From the Pulmonary, Critical Care, and Sleep
Division, Tufts-New England Medical Center, Tupper Research Institute,
Department of Medicine and § Jean Mayer United States
Department of Agriculture Human Nutrition Research Center on Aging,
Tufts University, Boston, Massachusetts 02111 and the
** Department of Pediatrics, Children's Hospital Medical
Center, University of Cincinnati, Cincinnati, Ohio 45229
Received for publication, October 11, 2002, and in revised form, February 24, 2003
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ABSTRACT |
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Serotonin (5-hydroxytryptamine (5-HT)) is a
mitogen of pulmonary artery smooth muscle cells (PASMC) and plays an
important role in the development of pulmonary hypertension. Signal
transduction initiated by 5-HT involves serotonin
transporter-dependent generation of reactive oxygen species
and activation of the MEK-ERK pathway. However, the downstream
transcriptional regulatory components have not been identified. In
systemic smooth muscle cells, GATA-6 has been shown to regulate
mitogenesis by driving cells into a quiescent state, and the
down-regulation of GATA-6 induces mitogenesis. Thus, the present study
tested the hypothesis that 5-HT induces mitogenesis of PASMC by
down-regulating GATA-6. Quiescent bovine PASMC were treated with 5-HT,
and the binding activity of nuclear extracts toward GATA DNA sequence
was monitored. Surprisingly, PASMC express GATA-4, and 5-HT
up-regulates the GATA DNA binding activity. Pretreatment of cells with
inhibitors of serotonin transporter, reactive oxygen species, and MEK
blocks GATA-4 activation by 5-HT. GATA-4 is not activated when the ERK
phosphorylation site is mutated, indicating that 5-HT phosphorylates
GATA-4 via the MEK/ERK pathway. GATA up-regulation is also induced by
other mitogens of PASMC such as endothelin-1 and platelet-derived
growth factor. Dominant negative mutants of GATA-4 suppress cyclin D2
expression and cell growth, indicating that GATA-4 activation regulates
PASMC proliferation. Thus, GATA-4 mediates 5-HT-induced growth of PASMC
and may be an important therapeutic target for the prevention of
pulmonary hypertension.
Exposure to chronic hypoxia leads to the development of
pulmonary hypertension through persistent vasoconstriction as well as
structural remodeling of pulmonary vessels. Proliferation of smooth
muscle cells is an important component of pulmonary vascular remodeling
that results in increased medial muscular wall thickness. Among a
number of mediators of pulmonary hypertension, serotonin (5-hydroxytryptamine (5-HT)1)
appears to play an important role in the remodeling of the pulmonary circulation (1-3).
Evidence for the role of 5-HT in the development of pulmonary
hypertension was first recognized in fawn-hooded rats, in which a
genetic deficit in 5-HT platelet storage and high plasma levels of 5-HT
are associated with a susceptibility to developing pulmonary hypertension in response to mild hypoxia (4). A further study showed
that a continuous intravenous infusion of 5-HT during a 2-week exposure
of rats to hypoxia potentiated the development of pulmonary
hypertension (2). The role of 5-HT in pulmonary hypertension appears to
be through the serotonin transporter (SERT), as mice deficient for SERT
developed less hypoxic pulmonary hypertension and vascular remodeling
than did control animals exposed to the same conditions (3).
High levels of plasma 5-HT have been observed in humans in association
with primary pulmonary hypertension (5, 6). More recently, Eddahibi
et al. (7) report that pulmonary artery smooth muscle cells
(PASMC) from patients with primary pulmonary hypertension grow faster
than the cells from control subjects due to increased expression of
SERT. The importance of 5-HT in human pulmonary hypertension is
supported by clinical observations that pulmonary hypertension is
associated with the use of amphetamine-like appetite suppressants,
fenfluramine, dexfenfluramine, and aminorex (8). The mechanism of these
appetite suppressants on PASMC may involve both direct interactions
with SERT and a stimulation of SERT expression (9-11).
5-HT can mediate cell signaling by interacting with several subtypes of
5-HT receptors or through SERT, which transports 5-HT across the
membrane using a Na+/Cl The GATA family of transcription factors includes six genes with a
highly conserved zinc finger DNA binding domain that interacts with DNA
regulatory elements containing consensus (A/T)GATA(A/G) sequence. The
GATA-1/2/3 genes are essential for hematopoietic cell development (19),
whereas the GATA-4/5/6 subfamily members are expressed in cells of the
cardiovascular system and endoderm-derived tissues (20). In aortic
smooth muscle cells, GATA-6 has been shown to play a role in
maintaining cells in a quiescent state, and mitogens down-regulate
GATA-6 to induce proliferation of cells (21). The mechanism appears to
involve p21Cip1, because the adenovirus-mediated
overexpression of GATA-6 was found to inhibit cellular entry into
S-phase by inducing this cyclin-dependent kinase inhibitor
(22). Furthermore, reversal of GATA-6 down-regulation in rats inhibited
intimal hyperplasia in the balloon-injured carotid artery (23).
The present study, therefore, tested the hypothesis that 5-HT may
down-regulate GATA-6 in PASMC. Surprisingly, however, our results show
that PASMC express GATA-4 that is activated by 5-HT and enhances PASMC growth.
Cell Culture and Reagents--
Bovine PASMC were isolated and
cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1%
penicillin/streptomycin, and 0.5% amphotericin B as previously
described (1). Cells (passages 0-6) were growth-arrested for 72 h
in medium containing 0.1% fetal bovine serum and treated with 5-HT
(Sigma-Aldrich). In some experiments, cells were treated with
imipramine, N-acetylcysteine, H2O2,
endothelin-1 (ET-1), or PDGF purchased from Sigma-Aldrich. Ebselen was
a kind gift from Dr. Helmut Sies (University of Dusseldorff). RPMI 1640 medium, penicillin/streptomycin, and amphotericin B were purchased from Invitrogen.
Adenoviral Infection--
The adenovirus-directed gene transfer
was implemented by adding the gene-carrying replication-deficient
adenovirus to cells grown in 6-well plates. Cells were incubated with
adenovirus in 0.75 ml of 0.1% fetal bovine serum-containing RPMI
medium with gentile shaking every 15 min. After 2 h, another 0.75 ml of medium was added, and cells were cultured for 48 h.
Adenovirus constructs expressing wild-type mouse GATA-4, dominant
negative GATA-4, and dominant negative MEK1 have been previously
described (24, 25). The site-specific mutation of serine 105 to alanine
was performed using a PCR-based strategy (26). The mutant was subcloned
as a 1.7-kilobase EcoRI fragment into the shuttle vector
pACCMCpLpA, and adenovirus was produced as previously described (24).
Adenovirus expressing GATA-6 was a kind gift from Dr. T. Evans (Albert
Einstein College of Medicine).
Electrophoretic Mobility Shift Assays (EMSAs)--
Cells were
washed in PBS and incubated in 10 mM Hepes (pH 7.8), 10 mM KCl, 2 mM MgCl2, 4 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride,
5 µg/ml leupeptin, 5 µg/ml aprotinin, 95 mM sodium
fluoride, 2.7 mM sodium orthovanadate, and 10 mM tetrasodium pyrophosphate for 15 min at 4 °C. Nonidet
P-40 was then added at a final concentration of 0.6%. Samples were
vigorously mixed and centrifuged. Pelleted nuclei were resuspended in
50 mM Hepes (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol and then mixed
and centrifuged, and the supernatant was harvested (27).
For EMSAs, the binding reactions were performed for 20 min in 5 mM Tris-HCl (pH 7.5), 37.5 mM KCl, 4% (w/v)
Ficoll 400, 0.2 mM EDTA, 0.5 mM dithiothreitol,
1 µg of poly(dI-dC)·poly(dI-dC), 0.25 ng (>20,000 cpm) of
32P-labeled double-stranded oligonucleotide, and 2 µg of
nuclear extract protein. Electrophoresis of samples through a
native 6% polyacrylamide gel was followed by autoradiography. The
double-stranded oligonucleotides containing two GATA consensus elements
5'-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3'
(Santa Cruz Biotechnology, Santa Cruz, CA) was used (27). The mutant
oligonucleotide had two GA to CT substitutions. Supershift experiments
were performed by incubating the nuclear extracts with 2 µg of
GATA-4, -5, or -6 antibody (Santa Cruz Biotechnology) at 4 °C for
1 h before the addition of 32P-labeled oligonucleotide.
Western Blot Analysis--
To prepare lysates, the cells were
washed in phosphate-buffered saline and solubilized with 50 mM Hepes solution (pH 7.4) containing 1% (v/v) Triton
X-100, 4 mM EDTA, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium
pyrophosphate, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Cell lysates (10 µg of
protein) were electrophoresed through a reducing SDS-polyacrylamide gel
and electroblotted onto a membrane. The membrane was blocked and
incubated with polyclonal IgG for phospho-specific MEK1/2 (Cell
Signaling Technology, Beverly, MA), ERK1/2, cyclin D2, or GATA-4 (Santa
Cruz Biotechnology). The levels of proteins and phospho-proteins were
detected with horseradish peroxide-linked secondary antibodies and the
ECL System (Amersham Biosciences).
Immunoprecipitation experiments were performed by incubating nuclear
extracts with 1 µg of rabbit anti-GATA-4 or GATA-6 IgG and 10 µl of
GammaBind G-Sepharose (Amersham Biosciences) overnight at 4 °C while
gently shaking. After washing twice, the pellet was boiled in Laemmli
buffer and centrifuged, and the supernatant was electrophoresed. A
Western blot was performed using goat anti-GATA-4 or GATA-6 IgG.
Statistical Analysis--
Means ± S.E. were calculated and
statistically significant differences between two groups were
determined by the Student's t test at p < 0.05.
5-HT Activates GATA DNA Binding Activity--
To test the
hypothesis that 5-HT may down-regulate GATA-6 activity, nuclear
extracts from 5-HT-treated PASMC were subjected to EMSA using
32P-labeled oligonucleotide probe containing the consensus
GATA sequence. Surprisingly, 5-HT was found to enhance GATA DNA binding activity (Fig. 1A). The
activation of GATA DNA binding activity by 5-HT was observed as early
as 3 h and was sustained for at least 20 h. The densitometry
analysis revealed that treatment of cells with 5-HT (1 µM) for 20 h caused a 5-fold increase in GATA DNA
binding activity. Experiments using cold competitor oligonucleotides containing wild-type and mutated GATA elements showed that the 5-HT-regulated GATA proteins specifically interacted with the consensus
GATA sequence (Fig. 1B). Fig. 1C shows that this
5-HT-induced GATA activation is blocked by pretreating cells with
imipramine, an inhibitor of SERT (14). Imipramine alone did not alter
the GATA DNA binding activity (data not shown).
Identification of GATA Transcription Factors Expressed in
PASMC--
To determine the identity of the GATA-binding protein(s) in
PASMC, supershift experiments were performed using antibodies against
GATA-4, -5, and -6. As shown in Fig.
2A, the antibody against
GATA-6 reduced the intensity of the GATA binding complex and caused a
supershift as shown by the arrow, indicating that GATA-6 is
expressed in PASMC. Surprisingly, the third and
fourth lanes of Fig. 2A show that two distinct
GATA-4 antibodies from goat (C-20) and rabbit (H-112) affected the GATA
binding complex. The C-20 antibody caused a supershift of the GATA
band, indicating that the antibody interacted with the
DNA·GATA-4 complex, resulting in a band with reduced mobility.
Although the H-112 antibody did not cause a supershift, it
reduced the intensity of the GATA band, indicating that the antibody
interacted with the GATA-4 molecule but also interfered with the
DNA-protein interactions. On the other hand, the effect of the GATA-5
antibody was minimal. These results indicate that GATA-4 and -6 contribute to the formation of the GATA-binding protein complex. To
confirm that GATA-4 is expressed in PASMC, nuclear extracts were
immunoprecipitated with rabbit anti-GATA-4 IgG (H-112) and blotted
with goat anti-GATA-4 IgG (C-20) in Western blot experiments. As shown
in Fig. 2B, a band was detected at ~50 kDa, the same
position as the control GATA-4 protein from adenovirally overexpressing
cells. This band was not observed when the samples were
immunoprecipitated with anti-GATA-6 IgG (data not shown).
Mechanism of 5-HT-induced GATA Activation--
Signal transduction
produced by 5-HT has been shown to be inhibited by antioxidants such as
N-acetylcysteine, indicating the role of ROS in the process
(18). Pretreatment of cells with this antioxidant consistently blocked
the 5-HT-induced GATA activation (Fig.
3A). Similar results were
obtained when cells were pretreated with ebselen, a selenium-containing
glutathione peroxidase mimic that scavenges
H2O2 (Fig. 3A). Antioxidants by
themselves did not alter the GATA DNA binding activity (data not
shown). Furthermore, H2O2 enhanced GATA DNA
binding activity (Fig. 3B). These results indicate that 5-HT
activates GATA DNA binding activity via the generation of ROS.
The mechanism of 5-HT-induced GATA activation could be via (i)
increased synthesis of GATA factors or (ii) post-translational modifications. To determine whether the gene transcription of GATA
factors may be enhanced in response to 5-HT, protein expressions of
GATA-4 and GATA-6 were determined. GATA-4- and GATA-6-enriched samples
were prepared from the nuclear extracts of cells with or without 5-HT
treatment via immunoprecipitation using rabbit anti-GATA-4 and GATA-6
IgG. As shown in the top panel of Fig. 4A, samples from 5-HT-treated
cells exhibited higher GATA DNA binding activity. However, Western
blotting of these samples with goat anti-GATA-4 or GATA-6 IgG showed
that 5-HT did not increase the expression of GATA-4 or GATA-6 (Fig.
4A). Furthermore, in cells pretreated with 15 µg/ml
actinomycin D (a general inhibitor of gene transcription), 5-HT still
enhanced the GATA DNA binding activity (data not shown). 5-HT also
enhanced the activity of exogenously expressed GATA-4 via
adenovirus-mediated gene transfer (Fig. 4B). The
5-HT-induced activation of exogenous GATA-4 was detectable as early as
10 min. In contrast, the activity of exogenously expressed GATA-6 was
not enhanced by 5-HT (Fig. 4B, lower panel). These results suggest that 5-HT exerts post-translational modifications of GATA-4 to enhance its activity.
Because 5-HT-induced mitogenesis has been shown to be blocked by
PD98059 (18) and GATA-4 can be phosphorylated via the MEK-ERK pathway
in cardiac muscle cells (25, 27, 28), 5-HT may activate GATA-4 through
ERK-dependent phosphorylation. Thus, the role of the
MEK-ERK pathway in the 5-HT-induced GATA-4 activation was tested. As
shown in Fig. 4C, 5-HT caused the activation of MEK as
determined using a phospho-specific antibody. The 5-HT-induced GATA
activation was blocked by pretreatment of cells with PD98059 (a MEK
inhibitor) (Fig. 4D) or with a dominant negative mutant of
MEK (Fig. 4E), suggesting that the signaling is dependent on MEK and perhaps ERK. PD98059 (data not shown) and adenovirus expressing a dominant negative mutant MEK (Fig. 4E) by themselves did
not alter the GATA DNA binding activity.
To determine the role of ERK-dependent phosphorylation in
the 5-HT-induced GATA-4 activation, cells were infected with adenovirus expressing mutant mouse GATA-4 in which serine 105 is replaced with
alanine (S105A). This serine is the preferential ERK phosphorylation site and has been shown to play a major role in the phosphorylation and
activation of GATA-4 in cardiac myocytes (25). The EMSA results showed
that, although 5-HT enhanced the DNA binding activity of wild-type
GATA-4, the S105A mutant of GATA-4 was not activated (Fig.
4F). Thus, the MEK/ERK-dependent phosphorylation
of the serine 105 residue of GATA-4 appears to be involved in the
5-HT-induced GATA-4 activation in PASMC.
Role of GATA-4 in PASMC Growth--
To characterize the functions
of 5-HT-induced activation of GATA-4 in PASMC, the potential role in
cell mitogenesis was addressed. We first asked whether other mitogens
of PASMC also activate GATA-4. An important mitogen of PASMC and a
mediator of pulmonary hypertension, ET-1 was also found to activate the
GATA DNA binding activity. As shown in Fig.
5A, the EMSA show that the
nuclear binding activity toward the GATA sequence was enhanced by ET-1.
The densitometric analysis revealed that a 20-h treatment with 30 nM ET-1 caused a 5-fold increase in the GATA activity.
Treatment of PASMC with PDGF also induced a 4-fold increase in the GATA
DNA binding activity (Fig. 5B). Furthermore,
PDGF plus 5-HT exerted synergistic enhancement (11-fold) of GATA DNA binding activity (Fig. 5B), consistent with the synergistic
ability of these agents to induce PASMC proliferation (1).
To determine the role of GATA-4 in mitogenesis of PASMC, we studied the
regulation of cyclin D2 expression because the promoter region of
cyclin D2 gene contains GATA elements (29, 30). In PASMC, both 5-HT and
ET-1 enhanced the protein expression of cyclin D2, suggesting that this
cell cycle regulator may play a role in the PASMC mitogenesis (Fig.
6A). Furthermore, forced expression of GATA-4 caused an increase in cyclin D2 expression (Fig.
6B), suggesting that gene regulation of this protein may be
GATA-4-dependent. An increase in cyclin D2 expression
alone, however, is not sufficient to elicit mitogenesis because forced expression of GATA-4 did not significantly increase the cell number (data not shown). To directly test the role of GATA-4 in 5-HT-induced PASMC growth, cells were infected with adenovirus expressing
GATA-4-engrailed fusion protein (AdG4-Engr) with dominant negative
GATA-4 activity (24). After 48 h of infection, the expression of
the dominant negative mutant of GATA-4 alone caused a reduction of the
cell number (Fig. 6C). Because this effect could be due to
suppressed cell growth or increased cell death, the effects of the
dominant negative GATA-4 on cyclin D2 expression was tested. The
results show that the expression of the dominant negative mutant GATA-4 alone down-regulated the expression of cyclin D2 without influencing ERK expression (Fig. 6D). Furthermore, 5-HT did not enhance
the cyclin D2 expression in dominant negative GATA-4-expressing cells (Fig. 6E). These results suggest that GATA-4 regulates the
expression of cyclin D2, and suppression of this transcription factor
affects the growth of PASMC.
To further explore the role of ERK-dependent
phosphorylation of GATA-4 in PASMC growth, adenovirus expressing mutant
GATA-4 with serine 105 replaced with alanine (S105A) was used. We found that this mutant also suppressed the constitutive cell number (Fig.
6F) as well as cyclin D2 expression (Fig. 6G).
These results suggest that the S105A GATA-4 mutant may serve as a
dominant negative mutant and that the phosphorylation of this site
within the GATA-4 molecule plays a role for cell growth and/or survival.
5-HT plays an important role in the development of pulmonary
hypertension, in part due to its ability to induce PASMC growth (1).
The 5-HT signaling has been identified to involve 5-HT transport
through SERT, tyrosine phosphorylation of GTPase-activating protein,
which may in turn activate Ras-NAD(P)H oxidase and result in production
of ROS and the activation of MEK and ERK (14-18). There is little
known about transcription factor activation by 5-HT. In skeletal muscle
myoblasts, 5-HT has been shown to activate 5-HT2A receptor to stimulate
the Jak-STAT (signal transducers and activators of transcription)
pathway (31), and 5-HT-dependent collagenase transcription
in myometrial cells requires an AP-1 site (32). Similarly,
5-HT-inducible interleukin-1 transcription in uterine smooth muscle
utilizes an AP-1 site (33). In the present study, we considered the
mechanism identified by Walsh and co-workers for aortic smooth muscle
cell proliferation, which involves the down-regulation of GATA-6 (21,
22). Thus, we initially hypothesized that 5-HT down-regulates GATA-6 in
PASMC. Surprisingly, we found that 5-HT increased the GATA DNA binding activity in PASMC and that these cells also express GATA-4
transcription factor that is regulated by 5-HT signal transduction.
Thus, GATA factors may respond differently in systemic and pulmonary
smooth muscle cell regulation.
Important GATA-binding sites have been identified in multiple
cardiac-specific transcriptional regulatory regions, and overexpression of GATA-4 has been shown to transactivate these elements. A number of
proteins that have been shown to be up-regulated during cardiac hypertrophy such as angiotensin II type 1a receptor, Functional roles of GATA-4 activation by 5-HT may include the
regulation of PASMC proliferation. GATA elements have been found in
promoter regions of cell growth-regulating proteins such as cyclin D2
(30) and cyclin D3 (35). Tanaka et al. (36) report that
GATA-1 induced the sustained expression of cyclin D1 in a murine
myeloid cell line M1. GATA-1 has also been shown to regulate the
proliferation of definitive erythroid and megakaryocytic cells (37).
Tsai and Orkin (38) report that GATA-2 is required for the
proliferation of hematopoietic cells. Results from the present study
showing that the specific suppression of GATA-4 activity by a dominant
negative mutant decreased the cell number and down-regulated the
expression of cyclin D2 indicate that GATA-4 regulates mitogenesis of
PASMC. Furthermore, other mitogens of PASMC such as ET-1 and PDGF also
activate GATA DNA binding activity, suggesting that GATA-4 may be a
universal mediator of PASMC proliferation.
Another possible role of GATA-4 activation is to regulate cell
apoptosis and survival. In cardiac myocytes, GATA-4 was recently identified as a cell survival factor, and down-regulation of this transcription factor resulted in the induction of apoptosis (39, 40). Consistently, we found that 5-HT inhibited apoptosis of PASMC
(41).
In summary, the present study demonstrates that PASMC express GATA-4
that is activated by 5-HT. From the data we have obtained, the 5-HT
signal transduction for GATA-4 activation appears to involve the SERT,
ROS, MEK/ERK pathway and the phosphorylation of serine 105, the
preferential ERK phosphorylation site within the GATA-4 molecule (Fig.
7). Furthermore, adenovirus-mediated gene
transfer of dominant negative GATA-4 mutants indicates that GATA-4
regulates the expression of cyclin D2 and PASMC growth. These results
suggest that GATA-4 may play a role in 5-HT-induced PASMC growth and
that this cascade of events may participate in the development of
pulmonary hypertension. Thus, therapeutic strategies targeting GATA-4
may be useful against pulmonary hypertension.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gradient. In bovine
PASMC, only one 5-HT receptor has been identified, which resembles the
5-HT1A or 5-HT4 receptor (12, 13). However, mitogenic and hypertrophic
responses by 5-HT are believed to be due to the action of SERT (14).
The mechanism of 5-HT signaling for PASMC growth through SERT has been
shown to involve tyrosine phosphorylation of GTPase-activating protein
(15) and the production of reactive oxygen species (ROS) such as
superoxide (16) and H2O2 (17), presumably via
the activation of NAD(P)H oxidase. These signaling events appear to
activate the MEK-ERK pathway as PD98059 blocked the 5-HT-induced PASMC
growth (18). However, downstream signaling components, which are
activated by ERK for the 5-HT-mediated induction of cell growth, have
not yet been identified.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
5-HT enhances the GATA DNA binding
activity. A, PASMC were treated with 5-HT (1 µM) for 20 h. Nuclear extracts were prepared, and
the GATA DNA binding activity was monitored by EMSA. The bar
graph indicates the means ± S.E. of the intensity of the
GATA band determined by densitometry in arbitrary units
(a.u.). The asterisk denotes a significant
difference from the untreated control value at p < 0.05 (n = 12). B, nuclear extracts were
incubated with 32P-labeled oligonucleotide containing
consensus GATA sequence in the presence of increasing amounts of cold
competitors containing wild-type or mutated GATA elements.
C, PASMC were pretreated with imipramine (100 µM) for 30 min and then treated with 5-HT (1 µM) for 20 h. Nuclear extracts were isolated, and
the GATA DNA binding activity was monitored.
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Fig. 2.
Identification of the GATA DNA-binding
proteins. A, nuclear extracts from PASMC were incubated
with 2 µg of antibodies (ab) against goat anti-GATA-6,
goat anti-GATA-4 (C-20), rabbit anti-GATA-4 (H-112), or rabbit
anti-GATA-5 before the addition of 32P-labeled
oligonucleotide for EMSAs. The arrow indicates the
supershifted bands. B, PASMC nuclear extracts
(NE) were immunoprecipitated (IP) with the GATA-4
(H-112) antibody. Samples were boiled, loaded on a 10% SDS-PAGE gel,
and blotted with the GATA-4 (C-20) antibody. Nuclear extracts from
cells infected with adenovirus expressing mouse GATA-4 (AdGATA-4) was
used as a control without immunoprecipitation.
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Fig. 3.
Role of reactive oxygen species in 5-HT
signaling for GATA activation. A, PASMC were pretreated
with N-acetylcysteine (1 mM) or ebselen (20 µM) and then treated with 5-HT (1 µM) for
20 h. Nuclear extracts were isolated, and the GATA DNA binding
activity was monitored. B, PASMC were treated with hydrogen
peroxide (H2O2) for 20 h. Nuclear extracts
were prepared, and the GATA DNA binding activity was monitored.
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Fig. 4.
Role of MEK/ERK-dependent
phosphorylation in the mechanism of 5-HT signaling for GATA
activation. A, nuclear extracts from PASMC with or
without treatment with 5-HT (1 µM) for 20 h were
immunoprecipitated with rabbit anti-GATA-4 or GATA-6 IgG. Samples were
subjected to EMSA to monitor GATA DNA binding activity. Samples were
boiled, loaded on a 10% SDS-PAGE gel, and blotted with goat
anti-GATA-4 or GATA-6 IgG in Western blot (WB) experiments.
The bar graphs indicate means ± S.E. of the
intensities of GATA-4 and GATA-6 bands in Western blot gels as
determined by densitometry (n = 4). a.u.,
arbitrary units. B, PASMC were infected with adenovirus
expressing GATA-4 (AdGATA4) or GATA-6 (AdGATA6)
and treated with 5-HT for 10 min. Nuclear extracts were isolated, and
the GATA DNA binding activity was monitored by EMSA, and protein
expression levels were determined by Western blot. C, PASMC
were treated with 5-HT (1 µM) for 5 min. Cell lysates
were prepared, and the activation of MEK was monitored by Western blot
using phospho-specific MEK antibody. D, PASMC were
pretreated with PD98059 (PD, 30 µM) and then
treated with 5-HT for 20 h. Nuclear extracts were isolated, and
the GATA DNA binding activity was monitored. E, PASMC were
infected with 50 plaque-forming units of adenovirus expressing
dominant negative MEK1 (AdDN-MEK) or control adenovirus
(AdCont) and then treated with 5-HT for 20 h. Nuclear
extracts were isolated, and the GATA DNA binding activity was
monitored. The bar graph indicates means ± S.E. of the
intensity of GATA band as determined by densitometry (n = 4). F, PASMC were infected with adenovirus (Ad)
expressing wild type GATA-4 (WT) or mutant GATA-4 with
serine 105 substituted with alanine (S105A) for 48 h and then
treated with 5-HT (1 µM) for 10 min. Nuclear extracts
were prepared, GATA DNA binding activity was monitored by EMSA, and
GATA-4 expression was determined by Western blot (WB). The
bar graph indicates means ± S.E. of fold-increase in
GATA DNA binding activity in response to 5-HT treatment as determined
by densitometry. The asterisk denotes significant difference
at p < 0.05 (n = 3).
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Fig. 5.
ET-1 and PDGF also activate GATA DNA binding
activity. A, PASMC were treated with ET-1 (30 nM) for 4-20 h. Nuclear extracts were prepared, and the
GATA DNA binding activity was monitored by EMSAs. The bar
graph indicates means ± S.E. of the intensity of GATA band
at 20 h post-treatment as determined by densitometry
(n = 9). The asterisk denotes the value that
is significantly different from untreated control value at
p < 0.05. B, PASMC were treated with a
combination of 5-HT (1 µM) and PDGF (10 ng/ml), PDGF
alone, or 5-HT alone for 20 h. Nuclear extracts were prepared, and
the GATA DNA binding activity was monitored by EMSAs. The bar
graph indicates means ± S.E. of the intensity of GATA band
as determined by densitometry. a and b denote
that the values are significantly different from the untreated value
and 5-HT + PDGF-treated value, respectively, at p < 0.05 (n = 4).
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Fig. 6.
Evidence for the role of GATA-4 in
mitogenesis. A, PASMC were treated with 5-HT (1 µM) or ET-1 (30 nM) for 20 h. Cell
lysates were prepared, and cyclin D2 protein expression was determined
by Western blot analysis. The bar graph shows the means ± S.E. of the intensity of cyclin D2 bands as determined by
densitometry analysis (n = 3). B, PASMC were
infected with control adenovirus without inserts (AdCont) or
adenovirus expressing GATA-4 (AdGATA4) for 48 h. Cell
lysates were prepared, and cyclin D2 protein expression was monitored
by Western blot analysis. The bar graph represents the
means ± S.E. of the intensity of cyclin D2 bands as determined by
densitometry analysis (n = 4). a.u.,
arbitrary units. C, PASMC were infected with AdG4-Engr or
control adenovirus for 48 h. Numbers of cells were determined by
trypan blue exclusion on a hematocytometer. The asterisk
denotes the value as significantly different from the control value at
p < 0.05 (n = 3). D, PASMC
were infected with control adenovirus (AdCont) or
GATA-4-engrailed fusion protein (AdG4-Engr) with dominant
negative GATA-4 activity for 48 h. The protein levels of cyclin D2
and ERK were monitored by Western blot in cell lysates. The bar
graph indicates the means ± S.E. of the intensity of cyclin
D2 band as determined by densitometry (n = 5).
E, PASMC were infected with control adenovirus
(AdCont) or GATA-4-engrailed fusion protein
(AdG4-Engr) with dominant negative GATA-4 activity for
48 h and then treated with 5-HT (1 µM) for 20 h. The protein levels of cyclin D2 and ERK were monitored by Western
blot in cell lysates. The ERK antibody was used for loading controls.
F, PASMC were infected with control adenovirus or adenovirus
expressing mutant GATA-4 with serine 105 replaced with alanine (AdGATA4
(S105A)) for 48 h. Cell number was determined by trypan blue
exclusion on a hematocytometer. The asterisk denotes the
value as significantly different from the control value at
p < 0.05 (n = 3). G, PASMC
were infected with control adenovirus or AdGATA4 (S105A) for 48 h.
The protein levels of cyclin D2 and ERK were monitored by Western blot
in cell lysates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-myosin heavy
chain,
-myosin heavy chain, myosin light chains, atrial natriuretic
factor, and brain natriuretic factor contain GATA regulatory sites in
their promoters (20). Thus, GATA-4 has been postulated to regulate
cardiac muscle cell hypertrophy. GATA-4 can be activated by a signal
transduction pathway involving calcineurin and NFAT3 (34). More
recently, GATA-4 has shown also to be phosphorylated via the MEK/ERK
pathway in response to ET-1 (27) or phenylephrine (28) in cardiac
myocytes. Serine 105, the preferential ERK phosphorylation site, was
identified to be phosphorylated in response to phenylephrine (25).
Consistent with these findings, 5-HT-mediated activation of GATA-4 in
PASMC requires MEK/ERK-dependent phosphorylation of serine
105 because MEK inhibitors blocked 5-HT-induced GATA activation, and
the substitution of the ERK phosphorylation site serine 105 with
alanine abolished the activation. This novel regulation for GATA-4 in
PASMC, however, does not necessarily preclude a role of GATA-6 in
mitogenesis as described in other vascular beds (21).
View larger version (13K):
[in a new window]
Fig. 7.
Proposed signal transduction pathway for the
5-HT-induced mitogenesis of PASMC. 5-HT, via SERT, induces the
generation of ROS, which in turn activate MEK and ERK. Phosphorylation
(P) and activation of GATA-4 enhances gene transcription of
cell cycle regulators such as cyclin D2 and promotes mitogenesis of
PASMC.
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ACKNOWLEDGEMENTS |
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We thank S. Fitch, Y. Kim, and J. Guo for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants R01-HL32723 (to B. L. F.) and R01-HL67340 (to Y. J. S.) and by the American Heart Association, New England Affiliate (to Y. J. S.), Massachusetts Thoracic Society/American Lung Association of Massachusetts (to Y. J. S.), and the American Lung Association (to R. M. D.). This material is based upon work supported by United States Department of Agriculture under cooperative agreement 58-1950-9-001.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.
¶ To whom correspondence should be addressed: USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington St., Boston, MA 02111. Tel.: 617-556-3148; Fax: 617-556-3344; E-mail: yuichiro.suzuki@tufts.edu.
Recipient of the Career Development Award from the American
Heart Association National Center.
Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M210465200
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ABBREVIATIONS |
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The abbreviations used are: 5-HT, 5-hydroxytryptamine; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; SERT, serotonin transporter; PASMC, pulmonary artery smooth muscle cells; ROS, reactive oxygen species; ET-1, endothelin-1; PDGF, platelet-derived growth factor; EMSA, Electrophoretic mobility shift assay.
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