Download GATA transcription factors in the developing and adult heart

Cardiovascular Research 63 (2004) 196 – 207
www.elsevier.com/locate/cardiores
Review
GATA transcription factors in the developing and adult heart
Sampsa Pikkarainen, Heikki Tokola, Risto Kerkelä, Heikki Ruskoaho *
Department of Pharmacology and Toxicology, Faculty of Medicine, Biocenter Oulu, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland
Received 8 November 2003; received in revised form 29 February 2004; accepted 18 March 2004
Available online 6 May 2004
Time for primary review 16 days
Abstract
During the past decade, emerging evidence has accumulated of different nuclear transcription factors in regulation of cardiac development
and growth as well as in cardiac hypertrophy and heart failure. GATA-4, -5 and -6 are zinc finger transcription factors that are expressed in
the developing heart and GATA-4 and -6 continue expression in the adult cardiac myocytes. GATA-4 and -6 regulate expression of several
cardiac-specific genes, and during murine embryonic development, GATA-4 is essential for proper cardiac morphogenesis. In support of this,
mutations of gene for GATA-4 or for its cofactors have been associated with human congenital heart disease. Pressure overload of the heart in
vivo as well as hypertrophic stimulation of cardiac myocytes in vitro provide adequate stimulus for activation of GATA-4. Activity of GATA4 transcription factor is subject to regulation at the level of gene expression and through post-translational modifications of GATA-4 protein.
A number of genes induced during cardiac hypertrophy possess functional GATA sites in their promoter region and cardiac-specific
overexpression of GATA-4 or -6 leads to cardiac hypertrophy. In addition, a pattern of interactions between GATA-4 and its numerous
cofactors have been identified, showing an increasing complexity in regulatory mechanisms. The present review discusses current evidence
of the role and regulation of GATA transcription factors in the heart, with an emphasis in the GATA-4 and development of cardiac
hypertrophy.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: GATA-4; GATA-6; Transcription factor; Mitogen-activated protein kinase; Signal transduction; Cardiac hypertrophy; Heart failure
1. Introduction
2. Expression pattern and cardiogenesis
Intracellular signals converge on a limited number of
regulatory elements to regulate expression of individual
genes. Thus, transcription factors that regulate expression
of other genes in embryonic and/or adult heart may
constitute a point of signal convergence for various
stimuli. The family of GATA transcription factors regulate
differentiation, growth and survival of a wide range of
cell types (for reviews see Refs. [1 –6]). This review
summarizes recent advances in understanding the role and
modulation of GATA transcription factors in the heart,
focusing especially on GATA-4 and on the development
of pathological cardiac hypertrophy.
The family of GATA transcription factors consists of six
proteins (GATA-1 – 6). GATA-1 – 2– 3 are important regulators of hematopoietic stem cells and their derivatives [1– 4],
whereas GATA-4, -5, and -6 genes are expressed in various
mesoderm and endoderm-derived tissues [7– 10]. GATA-4
exhibits a developmentally regulated and tissue-specific
expression pattern in mice, and during embryonic and fetal
development GATA-4 mRNA is found in the heart, gut,
gonads, liver, visceral endoderm and parietal endoderm
[7,8]. Interestingly, GATA-4 is one of the earliest transcription factors expressed in developing murine cardiac cells
[11]. Specifically, GATA-4 mRNA can be detected as early as
at 7.0– 7.5 days postcoitum (dpc) in the precardiac mesoderm
and both the transcript and protein are found during formation
and bending of the heart tube (8.0 dpc) in endocardium,
myocardium and precardiac mesoderm [11]. Abundant
GATA-4 mRNA continues to be expressed in cardiac myocytes throughout the life of the animal [7,11]. In adult mouse,
* Corresponding author. Tel.: +358-8-5375236; fax: +358-8-5375247.
E-mail address: [email protected] (H. Ruskoaho).
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.03.025
S. Pikkarainen et al. / Cardiovascular Research 63 (2004) 196–207
GATA-4 transcript is additionally found in gonads, lung,
liver and small intestine [7]. In contrast, GATA-5 is
detected during embryonic development in heart, lungs,
urogenital ridge, bladder and epithelium of the gut and,
during adulthood, in gastrointestinal tract, bladder, lungs
and endocardium, but not in myocardium [10,12]. GATA-6
mRNA is expressed during embryonic and fetal development in visceral endoderm, heart, lungs, urogenital ridge,
vascular smooth muscle cells, and in gastrointestinal tract
and, in adults, it is detected in myocardium, aorta, gastrointestinal tract and to a lesser extent in the liver and lungs
[10].
After the initial studies identifying GATA-4 as a major
GATA-binding factor in the developing heart, its role during
development has emerged. In P19 embryonal carcinoma
cells, overexpression of GATA-4 increases differentiation
of beating cardiocytes, while inhibition of GATA-4 by
antisense strategy prevents cardiocyte differentiation and
triggers extensive apoptosis [13,14]. GATA-4 deficient embryonic stem (ES) possess potential, though reduced, to
differentiate into cardiac myocytes in vitro [15], and are
partially defective in their ability to generate proper visceral
endoderm and definitive endoderm of the foregut [16,17].
Notably, transgenic mice with inactivation of the GATA-4
gene die during embryonic development due to the failure of
ventral morphogenesis and heart tube formation between 8.0
and 9.0 dpc [18,19]. In GATA-4 deficient mice the differentiation of cardiac myocytes is not impaired and cardiac atrial
natriuretic peptide (ANP) and a-myosin heavy chain (aMHC) genes are normally expressed, suggesting that upregulation of endogenous GATA-6 mRNA is potentially compensating the lack of GATA-4 [18,19]. The defect of
cardiogenesis in GATA-4 deficient mice may result from an
early endodermal defect: abnormal differentiation and ventral
197
migration of endodermal cells prevents concomitant movement of myocardial cells leading to cardia bifida (for review
see Ref. [20]). GATA-6 deficient mice die shortly after
implantation (at 5.5 – 7.5 dpc) due to abnormal visceral
endoderm function and defects in extraembryonic tissue
[21]. In addition, the lack of GATA-6 appears to prevent
generation of endodermally derived bronchial epithelium
[22]. In embryoid bodies derived from GATA-6 deficient
ES cells, abnormal differentiation is accompanied by attenuated levels of GATA-4 mRNA, suggesting that GATA-6
may regulate the expression of GATA-4 during differentiation [22]. Taken together, the expression of GATA-4 appears
to be indispensable for proper endodermal differentiation and
ventral morphogenesis, but not necessarily for differentiation
of cardiac myocytes, in which GATA-6 may be capable of
replacing GATA-4.
3. Protein structure
A characteristic feature of GATA factors is a domain
of two adjacent zinc fingers (Cys-X2-Cys-X17-Cys-X2Cys) that directs preferential binding to the nucleotide
sequence element 5V-(A/T)GATA(A/G)-3V of target gene
promoters [23]. GATA-4, -5 and -6 share a high homology ( f 80 – 90%) in the amino acid sequence within the
two zinc fingers and the DNA sequence recognition
domain of the C-terminal zinc finger is well-conserved
among all GATA proteins [7,9,10,24 – 26]. Given the
homology of DNA binding domains between different
GATA factors, the solution structure of GATA-1 bound to
DNA may elucidate also DNA binding mechanisms of
GATA factors in general. According to the solution
structure of C-terminal zinc finger of GATA-1 bound to
Fig. 1. Protein structure of murine GATA-4. Transcriptional activation domains are marked with red, zinc finger domains with blue and domain for nuclear
localization with green. Ser-105 is a phosphorylation target for mitogen-activated protein kinases (MAPK) and Ser-261 for protein kinase A (PKA). Single
amino acid mutations V217G and G295S abolish interaction with Friend of GATA-2 (FOG-2) and TBX-5, respectively.
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DNA, N-terminal and central part of GATA DNA binding
domain contact with major groove of DNA and Cterminal region interacts with minor groove [27]. Omichinski et al. [27] elucidated the three-dimensional
aspects of this interaction, suggesting that the complex
resembles a hand holding a rope with the palm and
fingers representing the protein core and the thumb, the
carboxyterminal tail. Similar to GATA-1, domain deletion
analysis of GATA-4 protein demonstrates that the Cterminal zinc finger is sufficient and necessary for
DNA binding [28]. N-terminal zinc finger, which is
unable to bind DNA by itself, influences stability and
specificity of DNA binding as well as transcriptional
activation by GATA factors [29,30]. Most of the protein – protein interactions of GATA factors are mediated
by its C-terminal zinc finger, while N-terminal zinc finger
interacts with Friend of GATA (FOG) transcription factors (see below). In addition, deletion analysis of GATA-4
shows that a nuclear localization sequence (NLS) is
present within the basic domain adjacent to the Cterminal zinc finger and two separate transcriptional
activation domains are present within the N terminus of
the protein [28] (Fig. 1). In comparison with GATA-5
and -6 these activation domains are partially conserved
[7,9,10], which may explain some functional redundancy
and the similarities with GATA-4 in transcriptional activation mechanisms.
4. Cardiac target genes
Accumulating evidence underscores the role of GATAbinding motifs in enhancer region of several cardiac-specific
genes. GATA binding elements of rat a-MHC promoter are
required for activity of the promoter in the heart, and aMHC promoter can be activated in skeletal muscle by cotransfection of GATA-4 expression plasmid [31]. Also, other
sarcomeric genes, such as cardiac troponin C and I and
myosin light chain-3, show a myocyte-enriched expression
pattern, at least in part, due to GATA elements in the
promoter region [32 – 36]. In addition to the sarcomeric
proteins, also genes for ANP, BNP and corin, a cardiac
enzyme involved in the processing of natriuretic peptides,
and multiple other genes including Na + /Ca2 + -exchanger,
acetylcholine receptor-M2, cardiac-restricted ankyrine repeat protein (CARP), adenosine receptor-A1 and carnitine
palmitoyltransferase-1b can be induced by GATA-4 [37 –
46]. Notably, genes of transcription factors that regulate
developmental expression pattern in vivo contain GATA
motifs in their promoter region: in the context of gene for
dHAND transcription factor, two conserved GATA elements are essential for right ventricle-specific activity of
the promoter during murine embryogenesis [47]. Murine
Nkx-2.5 gene promoter contains two GATA elements,
which bind GATA-4 and are necessary for the promoter
activity in the heart, pharynx and spleen at the early stages
of embryogenesis [48,49]. Interestingly, murine GATA-6
gene contains an Nkx-2.5 binding element (NKE), which is
essential for cardiac-specific expression of the promoter
during cardiogenesis [50], suggesting a reinforcing network of GATA factors and their cofactors at the level of
gene expression. Adenovirally delivered GATA-4 or
GATA-6 antisense cDNA treatment of neonatal rat cardiac
myocytes results in an inhibition of cardiac-restricted gene
expression, including BNP, h-MHC and cardiac troponin I,
while only diminution of GATA-4 attenuates a-MHC,
which is probably related to lower affinity of GATA-6
on GATA site of a-MHC promoter [51]. However, high
levels of GATA-4 are found in other tissues than the heart,
such as gonads [52,53], and thus additional mechanisms
likely contribute to the cardiac expression of these genes.
Given that GATA-4 induces several cardiac-specific promoters that are activated during both in cardiogenesis and
cardiac hypertrophy, GATA motifs of cardiac gene promoters may have an important role in these processes. In
support of this hypothesis, full induction of b-MHC or
angiotensin II type 1 receptor (AT1) genes in pressureoverloaded rat hearts requires intact GATA binding elements in the promoter regions [54,55]. Furthermore, functional GATA elements of gene promoters are required for
the activation of adenylosuccinate synthetase-1 transcription by pacing in neonatal rat cardiac myocytes [56] and
for rat BNP promoter induction in the hearts of nephrectomized rats [57]. GATA binding sites are necessary for
phenylephrine (PE) induced rat preproendothelin [58] as
well as isoproterenol (ISO) or PE induced human BNP
promoter [59,60] activities in neonatal rat cardiac myocytes. Endothelin-1 (ET-1) induces ANP and human BNP
promoter activities [61,62] in a GATA-site dependent
manner. In the context of rat BNP promoter, GATA sites
are required for transcriptional induction in mechanically
stretched or lipopolysaccharide stimulated neonatal rat
cardiac myocytes [63,64]. Interestingly, it is possible that
reinforcing loops may further amplify GATA-4 and/or
GATA-6 signaling during cardiomyocyte differentiation
and/or hypertrophy: once activated, GATA-4/-6 increase
transcription of bone morphogenetic protein-4 (BMP-4)
[12] and preproendothelin [58] via binding on GATA sites
of their promoters, and in turn, BMPs and ET-1 are
activators of GATA-4 [65,66].
5. Activity and expression of GATA-4 in hypertrophied
cardiomyocytes
The data on cis regulatory elements and trans-acting
factors responsible for expression of GATA factors in
cardiac myocytes is limited to GATA-6. 5V Flanking region
of the mouse GATA-6 gene contains several potential
consensus sites for Sp1 transcription factor (GC boxes), a
number of GATA elements, two E-box elements and NKE,
but no elements for serum response factor (SRF), myocyte
S. Pikkarainen et al. / Cardiovascular Research 63 (2004) 196–207
enhancer factor-2 (MEF-2) or transcription enhancer factor1 (TEF-1). While Nkx-2.5 is able to transactivate the
GATA-6, other potential cardiac transcription factors, including MEF-2C and -2B, SRF, eHAND, dHAND, and
TEF-1, do not activate GATA-6 promoter in vitro [50].
Therefore, the precise molecular mechanisms regulating the
transcription of GATA factors in response to hypertrophic
stimuli are currently unknown.
Cumulative evidence suggests that GATA-4 DNA
binding activity increases during initiation of hypertrophic
response of cardiac myocytes in vitro and in vivo.
Importantly, rats infused with arginine8-vasopressine
(AVP) demonstrate substantial pressure overload together
with activation of GATA-4 DNA binding in ventricles,
which can be blocked with bosentan, a mixed ETA/B
receptor antagonist [66]. In perfused isolated rat hearts,
mechanical loading activates GATA-4 binding through
endogenous ET-1 and angiotensin II (Ang II), and infusion of either of the substances is sufficient to induce
GATA-4 DNA binding activity [67]. In addition, various
hypertrophic agonists and cyclic mechanical stretch activate GATA-4 DNA binding in cultured cardiac myocytes
[63,68 –73]. The early activation of GATA-4 DNA binding is rapid in stretched and pressure-overloaded rat
hearts and peaks at 30 min [66,67], which is similar in
ET-1 (at 30 to 60 min [73]) and ISO (at 30 min [69])
stimulated or mechanically stretched (at 60 min [63])
neonatal rat cardiac myocytes. In immortalized adult
mouse atrial myocyte cell line (HL-1 [74]), activation
of GATA-4 DNA binding peaks even more rapidly (at 10
min) by the treatment with ET-1 or phorbol-12-myristate13-acetate (PMA) [70,71], but is maximal at 30 min in
Ang II stimulated cells [75]. In addition, significant
induction of cardiac GATA-4 DNA binding activity is
detected in bilaterally nephrectomized rats with robust
hemodynamic overload at 26– 28 h [57] and in aortic
banded rats at 2 days [54]. Taken together, GATA-4
DNA binding both in vitro and in vivo is initially
activated with relatively early time-course, and it may
additionally increase with a sustained time course in
overloaded myocardium in vivo.
The studies regarding the regulation of GATA-4 activity
at the mRNA or protein levels have produced variable
results depending on the hypertrophic stimulus. GATA-4
mRNA levels are not altered in isolated perfused rat hearts
by ventricular stretch (at 15 min to 2 h [67]) or in the hearts
of AVP infused (at 15 min to 4 h [66]) or nephrectomized
rats [57]. Similarly, GATA-4 mRNA levels remain stable in
cultured neonatal rat cardiac myocytes treated with ET-1
(from 0.5 to 4 h [66]). Therefore, given that AVP infusion
activates GATA-4 DNA binding via ET-1 and neither AVP
infusion in vivo or ET-1 treatment in vitro have effect on
GATA-4 gene expression, at least the ET-1-dependent
induction of GATA-4 DNA binding does not require increased synthesis or stabilization of GATA-4 mRNA. Accordingly, protein levels of GATA-4 or GATA-6 in neonatal
199
rat cardiac myocytes were not affected by treatment with PE
[68,76]. In contrast, mechanical stretch (at 4 h [63]), pacing
(from 1 to 12 h [56]) and ISO (from 6 to 12 h [69])
transiently elevate GATA-4 mRNA levels in neonatal rat
cardiac myocytes. Infusion of ISO, PE or both in adult mice
increases cardiac GATA-4 and Nkx-2.5 mRNA levels from
3rd day to at least for 2 weeks [77]. In this model,
normalization of blood pressure with hydralazine is not
sufficient to prevent upregulation of GATA-4 mRNA levels
or the increase in cardiac mass, while 1 week after withdrawal of the infusions the levels of GATA-4 mRNA return
to baseline together with normalization of cardiac index
[77]. In addition, pressure overload of the right ventricle in
pulmonary artery banded rats increases protein levels of
GATA-4 and some of its cofactors including Nkx-2.5, MEF2 and dHAND [78]. Collectively, it appears that GATA-4
gene may be regulated at the level of transcription and/or
transcript stability in certain experimental rodent models of
cardiac hypertrophy. Since the early activation of GATA-4
DNA binding peaks hours prior to possible increase in
GATA-4 mRNA levels, additional mechanisms are likely
to be involved.
6. Regulatory mechanisms
GATA-4 protein is subject to post-translational modifications, which affect its DNA binding activity, transactivation and/or localization within cardiac myocytes
(Fig. 2). Treatment of neonatal rat cardiac myocytes with
ET-1, PE or ISO increases GATA-4 phosphorylation
[69,72,76,79,80], which is accompanied by an increase
in GATA-4 DNA binding activity [72,76,80]. Similarly,
ET-1 and PMA induce phosphorylation of GATA-4 in
HL-1 cells [70,71]. In murine GATA-4, one key amino
acid for phosphorylation is Ser-105. Studies using fusion
protein of GATA-4 activation domain (amino acids 33–
227) with intact or mutated Ser-105 linked to GAL4
DNA binding domain show that Ser-105 is required for
GATA-4-mediated transcriptional activation [80]. In addition, despite not being located within the DNA binding
domain of GATA-4, mutation of Ser-105 reduces the
DNA binding activity [80]. It has also been suggested
that phosphorylation of GATA-4 on Ser-105 may increase
its stability within the cells [81].
Kinases shown to catalyze Ser-105-specific phosphorylation of GATA-4 in cardiac myocytes are extracellular
signal-regulated kinase (ERK) [80] and p38 mitogen-activated protein kinase (p38 MAPK) [79], two members of
the MAPK family (for review see Ref. [82]). GATA-4 has
been identified as a target for small GTPase RhoA activated p38 MAPK resulting in phosphorylation of Ser-105
of GATA-4 protein [79]. ERK may mediate phosphorylation of Ser-105 of GATA-4 protein in response to PE
treatment of cardiac myocytes [80]. Moreover, an inhibitor
(Y-27632) against a downstream kinase of RhoA (Rho-
200
S. Pikkarainen et al. / Cardiovascular Research 63 (2004) 196–207
Fig. 2. Schematic presentation of potential signal transduction mechanisms converging on GATA-4 and its cofactors. Ang II, angiotensin II; CaMK, calciumcalmodulin dependent kinase; CN, calcineurin; ERK, extracellular signal-regulated kinase; ET-1, endothelin-1; GPCR, G-protein coupled receptor; GSK-3,
glycogen synthase kinase-3h; HDAC, histone deacetylase; ISO, isoproterenol; MAPK, mitogen-activated protein kinase; MEF-2, myocyte enhancer factor-2;
MEK1, MAPK or ERK kinase-1; NFAT, nuclear factor of activated T-cells; PE, phenylephrine; PI3K, phosphatidylinositol 3-kinase; ROCK, Rho-associated
coiled-coil forming kinase; SRF, serum response factor.
associated coiled-coil forming kinase (ROCK)), prevents
both PE-induced increase in ERK phosphorylation and
GATA-4 DNA binding activity [83]. In cultured pulmonary
artery smooth muscle cells, serotonin activates GATA-4 via
similar mechanism including MEK/ERK-mediated induction of GATA-4 DNA binding activity [84]. However,
there is evidence suggesting that in neonatal rat cardiac
myocytes ERK may function as a regulator of basal
GATA-4 DNA binding activity, while p38 MAPK may
mediate activation of GATA-4 by ET-1 [72]. Given that
ET-1 activates both p38 and ERK and they are able to
phosphorylate Ser-105 of GATA-4, it is not clear which are
the additional mechanisms that modify activation of
GATA-4 and consequent gene expression. Also, the physiological role of this functional redundancy is unknown. Of
note, in addition to Ser-105, GATA-4 protein [7] contains
at least 6 other potential targets for MAPK mediated Serphosphorylation that could be involved in regulation of
GATA-4 DNA binding and GATA-dependent transcription.
In addition, ET-1 and MAPKs activate several other
nuclear mediators than GATA-4, such as ETS-like gene-1
(Elk-1) and nuclear factor of activated T-cells (NFAT), that
are involved in regulation of hypertrophic program of
cardiac myocytes [85,86]. Cardiac-specific activation of
ERK or p38 MAPK pathways appear to induce compensated cardiac hypertrophy [87] or restrictive cardiomyopathy [88], respectively. In view of these transgenic murine
models of GATA-4 regulating pathways, it is apparent that
partially overlapping regulation of GATA-4 is further
modified in the nucleus by other MAPK-dependent mechanisms and nuclear effectors.
In addition to p38 MAPK and ERK, GATA-4 has been
shown to be target of glycogen synthase kinase-3h (GSK3h). Recent evidence shows that GSK-3h functions as an
important negative regulator of cardiac hypertrophy (for
review see Ref. [89]). GSK-3h is negatively regulated by
protein kinase B (PKB)/Akt, a major kinase downstream
from phosphatidylinositol 3-kinase (PI3K). Therefore, stimuli that activate PI3K inhibit GSK-3h through PKB/Akt.
GATA-4 interacts physically with GSK-3h, which phosphorylates N-terminal domain of GATA-4 protein (within
2 – 205 amino acids). The increased phosphorylation inhibits
GATA-4-dependent transcription, at least in part, by decreasing nuclear levels of GATA-4 protein via a nuclear
S. Pikkarainen et al. / Cardiovascular Research 63 (2004) 196–207
exportin Crm1 [69]. Moreover, inactivation of GSK-3h
correlates with levels of nuclear translocation of NFAT, a
cofactor of GATA-4 [90]. Hence, GSK-3h may act in
parallel with the MAPKs to regulate GATA-4 dependent
gene expression and growth of cardiac myocytes.
Of note, at least in gonadal cells (MA-10 mouse Leydig
tumor cell line), dibuturyl cAMP treatment increases phosphorylation of GATA-4 via protein kinase A (PKA) at
evolutionary conserved Ser-261, which is located between
the two zinc finger domains of GATA-4 [91]. GATA-4
phosphorylation by PKA leads to recruitment of transcriptional co-activator, cAMP responsive element-binding protein-binding protein (CBP), and synergistic transcriptional
activation [91]. While CBP is capable to stimulate transcription by GATA-4 in transiently transfected NIH3T3
cells [92], it remains to be addressed whether similar
PKA dependent phosphorylation mechanism regulates
GATA-4 in cardiac myocytes.
Besides phosphorylation, GATA-4 activity can be further
influenced by other posttranslational modifications, such as
acetylation. Like CBP its close relative p300 possesses
intrinsic histone acetyltransferase activity that is required
201
for the synergistic transcriptional activation caused by
interaction of GATA-4 with p300/CBP [93]. However,
contradicting data exists whether p300/CBP directly acetylates GATA-4. While p300 overexpression failed to acetylate lysine residues of GATA-4 in NIH3T3 cells [93] similar
experiments in COS7 cells revealed clear acetylation of
GATA-4 [94]. Recent evidence shows that GATA-4 may be
regulated via acetylation in cardiac myocytes during hypertrophic growth process, possibly in a p300-dependent
mechanism. PE increases p300 expression as well as levels
of acetylated GATA-4 in cultured neonatal rat ventricular
myocytes [94]. Further, transgenic mice with cardiac-restricted overexpression of p300 develop cardiac hypertrophy, which is associated with increased GATA-4 acetylation
and DNA binding activity [94].
7. Interactions with cofactors in developing heart
GATA-4 has been shown to cooperate with a number of
other transcription factors and co-activators in vitro
[37,51,61,93,95– 106]. Specifically, the C-terminal zinc fin-
Table 1
Summary for interactions of GATA-4 and its cofactors
Cofactor Cofactors interactive region
GATA-4Vs interactive region
GATA-4Vs region for functional interaction Target promoters Ref.
dHAND basic helix – loop – helix
domain (bHLH)
C-terminal Zn-finger
FOG-2
N-terminal Zn-finger
C-terminal Zn-finger
(note: activation domains linked
to C-terminal Zn-finger N.D.)
N.D.
I Zn-finger or VI Zn-finger
are sufficient
GATA-6 N.D.
MEF2
N- and C-terminal Zn-fingers
and adjacent basic region
+ ANP
+ BNP
+ a-MHC
ANP
BNP
a-MHC
+ ANP
+ BNP
+
+
+
X
+
ANP
BNP
a-MHC
h-MHC
BNP
+
+
+
X
X
+
+
ANP
BNP
ca a-actin
a-MHC
h-MHC
ANP
h-MHC
ANP
C-terminal transactivation domain
(note: DNA binding of GATA-4 is
not necessary)
DNA-binding domain
C-terminal Zn-finger and adjacent C-terminal Zn-finger and transactivation
(MADS and MEF-2 domains) basic region
domains
NFATc4
C-terminal portion of the
Rel homology domain
Nkx-2.5 III helix of the homeodomain
C-terminal Zn-finger
N.D.
C-terminal Zn-finger
C-terminal Zn-finger and both C- and
N-terminal transactivation domains
p300
Cys/His-rich region (C/H3)
C-terminal Zn-finger domain
RXRa
two Zn-fingers of C-domain
SRF
MADS box domain
C-terminal Zn-finger and N-terminal
activation domain
C-terminal Zn-finger and adjacent N.D.
C-terminal region
C-terminal Zn-finger and adjacent C-terminal Zn-finger and transactivation
basic region
domains
+
+
+
+
+
+
X
ca a-actin
sk a-actin
sm 2a-actin
c-fos
a-MHC
CPT-Ih
BNP
[95]
[96 – 99]
[51]
[100]
[101]
[37,102,103]
[93]
[105]
[61,106]
ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; ca, cardiac; CPT-Ih, carnitine palmitoyltransferase-1h; MEF-2, myocyte enhancer factor-2;
MHC, myosin heavy chain; NFATc4, nuclear factor of activated T-cells-4; N.D., not determined; RXRa, retinoid X receptor-a; sk, skeletal; sm, smooth muscle;
SRF, serum response factor.
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S. Pikkarainen et al. / Cardiovascular Research 63 (2004) 196–207
ger domain of GATA-4 that is required for DNA binding has
been found to interact physically with several transcription
factors and co-activators in regulation of cardiac gene
promoters. However, a co-repressor protein FOG-2 interacts
with N-terminal zinc finger of GATA-4, and GATA-6 may
require both zinc fingers of GATA-4 for their interaction.
While the physical interaction with cofactors requires intact
zinc finger domains, the synergistic transcriptional activation
appears to require intact N-terminal and/or C-terminal activation domains of GATA-4 (see Table 1). There is evidence
that some of the interactions of GATA-4 may occur simultaneously with or even through p300/CBP to regulate transcriptional activation, including those with dHAND [95],
retinoid X receptor-a (RXRa) [105], and Yin Yang-1 [107].
In addition, GATA-4, SRF and Nkx-2.5 may generate
complex for synergistic transactivation of genes [108,109].
Interestingly, transgenic mice with a single amino acid
substitution on GATA-4 protein (V217G), which abolishes
interaction with FOG-2, have phenotypical similarities to
transgenic mice lacking the FOG-2 gene, such as absence of
coronary vasculature and defects in septation, suggesting
that this interaction is indispensable for proper heart morphogenesis and coronary vascular development [110]. In
human, mutations of Nkx-2.5 gene associate with development of congenital heart disease including cardiac septal
defects and atrioventricular conduction abnormalities [111,
112]. These mutations alter Nkx-2.5 DNA binding activity
[111]. In addition, the mutant Nkx-2.5 proteins and GATA-4
show abnormal ANP promoter transactivation, suggesting
that interaction with GATA-4 may play a role in the development of the cardiac phenotype [113]. Impaired synergistic
transcriptional activation by Nkx-2.5 and TBX-5, a member
of T-box transcription factor family, is detected, in turn, with
some of the mutations of TBX-5 gene that are associated with
Holt-Oram syndrome [114]. Holt-Oram syndrome is an
autosomal dominant syndrome characterized by cardiac
septation defects and upper extremity skeletal abnormalities
(for review see Ref. [115]). Notably, GATA-4 haploinsufficiency is associated with congenital heart defects in patients
with 8p23.1 monosomy [116]. Garg et al. [117] have recently
provided a potential linkage for a phenotypic similarity
(atrial septal defects) resulting from TBX-5 or GATA-4
haploinsufficiency: mutation of human GATA-4 gene resulting in a Gly to Ser substitution at codon 296 (adjacent to
NLS and C-terminal zinc finger) is associated with cardiac
septal defects. This mutation (mouse orthologue at codon
295) decreases GATA-4 DNA binding activity and abrogates
physical association with TBX-5, but not with Nkx-2.5
[117]. Moreover, a frame-shift mutation of human GATA-4
gene resulting in truncation of the last 40 amino acids of the
protein is associated with atrial septal defects [117]. This
mutation, in turn, renders GATA-4 transcriptionally inactive
and likely haploinsufficient [117]. Taken together, cardiac
malformations that associate with mutations of GATA-4,
Nkx-2.5 or TBX-5 genes may be in part due to disruption
of their combinatorial interactions.
8. Interactions with cofactors in hypertrophied
cardiomyocytes
Potential role of the interactions between GATA-4 and its
cofactors during cardiac hypertrophy is supported by circumstantial evidence. In support of a multiple line of
evidence that GATA-4 has a role in cardiac hypertrophy,
overexpression of GATA-4 or GATA-6 activates hypertrophic growth in cultured rat neonatal cardiac myocytes and in
the hearts of transgenic mice [68]. To examine the role
GATA-4 DNA binding in ET-1 stimulated neonatal rat
cardiac myocytes, GATA decoy oligonucleotides have been
used to competitively inhibit binding activity on GATAelements of target promoters [73]. GATA decoy oligonucleotide treatment decreases basal cardiac gene expression (i.e.
ANP and BNP) to similar extent as seen with adenoviral
antisense strategy, but in contrast, is insufficient to affect
ET-1 induced protein synthesis, hypertrophic morphology or
natriuretic peptide gene expression [73]. In contrast, depletion of GATA-4 protein in cultured neonatal cardiac myocytes by adenoviral antisense strategy inhibits hypertrophy
of neonatal rat cardiac myocytes in response to mechanical
stretch, PE and ET-1 [63,79]. Differential effects of decoy
oligonucleotide and adenoviral antisense treatments may be
related to complex formation of GATA proteins with other
transcription factors, which is likely more effectively crippled by depletion of GATA-4 protein levels than decoy
oligonucleotide which blocks GATA binding on target
promoters but does not necessarily affect interaction with
its cofactors. Given that various hypertrophic stimuli, including ET-1, have an effect on complex formation between
GATA-4 and its cofactors, the interactions may lead to
transactivation of gene expression via cis-elements of the
cofactors. In support of this, some cofactors of GATA-4,
such as Nkx-2.5 or SRF, have been shown to be able to
recruit GATA-4 to cardiac gene promoters [104,109]. In
stretched neonatal rat cardiac myocytes, increase in BNP
mRNA levels is completely inhibited by depletion of
GATA-4 protein by GATA-4 antisense adenovirus, but in
the context of BNP promoter delivered by liposome transfection, mutation of GATA elements alone inhibits stretch
inducibility only by 40%. Mutation of Nkx-2.5 binding
element (NKE) alone has no effect on BNP promoter
activation by stretch, but when combined with mutation of
GATA sites, almost completely abolishes the response,
suggesting that these factors may act in concert to regulate
hypertrophic gene program [63]. In addition, adenoviral
overexpression of a dominant negative fusion protein composed of GATA-4 DNA binding domain and a transcriptional repressor domain of Engrailed attenuates PE induced
hypertrophy of neonatal rat cardiac myocytes [68]. However, when comparing different inhibitory strategies targeted
on GATA-4 activity, it should be noted that higher transfection efficiency is obtained by adenoviral than liposomal
transfection and the effects on high order complex formation
by either approaches remains to be determined.
S. Pikkarainen et al. / Cardiovascular Research 63 (2004) 196–207
A number of GATA-4Vs cofactors have been implicated in
cardiac hypertrophy, most notably NFAT, MEF-2 and SRF.
These GATA-4-interacting factors may provide a point of
signal convergence for various hypertrophic stimuli and
signaling mechanisms. In the heart, calcium/calmodulin
complex (Ca2 +/CaM) activates phosphatases, including calcineurin [101]. Activation of calcineurin by the Ca2 +/CaM
results in dephosphorylation of its substrates including NFAT
[118]. Originally, by using GATA-4 as a bait (amino acids
130 –409 fused to yeast GAL4 protein) in the yeast twohybrid system, Molkentin et al. [101] identified NFATc4 (also
called as NFAT-3) as a novel GATA-4Vs cofactor. Importantly, they demonstrated that either calcineurin or the nuclear
localized mutant of its substrate (mutant of NFATc4) is
sufficient to produce cardiac hypertrophy when overexpressed in the heart of transgenic mice [101]. Moreover,
constitutively active calcineurin activates human BNP promoter synergistically with its substrate NFATc4 and GATA-4
[101], whereas inhibition of calcineurin prevents ET-1, 1%
serum, Ang II or PE induced hypertrophy in vitro [119,120].
Supporting the potential role of GATA-4 – NFAT interaction
during hypertrophic growth, physical interaction between
GATA-4 and NFATc increases in ET-1 treated neonatal rat
cardiac myocytes [121]. However, while numerous transgenic animal models provide strong evidence supporting the role
of calcineurin/NFAT-dependent signaling in regulation of
cardiac growth (for review see Ref. [122]), pharmacological
inhibitors of calcineurin have produced conflicting results in
various experimental rodent models (for reviews see Refs.
[123,124]).
MEF-2 transcription factor is an important downstream
mediator of Ca2 +/CaM dependent kinases (CaMK) and
MAPKs. Increased intracellular [Ca2 +] results in autophosphorylation of CaMKII, which switches it to a Ca2 +-independent state and prolongs its activation. CaMKI, which is
ubiquitously expressed, and CaMKIV, mainly expressed in
testis and brain, are activated by upstream Ca2 +/CaM dependent protein kinases [125,126]. Overexpression of CaMKIIy,
CaMKI or CaMKIV in the heart of transgenic mice is
sufficient to promote hypertrophic growth [127,128]. CaMK
stimulates MEF-2 activity by dissociating class II histone
deacetylases (HDACs) from the DNA-binding domain leading to nuclear export of HDAC with intracellular chaperone
14-3-3 [129]. In addition, MAPKs, which activate MEF-2 by
phosphorylation of the transcription activation domain, maximally stimulate MEF-2 activity only when repression by
HDACs is relieved by CaMK signaling [130]. In hematopoietic cells, the activities of GATA-1 and GATA-2 are repressed
by interaction with HDACs [131,132]. It remains to be
determined whether HDACs directly interact with GATA-4
in cardiac myocytes.
SRF activity is regulated by numerous mechanisms
ranging from expression of SRF to RhoA dependent
changes in actin dynamics, association with cofactors or
chromatin remodeling of its target promoter sequences (for
review see Ref. [133]). Analogically to MEF-2, CaMK
203
dependent dissociation of HDACs activates transcriptional
regulator SRF in cardiac myocytes [134]. In addition, SRF
may regulate cardiac gene expression via MAPK dependent
activation of ternary complex factors [135]. SRF and
GATA-4 and/or -6 form a ternary complex on a 30-bp
cis-element of ANP promoter containing juxtaposed GATA/
SRF binding motifs, which mediates activation of ANP
promoter in ET-1 stimulated neonatal cardiac myocytes
[61]. Notably, cardiac-restricted overexpression of SRF
results in robust cardiac hypertrophy in transgenic mice
[136], indicating a potential role for SRF in the development of pathological cardiac hypertrophy. Cardiac-specific
overexpression of upstream regulator RhoA results in
conduction system disturbances and consequent ventricular
dysfunction rather than cardiac hypertrophy [137]. Taken
together, while there is cumulative evidence for the role of
GATA-4Vs cofactors as well as GATA-4 itself in the
development of pathologic cardiac hypertrophy, it remains
to be addressed which of the interactions of GATA-4 are
crucial in vivo.
9. Future perspectives
Recent evidence suggests that the role of GATA transcription factors may extend to the regulation of cardiac
myocyte survival. In the context of erythropoiesis, deficiency of GATA-1 gene results in a developmental growth
arrest and apoptosis of erythroid precursors [138], which
may be linked to GATA-dependence of anti-apoptotic BclxL protein expression [139]. Similarly, inhibition of GATA4 protein levels increases apoptosis of P19 embryonal
carcinoma cells [13,14] and GATA-4 downregulation is
associated with increased apoptosis in cultured mouse
granulosa cells [140], while GATA-4 or GATA-6 overexpression protects cardiac myocytes from anthracyclineinduced apoptosis [141]. Expression of hepatocyte growth
factor (HGF), a potent mitogen for hepatocytes, is induced
by the ischemia – reperfusion in rat heart [142]. Administration of recombinant HGF reduces the size of infarct area
and improves cardiac function by suppressing apoptosis of
cardiac myocytes in the same experimental model [143].
Interestingly, in cultured cardiac myocytes HGF increases
Ser-105 phosphorylation and activation of GATA-4 protein
via MEK1/ERK cascade, which results in induction of the
levels of anti-apoptotic Bcl-xL protein, thus providing an
interesting insight for cardioprotective actions of GATA-4
[144]. Additionally, GATA-4 is among the earliest factors
known to bind the albumin gene promoter in liver precursor
cells, and it accesses sites in silent chromatin via novel
mechanism: GATA-4 appears to be capable of binding its
site in compacted chromatin and opening the local nucleosomal domain in the absence of ATP-dependent enzymes
[145]. It remains to be addressed whether GATA-4 might
act as a chromatin remodeling transcription factor in the
context of cardiac gene expression.
204
S. Pikkarainen et al. / Cardiovascular Research 63 (2004) 196–207
10. Concluding remarks
In summary, GATA-4 and -6 may play an important role
during early cardiac development and they may participate
in the development of cardiac hypertrophy during adulthood. GATA factors appear to function as trophic nuclear
factors in the postnatal heart, and while having several
target genes and cofactors, the hierarchy of the regulatory
mechanisms is yet to be elucidated. It will be of interest to
study these mechanisms that regulate utilization of GATA
factors in complexes of alternative cofactors in fetal and
adult tissues as well as those that govern the balance either
towards cardioprotection or detrimental cardiac hypertrophy. Furthermore, it is intriguing to speculate that specific
interactions between transcription factors could provide an
attractive therapeutic target in treatment of cardiovascular
diseases.
Acknowledgements
The original research was supported by grants from the
Academy of Finland, Sigrid Juselius Foundation, Finnish
Foundation for Cardiovascular Research, National Technology Center Tekes, Finnish Cultural Foundation, Ida
Montin Foundation, Aarne and Aili Turunen Foundation,
Maud Kuistila Foundation, Aarne Koskelo Foundation,
Paulo Foundation and Emil Aaltonen Foundation.
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