsed in PBS before lysis in RIPA-2 (150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM
Tris-HCl, pH 8.0) containing a mixture of protease inhibitors (90 µg/ml phenylmethylsulfonyl fluoride, 0.2 TIU/ml aprotinin, 0.1 mM leupeptin, 0.2 mM sodium orthovanadate, 5 µg/ml pepstatin A, and 5 µg/ml soybean trypsin inhibitor).
Insoluble debris was removed by centrifugation at 10,000 × g at 4 °C and discarded. Protein concentrations in the
supernatants were determined using a kit purchased from Bio-Rad, and
Immunohistochemistry--
VSMC (7 × 104 cells)
were plated in 35-mm dishes 24 h prior to drug treatments, and
changed to serum-free medium 1 h before the addition of drugs. CsA
was dissolved in 2.5% ethanol in cell culture medium containing 0.1%
Tween 80 to prepare a 100-fold concentrated stock solution. Ionomycin
was added to the cells directly from a 100-fold stock solution. The
cells received either CsA (1 µM final concentration) or
its vehicle for 10 min prior to the addition of ionomycin (1 µM final concentration) or its vehicle. After 15 min, the
medium was aspirated and the cells were rinsed with PBS before fixation
for 10 min using cold methanol/0.3% H2O2
before storing at 4 °C in PBS. Antibody staining was performed at
room temperature within 1 week of these treatments. The samples were
first blocked against avidin/biotin using the AVB kit (Vector Laboratories), according to directions supplied by the manufacturer. After rinsing with PBS, nonspecific protein binding was blocked by
incubating the cells with PBS containing 2% horse serum (Atlanta Biologicals, Norcross, GA) and 0.5% Triton X-100 for 30 min at 22 °C. All primary antibodies were diluted in this blocking solution at a 1:250 dilution and applied to the cells for 60 min at 22 °C. Following three washes with PBS containing 0.2% Tween 20, the cells
were incubated for 60 min with a 1:10,000 dilution of biotinylated anti-mouse IgG (Jackson Immunochemicals, Inc., West Grove, PA). After
washing as above, antibody binding was visualized using a catalyzed
reporter deposition kit (T.S.A. Direct Green, NEN Life Science Products
according to the manufacturers directions but with a 1:100 dilution of
the streptavidin HRP conjugate. After the final rinse, the cells were
incubated for 10 min at 22 °C with a Hoechst stain (H33258) in PBS,
then washed three times with PBS. After coverslipping using
Vectashield, all analyses and photography were performed on a Zeiss
Axiovert microscope equipped with an Optronics video camera, which was
in line with a Power Macintosh 7600 computer equipped with NIH
Image.
Fura-2 Assays--
Confluent 10-cm plates of VSMC were washed
three times with Ca2+/Mg2+-free Hank's
balanced salt solution (HBSS) and treated with 1 mg/ml type-1
collagenase (178 units/ml; Worthington Biochemical, Inc., Freehold, NJ)
at 37 °C. The dispersed cells were collected, washed in
Ca2+/Mg2+- replete HBSS
(Ca2+/Mg2+ HBSS), counted, and then resuspended
to 3-4 × 106 cells/ml in HEPES-buffered DMEM (pH
7.4) containing 0.5 mg/ml bovine serum albumin (DMEM+BSA). Fura-2/AM (1 mM stock in Me2SO) was added to a final
concentration of 2 µM, and the cells were incubated for
15 min at 37 °C in the dark. The cells were brought to a final
volume of 40 ml in DMEM+BSA, spun at 500 rpm for 10 min at 4 °C in a
benchtop centrifuge, and the cell pellet was resuspended in 10 ml of
Ca2+/Mg2+ HBSS. After counting, the cells were
diluted on ice into 3-ml aliquots containing 2 × 106
cells/ml. Just prior to assay, each aliquot was centrifuged briefly and
the pellet resuspended gently in 1 ml of
Ca2+/Mg2+ HBSS that was added to stirred
cuvettes containing 2 ml of pre-warmed Ca2+/Mg2+ HBSS. Agonists were added from
100-fold concentrated stocks, and the excitation ratio (340/380 nm) was
measured for emission intensity at 510 nm on a Perkin Elmer LS50
spectrometer.
Gel Mobility Shift Assays--
VSMC, or control 293 cells
transfected with pSH210, were treated with drugs as described in legend
to Fig. 5 before nuclei were harvested as described previously (15).
Nuclear extracts were prepared by gently resuspending the nuclei in 0.5 volumes of low salt buffer (20 mM HEPES, pH 7.9 at 4 °C,
1.5 mM MgCl2, 25% glycerol, 0.2 mM
EDTA, 20 mM KCl). An equivalent volume of high salt buffer
(low salt buffer except with 0.8 M KCl) was next added
dropwise with gentle mixing, and the nuclei were incubated on ice for
30 min. After centrifugation in a Beckman TLS55 rotor at 25,000 × g for 30 min at 4 °C, the supernatant was dialyzed against 500 volumes of a buffer identical to the low salt buffer above
except containing 100 mM KCl. Aliquots of the dialyzed
sample were frozen at -80 °C until use. Binding assays were
performed at 22 °C for 30 min in a 20-µl volume composed of
nuclear extract (containing 10 µg of VSMC nuclear protein or 1 µg
of 293/NFAT nuclear protein), 4.5 µg of bovine serum albumin, 2 µg
of poly(dI-dC), 12 mM Hepes (pH 7.9), 4 mM
Tris-HCl (pH 7.9), 80 mM KCl, 13% glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The probe used in binding
assays (wtNFRE) was derived from the distal NFRE in the human IL-2 gene
and formed by the oligonucleotides D79
(5'-GAAAGGAGGAAAAACTGTTTCATACAGAAGGCGT) and D80
(5'-GAACGCCTTCTGTATGAAACAGTTTTTCCTCCTT), which were end-labeled with 32P using T4 polynucleotide kinase. Unlabeled
competitor probes used in binding assays include mutNFRE, which was
formed by annealing oligonucleotides F87 and F88 (see above), and the
wtNFRE composed of oligonucleotides D79 and D80. After the binding
incubation period, the samples were loaded onto Tris-glycine
polyacrylamide gels, resolved by electrophoresis, and subjected to
autoradiography.
Western blot analysis indicates that a protein in VSMC reacts with
monoclonal antibody 7A6 (23), which was prepared against a polypeptide
from the NFATc1 isoform (Fig. 1). The
antibody specificity for the NFATc1 isoform is indicated by
immunoreactivity in extracts of COS-7 cells transfected with a human
cDNA for NFATc1, but not in extracts transfected with sham plasmid
or with a plasmid encoding the NFATc2 isoform (23). The heterogeneous
mobility of the immunoreactive VSMC protein is typically seen in
NFAT-expressing cells, and might result from either alternate exon
usage or various post-translational modifications, including
phosphorylation (24). No other currently available antibody directed
against the three other NFAT isoforms (12, 23, 25) is compatible with
our Western blot protocol, including the GH8-G1-G9 monoclonal antibody
directed against NFATc2 that is described below (data not shown).
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
q-coupled receptors, are significantly weaker
inducers of NFAT-mediated luciferase expression than is PDGF-BB.
However, co-stimulation studies show that G
q receptor
agonists augment the NFAT-mediated transcriptional response to PDGF-BB.
This synergy can be explained in part by augmented intracellular
Ca2+ transients elicited by multiple agonist challenges.
These data indicate that agonists for phospholipase C-coupled receptors
stimulate NFAT-mediated transcription in VSMC differentially, and that
NFAT can function to integrate co-activating signals in the
extracellular environment.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
activity
by coupling through the G
q class of heterotrimeric GTP-binding proteins. However, these latter agonists are weaker inducers of NFAT-mediated transcription than is PDGF-BB.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
globulin as standard. Samples were dissolved in SDS loading buffer,
heated for 5 min at 95 °C before separation on 10%
SDS-polyacrylamide gels, and transferred to Immobilon P membranes that
were blocked in 0.5% nonfat dried milk and incubated overnight with a
1:1000 dilution of either the anti-NFATc1 or anti-NFATc2 monoclonal
antibodies. The blots were developed using a chemiluminescent detection
kit (Phototope-HRP, New England Biolabs, Natick, MA) using 1:20,000 horseradish peroxidase (HRP)-labeled anti-mouse IgG secondary.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
NFATc1 immunoreactivity in VSMC
extracts. Figure shows 50 µg of VSMC protein (lanes 1 and 5), or 5 µg of extracts of COS cells transfected with
vector pCDM8 as sham, (lanes 2 and 6), an
expression vector encoding human NFATc2 (lanes 3 and
7), or a vector encoding human NFATc1 (lanes 4 and 8). Lanes 1-4 were developed after
incubating with monoclonal antibody 7A6 which binds NFATc1. Lanes
5-8 show background staining of a parallel blot incubated with
control mouse ascites IgG instead of 7A6. The result is representative
of three experiments.
Immunohistochemical analysis of cultured VSMC provides evidence that, in addition to NFATc1, NFATc2 is not only expressed in these cells, but has a subcellular pattern of expression distinct from that of NFATc1. In unstimulated cells, NFATc1 immunoreactivity is predominantly cytoplasmic, but occurs in a clustered pattern, consistent with vesicular localization (Fig. 2a). This basal pattern contrasts sharply with the more pronounced nuclear localization observed after a 10-min exposure to 1.0 µM ionomycin (Fig. 2c). Treatment of the cells with 1 µM CsA before and during challenge with ionomycin prevents ionomycin-induced nuclear translocation of NFATc1 (Fig. 2e). This calcium-dependent, CsA-sensitive nuclear transport is a characteristic property of NFAT (2, 24, 26). In striking contrast, NFATc2 immunoreactivity is predominantly nuclear in unstimulated cells (Fig. 2g), and this localization is unaffected by ionomycin treatment (Fig. 2i) or by ionomycin treatment in the presence of CsA (Fig. 2k). The specificity of these antibodies was confirmed in immunohistochemical control experiments, in which 293 cells were transfected with plasmids encoding each of the four major NFAT isoforms. Furthermore, there is no detectable staining in VSMC incubated with non-immune IgG as a negative control (data not shown). From these controls and the staining shown in VSMC, these data indicate that localization of various endogenously expressed NFAT isoforms differs in VSMC, suggesting they may be under control of different regulatory processes.
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In order to study control of NFAT-mediated transcription in VSMC, a retroviral-based luciferase transcriptional reporter system was developed to take advantage of the higher efficiency of retroviral gene transfer than is possible in VSMC using plasmid transfection approaches. The retroviral NFAT/luciferase reporter vector pKA7 is depicted in Fig. 3A. This vector contains a luciferase coding sequence under the control of a minimal human IL-2 promoter with an upstream triplex of the distal IL-2 gene NFRE. These elements were derived from a plasmid NFAT-responsive reporter vector whose fidelity has been characterized extensively, by ourselves and others (13, 19). A neomycin resistance gene is expressed from the viral 5'-LTR promoter, and selection for cells resistant to this antibiotic ensures that all cells in the culture have been infected with a retrovirus, which is important for comparing responses among the various control reporter vectors. In preliminary experiments, we found that NFAT-luciferase reporter vectors are substantially more responsive to stimuli when the orientation of luciferase transcription is directed toward the retroviral 5'-LTR, rather than when transcription of the two promoters are driven off of the same strand (data not shown). The control reporter vectors are shown in Fig. 3B and include a promoterless luciferase (pKA8), a luciferase driven by a minimal IL-2 promoter but lacking an NFRE (pKA9), and a minimal promoter with a NFRE possessing mutations that disrupt the NFAT-binding site within the NFRE (pKA10).
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The data shown in Fig. 3C indicate that highly inducible and synergistic luciferase expression consistent with NFAT-mediated transcription occurred only in VSMC infected with retrovirus prepared from plasmid pKA7, and that no responses were observed in cells infected with any of the other reporter viruses. Stimulation for 4 h with 100 nM PMA alone yielded a response that was 28 ± 1-fold over basal (mean ± S.E., n = 3), whereas the response to 1 µM ionomycin (1.3 ± 0.2-fold; mean ± S.E., n = 3) was no different from that of vehicle. Co-stimulation of VSMC with ionomycin and PMA resulted in a synergistic response that was 86 ± 10-fold over basal (mean ± S.E., n = 3), and was consistent with the known co-dependence of NFAT-mediated transcription upon simultaneous Ca2+ and protein kinase C signaling (27). The lack of response in VSMC harboring virus prepared from pKA10, which has mutations in the NFAT binding site in the NFRE, indicates that luciferase activity transcribed from the pKA7 vector is strictly dependent upon the binding of a protein to the well characterized NFAT binding site in the NFRE.
NFRE DNA binding assays were performed using nuclear extracts prepared from both VSMC and from 293 cells transfected with a plasmid encoding NFATc1 (Fig. 4). Both high and low mobility complexes (HMv and LMv, respectively) in VSMC extracts bound to this probe. Binding in the LMv was enhanced in extracts from cells co-stimulated with ionomycin and PMA, compared with either vehicle or drug treatment alone (compare lane 4 to lanes 1, 2, or 3). We interpret the enhanced presence of the LMv in co-stimulated cell extracts as indicative of an assembly of proteins, which is consistent with the formation of an NFAT transcriptional complex. As shown by comparing lanes 5, 6, and 7 to lanes 9, 10, and 11 in Fig. 5, lower concentrations of the NFRE containing an intact NFAT binding site (wtNFRE) competed more effectively for LMv binding than do lower concentrations the mutant NFRE (mutNFRE). Since the mutNFRE competitor contains mutations only in the NFAT binding site, and not the AP1-like site of the NFRE, it is also capable of inhibiting both LMv and HMv binding to the labeled probe, albeit at higher concentrations. However, the NFRE binding complexes are difficult to study at the relatively low levels of endogenous NFAT expression that occur in VSMC. The use of relatively high levels of nuclear proteins necessary in the binding assays precludes a definitive evaluation of what proteins in VSMC bind to this probe.
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For this reason, we also studied binding in extracts of 293 cells transfected with an NFATc1 expression vector, which is also shown in Fig. 4. No binding activity was observed in extracts prepared from untransfected 293 cells, whether or not they were treated with drugs (data not shown). However, both low and high mobility complexes formed on the radiolabeled wtNFRE probe (lane 13; arrows 3 and 4, respectively) in extracts prepared from transfected cells treated with ionomycin and PMA. An excess of the unlabeled wtNFRE competed for binding to both of these complexes quite effectively (lane 14). It is likely that both 293/NFAT complexes contained NFAT bound to the probe, with the low mobility complex (arrow 3) representing co-assembly of NFAT and an AP1-like dimer, whereas the high mobility complex represented binding of monomeric NFAT to the probe. This is consistent with the observation that the high mobility complex was resistant to a competitor DNA (mutNFRE) harboring mutations only in the NFAT binding site of the NFRE (lane 15, arrow 4). Thus, the mutNFRE appears capable of inhibiting binding of the NFAT transcriptional partner, presumably AP1, to the probe, but not NFAT. These data provide evidence that NFAT is incapable of binding to the mutNFRE, and are consistent with the lack of response in cells infected with retroviral reporter virus prepared from the vector pKA10 (Fig. 3C).
As shown in Fig. 5, stimulation of VSMC that were infected with retrovirus prepared from the pKA7 plasmid with PMA alone induces a luciferase response dose-dependently, representing a 21-fold maximal effect at 100 nM. Although ionomycin is itself a very weak activator, it synergized markedly the luciferase response of 100 nM PMA in a dose-dependent manner. A maximally effective concentration of CsA (1 µM) blocked completely this synergy between 100 nM PMA and ionomycin. This particular observation is characteristic of an NFAT-mediated transcriptional response and indicates that the retroviral reporter cassette mimics NFAT-mediated transcription faithfully. Although the 20-fold induction of luciferase in response to 100 nM PMA in the presence of low concentrations of ionomycin was unaffected by CsA treatment, it was decreased in the presence of CsA with increasing ionomycin concentrations. This indicates that the NFAT transcriptional response to PMA alone is regulated negatively by Ca2+, a feature that is revealed only when CsA blocks nuclear import of NFAT.
The influence of cAMP elevation on NFAT transcriptional activity in VSMC was investigated by examining the effects of forskolin on luciferase expression (Fig. 6). Unlike for PMA, forskolin (10 µM) alone did not stimulate luciferase above basal levels in VSMC infected with the NFAT reporter virus. However, luciferase levels were induced synergistically by increasing doses of ionomycin in the presence of 10 µM forskolin. The maximal response was 45 ± 8-fold over basal (mean ± S.E., n = 3) at 10 µM forskolin with 100 nM ionomycin. Since this effect was completely abolished in the presence of 1 µM CsA, it suggests that forskolin synergy with ionomycin represents NFAT-mediated transcription. At maximally effective concentrations of ionomycin, 10 µM forskolin co-treatment with 100 nM PMA gave a 111 ± 6-fold over basal response (mean ± S.E., n = 3), which was not different from the maximal effect of ionomycin and PMA treatment. However, the ionomycin dose-effect curve in the presence of forskolin and PMA was shifted leftward relative to that in the presence of PMA alone, indicating forskolin and PMA have either additive or synergistic effects on luciferase transcription at submaximal concentrations of ionomycin.
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It is of interest to understand what physiologic activators of VSMC
might be capable of controlling NFAT-mediated transcription in VSMC.
Fig. 7 shows dose-dependent
luciferase induction by the mitogen PDGF-BB in cultures of VSMC
infected with the NFAT reporter virus. PDGF-BB activated a tyrosine
kinase receptor leading to activation of signaling molecules including
PLC (28). The luciferase induction in the KA7 cells was blocked by 1 µM CsA, indicating the response to PDGF-BB was also
NFAT-mediated. We also sought to determine if agonists for
G
q-coupled receptors, which activate PLC
isoforms,
could stimulate NFAT-mediated transcription by stimulating NFAT
reporter virus infected VSMC with angiotensin II and thrombin. As shown
in Fig. 8, 100 nM angiotensin
II alone induced NFAT-mediated transcription approximately 1.6 ± 0.2-fold over basal (mean ± S.E., n = 3), whereas
the response to 100 nM thrombin was 7.4 ± 0.8-fold
over basal. These responses were significantly less than those elicited
by the growth factor PDGF-BB (24 ± 2-fold over basal).
Co-stimulation with PDGF-BB and either angiotensin II (38 ± 6-fold over basal), or thrombin (43 ± 5-fold over basal), or both
of these G
q-coupled receptor agonists (49 ± 6-fold
over basal) yielded a greater response than the sum of the agonists alone, indicating NFAT-mediated transcription in VSMC is synergized modestly by multiple agonist stimuli.
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Recent studies in T cells have suggested that Ca2+
transient duration is a critical determinant for committing NFAT to
transcription (26). The data shown in Fig.
9 compare the Ca2+ transients
elicited in VSMC by agonists alone and in combination, using Fura-2
spectrofluorometry. The Ca2+ transients elicited by the
Gq-coupled receptor agonists angiotensin II and thrombin
peak within 30 s after agonist addition and are for the most part
exhausted an additional 30 s later. These contrast with the
Ca2+ transient elicited by PDGF-BB, which is slower in
onset but significantly more prolonged than for the
G
q-coupled receptor agonists. The response to the
simultaneous addition of agonist mixtures behaved as a composite of
their individual effects. In particular, the rise of intracellular
Ca2+ elicited by co-stimulation of VSMC with PDGF-BB and
angiotensin II was more rapid than that seen with PDGF-BB alone, and
more sustained above basal levels than that seen with angiotensin II alone. The transient was even greater when the cells are stimulated with all three agonists. This augmented release of intracellular Ca2+ by two or more agonists provides one reasonable
explanation for the ability of G
q-coupled receptor
agonists to induce a synergistic effect on NFAT-mediated
transcriptional responses from the growth factor alone. However,
although angiotensin II mobilized Ca2+ more robustly than
did thrombin, the latter was a stronger activator of NFAT-mediated
transcription (see Fig. 8). Thus, the amplitude of the immediate-early
Ca2+ transient elicited by G
q-coupled
receptor activation is not a sufficient predictor of agonist induction
of NFAT-mediated transcription in VSMC.
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DISCUSSION |
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The use of a heterologous promoter reporter in this study, reconstructed from a T-cell-derived NFAT-responsive gene, was necessary because the genes which might be regulated by NFAT in VSMC are currently unknown. To our knowledge, the use of retroviral-based inducible promoter system to study the control of transcription by cell surface signaling has not been reported previously. This study shows the promise of this approach for analysis of transcriptional function in cultured cells that are otherwise difficult to transfect with plasmid DNA. The pattern of luciferase expression in response to PMA, ionomycin, and CsA in VSMC from this reporter vector is consistent with that known for PLC-mediated NFAT induction in other cells (2, 3, 19, 27), and serves to demonstrate that the retroviral reporter vector mimics NFAT-mediated transcriptional responses with fidelity.
With the exception of antigen receptors on lymphocytes, little is known
about how NFAT-mediated transcription might be controlled by endogenous
receptors for physiological agonists. This report shows that growth
factors can serve as strong physiological activators of NFAT-mediated
transcription in VSMC, whereas in comparison, agonists of
Gq-coupled receptors are relatively weaker activators of
NFAT. Previous studies have shown that NFAT-mediated transcription can
be strongly induced in both PC12 pheochromocytoma and Jurkat T cells
transfected with recombinant G
q-coupled muscarinic
receptors (13, 29). On the basis of these previous observations, we initially predicted that angiotensin II and thrombin treatment would be
more powerful inducers of NFAT-mediated transcription in VSMC than what
is in fact observed. There are several potential explanations for why
this is so. First, various cell phenotypes may differ in
receptor-mediated control of capacitive Ca2+ entry. A
proposal from previous studies suggests that a requirement for
prolonged cytosolic Ca2+ transients in NFAT-mediated
signaling may provide an important mechanism to prevent NFAT from
committing to gene induction in response to the relatively minor and
the brief Ca2+ transients elicited by various stimulators
of cell surface receptors in the course of normal cellular function
(26). The present results provide support for this hypothesis in that
PDGF-BB, which alone elicits a more sustained Ca2+
transient than either angiotensin II or thrombin, is a stronger activator of NFAT-mediated transcription than the latter agonists. Consistent with this is our observation that co-stimulation of the
cells with PDGF-BB and one or more agonists results in both more
pronounced Ca2+ release and also augmented NFAT-mediated
transcription.
The observation that thrombin is a stronger inducer of NFAT-mediated transcription but a weaker inducer of Ca2+ release than is angiotensin II (Fig. 8) suggests additional complexities are responsible for dictating NFAT responsiveness to receptor stimulation. Differential regulation of the receptors at the cell surface may in part explain how angiotensin II and thrombin elicit different magnitudes of NFAT responsiveness, perhaps reflecting that angiotensin II receptors desensitize more quickly than do thrombin receptors. Alternately, angiotensin II may be a weaker activator than thrombin of the signaling pathways responsible for inducing AP1 complexes, the NFAT transcriptional partner. Another possibility for the differential responsiveness to the two agonists is that thrombin and angiotensin II receptors may be expressed on distinct classes of cells in this primary culture, which differ in their intrinsic degree of NFAT-responsiveness.
Current evidence indicates that both nuclear import and export of NFAT
can be regulated dynamically (24, 26). Signals downstream of
Gq-coupled receptor activation may influence both of
these processes differentially and explain in part how these agonists
are weaker inducers of NFAT-mediated transcription. In T cells,
relatively profound and sustained cytosolic Ca2+ transients
such as those that occur following antigen receptor engagement appear
to be necessary to activate calcineurin and counterbalance the effects
of processes that effect nuclear export of NFAT (30). The short
duration Ca2+ transient elicited by agonists of
G
q-coupled receptors may provide a signal to stimulate
the export of nuclear NFAT; a signal which we suggest would serve to
compete against the effect of Ca2+ on the
calcineurin-dependent nuclear import pathway. One basis for
this postulate is the observation shown in Fig. 5 in which PMA-stimulated luciferase levels are reduced
dose-dependently by ionomycin in the presence of CsA.
Presumably, the 20-fold transcriptional response following PMA
treatment in the absence of Ca2+ ionophore reflects that
some level of NFAT pre-exists in the VSMC nuclei. Our data showing
nuclear localization of NFATc2 under basal conditions (Fig. 2) is
consistent with these functional responses and supports this
interpretation. CsA treatment does not lower the effect of PMA
stimulation alone, as anticipated since CsA only serves to block the
calcineurin-mediated nuclear import of NFAT. However, since increasing
ionomycin doses in the presence of CsA lead to a reduction in the
responsiveness to PMA, this result at face value indicates that a
Ca2+ signal can negatively regulate NFAT-mediated
transcription in VSMC. One postulate is that this might involve
activating some process stimulating the nuclear export of NFAT. As
shown in Fig. 2, brief ionomycin stimulation in the presence of CsA
does not yield any obvious export of the NFATc2 immunoreactivity from
the nucleus, which argues against this possibility. However, it would be premature to rule out this possibility for now given the relatively static nature of this experiment. The possibility remains that the rate
of nuclear export of either an NFAT isoform or a crucial component of
the complex necessary for NFAT-mediated transcription is enhanced by
Ca2+ signaling alone or by other kinases activated in this
experiment
We speculate that the Ca2+ signals of shorter duration
elicited by activation of the Gq receptors may
preferentially activate this putative negative regulatory processes,
whereas activation of calcineurin, dephosphorylation of NFAT and its
subsequent nuclear import require Ca2+ transients of longer
duration than these agonists can achieve alone. This notion is
consistent with recent studies providing evidence that a nuclear kinase
activity is involved in re-phosphorylating NFAT and exporting it to the
cytosol as a means for terminating its transcriptional activity (31).
Although protein kinase A (PKA) and glycogen synthase kinase-3 have
been implicated as the major NFAT kinases in Jurkat T-cells,
calmodulin-dependent kinases appear to have some NFAT
nuclear export activity as well in an heterotopic expression system
(30).
The novel finding shown here that ionomycin and forskolin act
synergistically to stimulate NFAT-mediated transcription suggests that
a down stream effector of cAMP-dependent signaling may be capable of partnering with NFAT isoforms on the NFRE. To our knowledge, co-activation of NFAT-mediated transcription in other cells through simultaneous stimulation of PKA and Ca2+ pathways has not
been reported. In general, the effects of stimulating PKA activity in
lymphoid cells is held to be immunosuppressive, although a recent study
both provides and extensively documents evidence suggesting otherwise
(32). The synergy between ionomycin and forskolin in VSMC suggests that
PKA activation is capable of bypassing the protein kinase C arm of the
signaling pathway. NFAT has been shown to cooperate with ATF-2/Jun
dimer binding to a closely spaced cAMP response element in the tumor
necrosis factor promoter to mediate transcription in response to
Ca2+ ionophore, but it is unknown to what degree
cAMP-dependent processes are involved in this effect (33).
Because little is understood generally about how cAMP-regulated
transcription factors function in VSMC, discovering whether NFAT
assembles with PKA-regulated transcription factors of the CREB/ATF
family (34) on the enhancer used in these studies in the VSMC cellular
context is of interest. Alternately, PKA may induce other AP1 proteins
via CREB-mediated effects and these, rather than CREB/ATF proteins,
assemble with NFAT (35, 36). No matter which of these possibilities
proves true, these observations highlight the attractiveness of NFAT mediated transcription in VSMC as a model for understanding mechanisms for integration of multiple co-activating cell surface signals into
defined gene expression control regimes.
The present finding that NFAT is expressed and regulated by physiological agonists in VSMC follows a recent observation of NFAT expression in cultured vascular endothelial cells (10), which together may have important implications. Atherosclerotic vascular lesions are composed of VSMC resembling those used in cell culture (37) and are exposed to episodic inflammatory insults (38) involving numerous autacoids, cytokines and growth factors which activate the expression of several types of genes (39-42). Since the present results demonstrate that NFAT is not only expressed in vascular smooth muscle cells, but is potentially a downstream coordinator of multiple signaling pathways, NFAT may participate in the expression of genes in pathological segments of the arterial wall. Furthermore, hypertension is among the more serious and common side effects of chronic CsA immunosuppressive therapy, and is thought to be secondary to CsA-mediated damage of renal vascular structures (43). To the extent that chronic inhibition of NFAT in these structures might account for this pathology, this might predict a subtle role for NFAT in the coordination of gene expression within normal VSMC in vivo. For instance, the organ damage leading to hypertension might reflect CsA-mediated interference with the ability of NFAT to modulate putative genes involved in feedback homeostatic control responses. Identification of the genes controlled by NFAT in VSMC will provide insight not only into the mechanisms of cardiovascular toxicity associated with immunosuppressive therapy, but perhaps also into mechanisms of normal adaptation in cardiovascular tone.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL52801, AR42687, AR43410, and DE11987 and by a grant-in-aid from the American Heart Association and Sanofi-Winthrop.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.
¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pharmacology, Emory University School of Medicine, Rm. 5031, O. W. Rollins Research Bldg., Atlanta, GA 30322. Tel.: 404-727-2467; Fax: 404-727-0365; E-mail: medtjm{at}bimcore.emory.edu.
1 The abbreviations used are: NFAT, nuclear factor of activated T cells; PLC, phospholipase C; CsA, cyclosporin A; VSMC, vascular smooth muscle cells; PMA, phorbol myristate acetate; PDGF-BB, platelet-derived growth factor BB chain; IL-2, the cytokine interleukin-2; HRP, horseradish peroxidase; PKA, protein kinase A; NFRE, NFAT response element; CREB, cAMP response element-binding protein; ATF, activating transcription factor; HBSS, Hank's balanced salt solution; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; LTR, long terminal repeat; GM, growth medium.
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REFERENCES |
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