From the Program in Cell, Molecular, and
Developmental Biology, Tufts University, Sackler School of Biomedical
Studies, Boston, Massachusetts 02111, the § Division of
Cardiovascular Research, St. Elizabeth's Medical Center, Tufts
University School of Medicine, Boston, Massachusetts 02135, and the
Division of Nephrology, Department of Medicine, Case
Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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GATA transcription factors represent a family of highly conserved zinc finger proteins with tissue-specific expression patterns. Previous studies have shown that GATA-6 is expressed in vascular smooth muscle cells (VSMCs) and rapidly down-regulated when VSMCs are induced to proliferate. Here we investigated whether the GATA-6 transcription factor can modulate cellular proliferation. Transient transfection with a GATA-6 expression vector inhibited S-phase entry in VSMCs and in mouse embryonic fibroblasts (MEFs) lacking both p53 alleles. The GATA-6-induced growth arrest correlated with a marked increase in the expression of the general cyclin-dependent kinase (Cdk) inhibitor p21. In contrast to p53-deficient MEFs and VSMCs, MEFs null for both p21 alleles were refractory to the GATA-6-induced growth inhibition. These data demonstrate that elevated GATA-6 expression can promote the quiescent phenotype in VSMCs.
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INTRODUCTION |
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The GATA transcription factors are required for establishing hematopoietic cell lineages and for the proper formation of the cardiovascular system. GATA-1/2/3 isoforms are predominantly expressed in hematopoietic cells, and disruption of each of these genes results in deficiencies in hematopoiesis and embryonic lethality (1). In contrast, the more recently identified GATA-4/5/6 genes represent a subfamily of factors that are expressed in cardiovascular tissues (2, 3). Embryos that lack GATA-4 fail to develop a centralized heart tube and foregut (4, 5), and antisense oligonucleotides to GATA-4 inhibit the differentiation of P19 embryonic carcinoma cells into beating cardiac myocytes (6). Little is known about the functions of GATA-5 and -6, but their expression patterns overlap with that of GATA-4 in the developing cardiovasculature. Both GATA-4 and -6 are expressed in heart throughout development, while GATA-5 is expressed during early heart development (7, 8). Of note, GATA-4 and -6 are co-expressed in the vasculature during early development, but only GATA-6 is detected in vessels post-day 13.5 of mouse embryonic development (7-9). Moreover, GATA-6 mRNA expression is also detected in cultured rat and human VSMCs1 (7, 10).
Unlike cardiac and skeletal muscle cells, VSMCs are not terminally differentiated and can dedifferentiate and reenter the cell cycle in response to multiple stimuli (11, 12). The proliferation of VSMCs contributes to neointimal lesion formation in response to chronic or acute vessel wall injury (13, 14). Following acute injury, the quiescent VSMCs of the vessel wall up-regulate cyclin and Cdk expression and rapidly re-enter cell cycle in a synchronous manner (15). As lesion size increases the proliferative capacity of VSMCs decreases, and this correlates with an up-regulation of the Cdk inhibitors p21Cip1 and p27Kip1 (16-18).
In cultured VSMCs, GATA-6 expression is rapidly and transiently down-regulated following mitogen stimulation, suggesting that GATA-6 might have a role maintaining VSMCs in a quiescent state (10). In this study, we analyzed the cell cycle regulatory properties of the human GATA-6 factor. The forced expression of GATA-6 inhibited S-phase entry in VSMCs and non-muscle cells. GATA-6 overexpression resulted in the induction of the Cdk inhibitor p21, and the cell cycle inhibitory activity of GATA-6 was markedly attenuated in p21-deficient cells. These data suggest that GATA-6 may function in VSMCs to coordinate cell cycle activity with the state of cellular differentiation.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
Cells were incubated at 37 °C in
Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine
serum (FBS) and penicillin/streptomycin. Primary cultures of rat smooth
muscle cells were prepared from thoracic aortas of adult male
Sprague-Dawley rats according to Mader et al. (19). COS1
cells were cultured in DMEM with 10% FBS. Mouse embryonic fibroblasts
with a homozygous deletion of both p53 alleles designated p53/
MEFs
were a generous gift from Dr. Arnold Levine (20). Both p21 knockout
mice and passage 28 mouse embryonic fibroblasts that contain a
homozygous disruption of both p21 alleles (p21
/
MEFs) were generous
gifts from Dr. Philip Leder (21). Early passage embryonic fibroblasts
derived from p21 knockout mice (21) were prepared from day 13.5 embryos as described previously (22). p53
/
MEFs were induced to the quiescent state by serum starvation for 3 days in 0.5% FBS/DMEM, while
early and late passage p21
/
MEFs cells were serum-starved in 0.2%
FBS/DMEM for 4 days.
Adenovirus, Plasmids, and Transfections--
Human GATA-6 wild
type cDNA was subcloned into pCDNA1/Amp vector (Invitrogen) at
HindIII and XbaI sites (pCDNA1-hGATA-6wt). The zinc finger domains deletion mutant, which lacked codons 244 to
306, was prepared by digesting pBS-hGATA-6wt with EcoRI and PflmI and ligating with a double-stranded oligonucleotide
(ZF1: sense strand,
ZF1: antisense strand).
Electrophoretic Mobility Shift Assay-- Whole-cell extracts were prepared from COS1 cells. In brief, cells were washed twice in PBS, removed from culture dishes by scraping, and collected by centrifugation. The pellet was resuspended in an equal volume of 2 × lysis buffer (20 mM HEPES-KOH (pH 7.8), 0.6 M KCl, 1 mM dithiothreitol, 20% glycerol, 2 mM EDTA, 2 µg/ml leupeptin) and subjected to three cycles of freezing and thawing. After centrifugation at 16,000 × g for 10 min at 4 °C, protein concentration was measured by the Bradford method according to the direction of the manufacturer (Bio-Rad). Electrophorectic mobility shift assays were carried out in reaction mixtures containing 5-20 µg of extract, 20 fmol of probe, 1 µg of poly(dL-dC), and when indicated 200 ng of single-stranded oligonucleotide as nonspecific DNA competitors. Electrophoresis was carried out on 5% nondenaturing polyacrylamide gels with 0.5 × TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA) in a circulating water-cooled gel box. A probe to the GATA site was prepared from the mouse globin gene. Probes, and competitor DNAs were double-stranded synthetic oligonucleotides, and the coding-strand sequences are as follows: GATA, 5'-GCCGGGCAACTGATAAGGATTCCCA-3'; GATA mutant, 5'-GACCGGGCAACTGcgAAGGATTCCCA-3'.
Nucleotides in lowercase letters designate the mutated nucleotides. One pmol of each probe was labeled at the 5' terminus with [Antibodies-- Three peptide sequences were synthesized based on the human GATA-6 sequence (amino acids 8-22, 373, 387, and 428-442) and used to raise antibodies. These peptides were conjugated to KLH and injected into New Zealand White rabbits subcutaneously. The immunoglobin fraction of the antisera was purified with protein A-Sepharose CL-4B according to the instructions of the manufacturer (Amersham Pharmacia Biotech).
Western Blot Analysis-- Whole-cell extracts prepared from COS1 cells (50 µg) were subjected to a 10% SDS-polyacrylamide gel electrophoresis, and transferred to Immobilon-P (Millipore) by semidry blotting. Filters were blocked for 1 h at room temperature in PBS, 0.2% Tween 20, 5% nonfat dry milk. The filters were then incubated with anti-human GATA-6 antibody (1 µg of immunoglobin/ml) overnight at 4 °C in PBS, 0.2% Tween 20, 2% non-fat dry milk. Filters were washed in PBS, 0.2% Tween 20, 2% non-fat dry milk and incubated with anti-rabbit antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Visualization of the immunocomplexes was performed as recommended by the manufacturer (Enhanced Chemiluminescence kit; Amersham Pharmacia Biotech).
Growth Arrest Assays--
Cultured rat VSMCs (A7r5) were seeded
in six-well (20-mm) dishes for the assay. 2.5 µg of
pMSV--galactosidase, which expresses the bacterial lacZ
gene under transcriptional control of the murine sarcoma virus long
terminal repeat and either GATA-6,
ZF-GATA-6, Gax, or p21 expression
plasmids at a ratio of 1:5 (w/w) were mixed, and 5 µg/well of this
mixture was used for transfection by the calcium phosphate method.
After 24 h, the cells were washed three times with DMEM and
incubated with 20 µM 5-bromo-2-deoxyuridine (BrdUrd) for
another 24 h in high serum medium. The cultures were then washed
twice with ice-cold Dulbecco's phosphate-buffered saline (DPBS) and
fixed for 5 min on ice with 2% formaldehyde, 0.2% glutaraldehyde in
DPBS.
Double Immunofluorescence--
p53/
MEFs were cultured on
60-mm plates containing 1.5% gelatin-coated glass coverslips and serum
starved for 3 days in 0.5% FBS/DMEM. 5 µg of plasmid was transfected
using the LipofectAMINE procedure and, following transfection, was
serum-stimulated (10% FBS for 24 h). Cultures were then fixed in
4% neutral buffered formalin for 10 min, permeabilized for 5 min using
0.1% Nonidet P-40 and blocked in 2% goat serum. A mixture of 2.5 µg/ml mouse anti-p21 antibody (Calbiochem) and 1 µg/ml rabbit
anti-GATA-6 was applied at 4 °C overnight. Coverslips were washed
and incubated with 1:200 dilution of fluorescein
isothiocyante-conjugated goat anti-mouse antibody and 1:800
dilution of goat anti-rabbit rhodamine-conjugated antibody.
Nuclei were counter stained with Hoechst 33258 and mounted on glass
slides.
Colony Formation Assay--
Triplicate 100-mm cultures of
p21/
MEFs (passage 3) were co-transfected with 10.8 µg of
pCDNA-p21 or pCDNA-GATA-6 expression vector and 1.2 µg of
pZEOSV2 expression vector (Invitrogen) using LipofectAMINE. 24 h
following transfection cultures, 500 µg of zeocin/ml was added to the
medium to select for stably transfected cells. Two weeks following
initial selection, cultures were fixed in methanol, stained with trypan
blue, and quantified by visual inspection.
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RESULTS |
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Deletion of the Zinc Finger Domain Abrogates GATA-6 DNA Binding
Activity--
We compared the functionality of two plasmid constructs
that express either wild type GATA-6 or a mutant GATA-6 that lacks the
conserved zinc finger binding domain, ZF-GATA-6 (Fig.
1A). The wild-type and mutant
GATA-6 proteins were comparably expressed in transfected COS1 cells as
determined by Western blot analysis (Fig. 1B). The
specificity of the anti-GATA-6 antibody was indicated by an abrogation
of signal when the primary antibody was preabsorbed with a molar excess
of immunogenic peptide (Fig. 1C). Recombinant GATA-6 protein
formed a nucleoprotein complex when nuclear extracts from transfected
cells were incubated with a double-stranded radiolabeled oligonucleotide containing the consensus GATA site from the mouse globin gene, and this complex was sensitive to competition by a molar
excess of nonlabeled wild type probe, while a mutant oligonucleotide had no effect (Fig. 1D). In addition, preincubation of
rabbit anti-GATA-6 antibody with the nuclear extract induced a
supershifted band (not shown). In contrast to wild type GATA-6, nuclear
extracts from
ZF-GATA-6-transfected cells were unable to bind to the
wild-type oligonucleotide (Fig. 1D).
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GATA-6 Overexpression Inhibits DNA Synthesis in VSMCs-- Immunofluorescence was performed on rat VSMCs to determine the relative levels of endogenous and transiently-transfected GATA-6 protein. GATA-6 mRNA has been shown by immunoblot analysis to be highly expressed in quiescent VSMCs and down-regulated upon serum stimulation (10). As shown in Fig. 2A, GATA-6 protein is detected in the nuclei of quiescent VSMCs and is less abundant in proliferating VSMCs. However, serum-stimulated VSMCs transfected with the wild-type GATA-6 vector express GATA-6 protein at levels comparable with quiescent VSMCs.
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Ectopic GATA-6 Induces Cell Cycle Arrest in Fibroblasts Lacking
p53--
Since mouse embryonic fibroblasts (MEFs) deficient for p53 do
not express GATA-6, ectopic expression of this factor could be directly
analyzed with anti-GATA-6 antibodies (Fig.
3A). In addition, analyses of
growth control in this cell type are not confounded by p53-induced p21
expression (see below). In these experiments, quiescent p53/
MEFs
were transfected with GATA-6 expression vectors and labeled with BrdUrd
following serum stimulation. Immunofluorescence analysis of both BrdUrd
and GATA-6 at 24 h post-transfection revealed that wild type
GATA-6 inhibited S-phase entry by 95 ± 1% (Fig. 3B).
This inhibition was not due to a delay in S-phase entry since similar
results were obtained when GATA-6 transfected cultures were assessed at
48 h (not shown). In comparison, the VSMC transcription factor Gax
also inhibited BrdUrd incorporation in this assay consistent with its
known cell cycle inhibitory properties (24). As anticipated by its lack
of activity in VSMCs, the
ZF-GATA-6 mutant construct had no effect
on cell cycle activity in the p53
/
MEFs (Fig. 3B).
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Role of p21 in GATA-6-mediated Growth Arrest--
Since p21 Cdk
inhibitor has been implicated in G1 cell cycle arrest in VSMCs and
other cell types (17, 24, 30), we examined the role of p21 in
GATA-6-transduced cells. Double immunofluorescence assays revealed that
nontransfected p53/
MEFs do not express detectable levels of p21,
presumably due the lack of p53 activation of the p21 promoter (23), but
an intense p21 signal was detected in cells that were positive for wild
type GATA-6 expression (Fig. 4A). High levels of p21
expression occurred in all GATA-6-positive cells examined (>100
transfected cells). In contrast, no detectable p21 expression was found
in any of the
ZF-GATA-6-positive cells examined.
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DISCUSSION |
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VSMCs are of interest because of their ability to undergo a dedifferentiation process referred to as phenotypic modulation in response to mitogen stimulation, vascular injury, or other environmental stimuli (11, 12). Phenotypically modulated VSMCs are proliferative and migratory and resemble their fibroblast-like precursor cells in that they express low levels of contractile proteins and high levels of extracellular matrix. Thus, it is likely that VSMCs contain mitogen-regulated transcription factors that function to coordinate the expression of genes required for cell cycle activity with those encoding smooth muscle contractile proteins (31, 32). A number of mitogen-regulated transcription factors have been identified in VSMCs, including the homeoprotein Gax (33-35), MEF2 (36, 37), and Ets-1 (38). Recently, GATA-6 expression was shown to be rapidly down-regulated when quiescent human or rat VSMCs were stimulated to proliferate with mitogens (10). This expression pattern is reminiscent of that described for the gas and gadd family genes (39, 40), some of which have been shown to inhibit cell growth when overexpressed (41, 42).
In this study we explored the growth regulatory properties of GATA-6 in VSMCs and fibroblasts. The forced expression of GATA-6 in VSMCs inhibited the incorporation of BrdUrd following mitogen stimulation, but a GATA-6 mutant lacking the zinc finger domains exhibited no detectable effect on DNA synthesis. The wild type GATA-6, but not mutant GATA-6, also inhibited the mitogen-induced proliferation of a fibroblast line that does not normally express GATA-6. These results demonstrate that GATA-6-induced growth arrest is not cell type-specific and that it may control cell cycle activity through the modulation of a ubiquitously expressed cell cycle regulatory protein.
Ectopic GATA-6 expression induced the expression of p21, a general Cdk
inhibitor induced in skeletal myoblasts and other cell types during
differentiation (reviewed in Ref. 30) and whose expression is modulated
by the tumor suppressor, p53 (23). GATA-6-mediated growth arrest and
p21 induction are independent of p53 function as indicated by the
ability of ectopic GATA-6 to fulfill these functions in fibroblasts
deficient for p53. Other factors that activate p21 expression in a
p53-independent manner include the transcriptional regulators MyoD
(43), C/EBP- (44), Gax (24), and the vitamin D3 nuclear hormone
receptor (45) and the tumor suppressor BRCA-1(46). Thus, up-regulation
of p21 by these developmental regulators may represent a general
mechanism by which cell growth and phenotypic differentiation are
coordinated during embryogenesis and tumorigenesis. Similar mechanisms
may also be involved in regulating VSMC phenotypic modulation in
response to injury in adult vessels.
Our data indicate that the up-regulation of p21 is a functionally
significant feature of growth arrest induced by GATA-6. In contrast to
results with p53-deficient MEFs, p21-deficient low passage MEFs
expressing GATA-6 incorporated the S-phase marker BrdUrd at a high
frequency (91% for p21/
MEFs versus 5% for p53
/
MEFs). These data suggest that the up-regulation of p21 by GATA-6
overexpression largely accounts for the G1 cell cycle arrest under
these conditions. Consistent with this proposed mechanism GATA-6-transfected p21
/
MEFs form stable colonies at a high frequency, while the restoration of p21 decreases colony formation. In
separate experiments, GATA-6 was unable to transactivate expression from a 2.4-kilobase pair fragment of the p21 promoter (not shown), and
these data suggest that GATA-6 either acts on an unidentified p21 gene
regulatory element at a distant site or increases p21 expression
through a post-transcriptional mechanism. Post-transcriptional regulation of p21 was recently reported for C/EBP-
, which increases the stability of the p21 protein (47).
In summary, we have shown that GATA-6 overexpression can up-regulate the expression of the p21 Cdk inhibitor and inhibit S-phase entry in VSMCs and fibroblasts. Though VSMC-specific gene targets for GATA-6 are currently unknown, it is reasonable to speculate that in VSMCs GATA-6 functions, like GATA-4 in heart, to activate the expression of contractile protein genes associated with the differentiated phenotype. Therefore, GATA-6 may serve multiple regulatory roles in VSMCs and function, along with other mitogen-regulated transcription factors, to coordinate VSMC proliferation and differentiation during normal vessel development. The identification of GATA-6 as a regulator of VSMC proliferation may also have implications in the development of molecular therapies to treat proliferative vessel wall disorders.
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ACKNOWLEDGEMENT |
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We thank Linda Whittaker for assistance in the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR-40197 and HL-50692 (to K. W.).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.
¶ Current address: The Second Dept. of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113, Japan.
** To whom correspondence should be addressed: Division of Cardiovascular Research, St. Elizabeth's Medical Center, 736 Cambridge St., Boston, MA 02135. Tel.: 617-562-7501; Fax: 617-562-7506; E-mail: kwalsh{at}opal.tufts.edu.
1 The abbreviations used are: VSMC(s), vascular smooth muscle cell(s); DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; BrdUrd, 5-bromo-2-deoxyuridine; MEF(s), mouse embryonic fibroblast(s).
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REFERENCES |
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