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Cardiovascular Research 2006 69(1):15-25; doi:10.1016/j.cardiores.2005.07.007
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Copyright © 2005, European Society of Cardiology

The complex pattern of SMAD signaling in the cardiovascular system

Gerhild Euler-Taimor* and Jacqueline Heger

Physiologisches Institut, Justus-Liebig Universität Giessen, Aulweg 129, 35392 Gießen, Germany

* Corresponding author. Tel.: +49 641 9947246; fax: +49 641 9947219. Email address: Gerhild.Taimor{at}physiologie.med.uni-giessen.de jacqueline.heger{at}physiologie.med.uni-giessen.de

Received 25 April 2005; revised 23 June 2005; accepted 7 July 2005


   Abstract
Top
 Abstract
1. Introduction
 2. SMAD activation in...
 3. Structure and interaction...
 4. Receptor activation of...
 5. Transcriptional control by...
 6. Cardiac effects of...
 7. Conclusions
 References
 
The SMAD (small mother against decapentaplegic) family comprises transcription factors that function as signal transducers of TGFβ (transforming growth factor) superfamily members. The number of studies showing expression, activation or involvement of both SMAD and TGFβ family members in cardiovascular diseases is constantly rising. In this context, the position of SMADs in the diseased heart is particularly interesting because, besides their well-known fibrotic effects, increasing evidence demonstrates direct action of SMADs on cardiomyocytes as well as on the vascular system. In these systems, SMAD proteins are described to have effects on heart development, cell proliferation, cell growth, and apoptosis. As will be discussed in this review, these different consequences of SMAD activation are dependent on different SMAD isoforms, interaction of SMAD with other transcription factors in the particular situation, and modulation of SMAD activity by various kinases. As a result of all these influences, it turns out that activation of SMAD by members of the BMP (bone morphogenetic protein) family, which is a subfamily of the TGFβ superfamily, is necessary for correct heart development. On the other hand, activation of SMADs by TGFβ family members results in fibrotic, apoptotic, and anti-hypertrophic processes that are related to a detrimental cardiac remodeling and progression to heart failure.

KEYWORDS Transcription factors; TGFβ; BMP; Hypertrophy; Apoptosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. SMAD activation in...
 3. Structure and interaction...
 4. Receptor activation of...
 5. Transcriptional control by...
 6. Cardiac effects of...
 7. Conclusions
 References
 
In recent years, elevated expression of proteins of the transcription factor family SMAD was found under several pathophysiological situations in the heart, i.e. after myocardial infarction or in diverse forms of cardiomyopathy. In this context, SMAD proteins are discussed mostly as being involved in cardiac fibrosis [1–3]. However, there is forthcoming evidence that they also have lethal effects on cardiomyocytes [4] and that they can reduce hypertrophic growth of these cells or modulate vascular growth [5]. Reasons for these different effects of SMAD proteins on cardiovascular function may lie in the complex pattern of SMAD signaling. First of all, different SMAD isoforms, which have different targets in the cell, can be activated. The particular SMAD isoform activated is mainly dependent on receptor activation by either of the two subfamilies of the TGFβ superfamily, i.e. BMP or TGFβ. Secondly, since DNA-binding affinity of SMAD proteins is very low, interaction of SMAD proteins with binding partners present in a particular cell type increases the binding affinity and changes the binding specificity of SMADs. Thus, depending on the set of binding partners, SMADs will be attracted to different promoter regions. This binding can additionally be modulated by kinases or phosphatases. Because of this, SMAD proteins control the expression of different genes as a function of cell type and the environmental influences affecting the cells, and this will determine the outcome of SMAD activation. In this review, TGFβ superfamily members and SMAD isoforms that are found in the cardiovascular system will be summarized, influences of other transcription factors and kinases on SMADs in the heart will be presented, and functional involvement of SMAD proteins in heart development and in cardiac remodeling will be discussed.


   2. SMAD activation in the heart
Top
 Abstract
 1. Introduction
 2. SMAD activation in...
3. Structure and interaction...
 4. Receptor activation of...
 5. Transcriptional control by...
 6. Cardiac effects of...
 7. Conclusions
 References
 
Members of the TGFβ superfamily, comprising the TGFβ and BMP family, are the classical activators of SMAD proteins. Thus, enhanced expression of TGFβ1 and other family members under pathophysiologic conditions in the heart is the first indication that SMAD signaling is also activated. And indeed, besides elevated levels of TGFβ, activin, and myostatin after myocardial infarction [6–8], activation of SMAD2, 3,and 4 is present at the same time [2,6]. Expression of these factors is always related to deleterious development of cardiac failure. Evidence for this comes from studies in which TGFβ activity was suppressed after myocardial infarction by transfection of cells with the extracellular domain of TGFβ type II receptor. This TGFβ inhibition decreased left ventricular hypertrophy and interstitial fibrosis, so that left ventricular remodeling and heart failure did not proceed [9]. Also, inhibition of the renin–angiotensin pathway by losartan, an angiotensin I receptor antagonist, reduced TGFβ, and SMAD activity and decreased fibrosis in reperfused hearts [2]. From these studies, detrimental effects of TGFβ/SMAD signaling can clearly be attributed to enhancement of cardiac fibrosis. Overexpression of SMAD7, which is an inhibitory SMAD protein, reduces collagen synthesis, thus confirming that collagen synthesis is mediated via SMAD signaling [10]. This collagen production determines tissue stiffness and impairs function of the heart.

Besides these fibrotic effects, however, SMAD signaling can induce apoptosis in cardiomyocytes [4], and this may accelerate the destructive effects of TGFβ. These apoptotic effects may be mediated by stimulation of pro- and inhibition of anti-apoptotic Bcl family members, which have been implicated in TGFβ-induced apoptosis [11,12]. Death-associating protein (DAP) kinase, which induces apoptosis in certain cell types, was found to be transcriptionally induced by the TGFβ/SMAD signaling pathway. Furthermore, inhibition of DAP kinase activity protected the cells from TGFβ-induced apoptosis [13]. Whether this pathway is relevant in the heart has yet to be proven.

Another aspect of TGFβ signaling is the coupling of hypertrophic responsiveness to β-adrenoceptor stimulation [14]. This step is a prerequisite for enhanced hypertrophic responsiveness of cardiomyocytes and may thus exaggerate hypertrophic growth after myocardial infarction and cause the transition from adaptive to decompensated hypertrophy.

These studies suggest that elevated levels of TGFβ and activation the SMAD2/3 and 4 complexes in the heart are related to a poor prognosis for ventricular function. For the other TGFβ family members, myostatin and activin, similar correlations between their activation and the development of ventricular remodeling have also been found, but direct functional involvement in ventricular remodeling has not been shown. However, since the same SMAD isoforms are activated by these proteins, equal functions can be assumed (Fig. 1).


Figure 1
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  		Fig. 1 Central role of SMADs in angiogenesis, heart failure, and cardiac development. SMADs are activated during cardiomyopathy or after myocardial infarction by different members of the TGFβ superfamily. Simultaneously, other transcription factors are activated that cooperate with SMADs in order to either enhance cardiac apoptosis and fibrosis, resulting in heart failure progression, or to promote angiogenesis in the vascular system. During {alpha}-adrenoceptor stimulation of cardiomyocytes, hypertrophy develops in the absence of SMAD proteins due to activation of AP-1, GATA, Akt, and calmodulin. Even if additional stress, e.g. ischemia, induces SMADs this activation can be counteracted by Akt, calmodulin, or ERK and may protect cardiomyocytes against apoptosis. BMP, signaling via distinct SMAD isoforms, plays a major role in the development of embryonic hearts and has anti-apoptotic effects in the adult heart.

 
Further evidence for detrimental effects of the TGFβ family for heart functions comes from the observation that elevated TGFβ/SMAD levels are also found in other disease states of the heart: a mouse model carrying a mutation in the LMNA gene encoding A-type lamins displays dilative cardiomyopathy, which goes along with an abnormal nuclear accumulation of SMAD2 and 3 [15], and elevated levels of SMAD2 and 4 are found in hereditary cardiomyopathic hamsters [1]. Elevated TGFβ levels are found in patients with idiopathic hypertrophic cardiomyopathy [16]. In homozygous REN2 rats, AngII-induced LV remodeling and fibrosis are both dependent on ERK and SMAD2 activation, the major signaling molecules of TGFβ1, and mediate the progression towards overt heart failure [17].

In contrast to the deleterious effects of the TGFβ/activin subgroup, the BMP subfamily has beneficial effects in the heart. The primary function of BMP is in heart development (Fig. 1). Addition of BMP to embryonic cells induces differentiation into cardiomyocytes via SMAD1/4 signaling [18]. Overexpression of SMAD6, an inhibitory SMAD protein that specifically inhibits BMP-mediated SMAD activation [19], prevents myocyte differentiation, and SMAD6 knockout mice have several cardiovascular abnormalities [20]. A crucial role for cardiac morphogenesis is the activation of the transcription factor Nkx2-5 by BMP, because Nkx2-5 regulates early cardiac gene expression. The control of Nkx2-5 is mediated via SMAD1/4 and GATA binding [21].

In the adult heart, only a few functions of BMP have been analyzed. Addition of BMP-2 to adult cardiomyocytes increases PI 3-kinase activity and enhances fractional shortening of myocytes [22]. In other studies, PI 3-kinase activation has been characterized as a pro-hypertrophic stimulus, and it remains to be elucidated if BMP exerts hypertrophic effects on the heart. An anti-apoptotic action of BMP-2 in neonatal cardiomyocytes mediated via SMAD1 could be demonstrated [23]. This anti-apoptotic effect of BMP-2/SMAD1 signaling can be repressed by SMAD6. In summary, BMP action has primarily beneficial effects for heart function.

Explanations for these broad and partially contrasting effects of TGFβ superfamily members in the heart, including cardiac development, fibrosis, apoptosis and growth, can be found (i) in the induction of specific SMAD isoforms, (ii) in the additional stimulation of different transcription factors that interact with SMAD, or (iii) in the presence of SMAD activity-modulating kinases.


   3. Structure and interaction of SMAD isoforms
Top
 Abstract
 1. Introduction
 2. SMAD activation in...
 3. Structure and interaction...
4. Receptor activation of...
 5. Transcriptional control by...
 6. Cardiac effects of...
 7. Conclusions
 References
 
At present eight distinct SMAD proteins are known. They can be divided into three different functional classes: (i) the receptor-activated R-SMADs (SMAD 1, 2, 3, 5, and 8), (ii) the co-mediator Co-SMADs (SMAD 4), and (iii) the inhibitory I-SMADs (SMAD 6 and 7). All three functional classes of SMADs and nearly all isoforms have been detected in the cardiovascular system.

In non-activated cells, R-SMADs are predominantly localized in the cytoplasm, Co-SMADs are equally distributed in the cytoplasm and the nucleus, and I-SMADs are found mostly in the nucleus. Upon stimulation of receptors of the TGFβ superfamily, R-SMADs become phosphorylated and activated, undergoing dimerization and thereafter forming heterotrimers with Co-SMADs. This complex then translocates to the nucleus and influences transcriptional regulation [24].

The C-terminus of all SMAD isoforms, with its MH2 region, is highly conserved (Fig. 2). This region is necessary for dimerization of SMAD proteins and their nuclear transport [25,26] and the main transcriptional activation domain is located in this region. DNA-binding co-factors and transcriptional co-activators and repressors also bind in this area. R-SMADs additionally have a C-terminal SSxS motif at which they become serine phosphorylated after ligand binding to their specific receptors. This phosphorylation is the signal for complexation of SMAD proteins. R-SMADs and Co-SMADs also possess an MH1 region at the N-terminus. This domain enables sequence-specific binding of SMADs to DNA. Additionally, other transcription factors interact with SMAD at this site. MH1 and MH2 are joined via a linker region that contains a transcriptional activation motif and phosphorylation sites for diverse kinases [27]. These phosphorylation sites allow crosstalk between SMAD proteins and their environment. The linker region also determines the stability of SMADs via a PY motif, which mediates interaction with the ubiquitin ligases targeting SMADs to degradation [28].


Figure 2
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  		Fig. 2 Functional domains in SMAD isoforms.

 
The N-terminal region of I-SMADs reveals low homologies to the MH1 domain of other family members. Since this DNA-binding region is missing in I-SMADs, binding of R- or Co-SMADs with I-SMADs results in a binding- and therefore transcription-inactive complex. SMAD7 acts as a general inhibitor of all TGFβ family members whereas SMAD6 preferentially blocks BMP signaling [19,29,30].


   4. Receptor activation of SMADs
Top
 Abstract
 1. Introduction
 2. SMAD activation in...
 3. Structure and interaction...
 4. Receptor activation of...
5. Transcriptional control by...
 6. Cardiac effects of...
 7. Conclusions
 References
 
Although SMAD signaling can be regulated by kinases and cooperating transcription factors in the cells, C-terminal phosphorylation by a specific receptor is the key event in SMAD activation [31]: Hence the binding of ligands to receptors of the TGFβ family activates R-SMADs. This is the first step that will decide the outcome of SMAD activation.

The TGFβ-family members initiate their intracellular action through formation of heteromeric receptor complexes of specific type I and type II serine/threonine kinases [32]. Type II receptors are constitutively active kinases and phosphorylate type I receptors. Type I receptors, in turn, mediate specific intracellular signaling pathways and therefore determine the specificity of the downstream signaling. Seven mammalian type I receptors (ALKs=activin receptor-like kinases) and five type II receptors have been identified [33,34].

The TGFβfamily comprises around 30 members in the mammalian system and can be divided into two groups: the TGFβ/activin family and the BMP group. Each group is responsible for activation of different SMAD isoforms (Fig. 3). Binding of the ligand results in type I and II receptor assembly. Different combinations in the subgroup composition of the receptors will finally determine by which ligand the receptor is bound and which SMAD isoform will be activated [35]. Important ligands in the cardiovascular system are TGFβ, which binds to a complex of type II-R and type I-R (=ALK5), and activin or myostatin, which bind to ActR-IIB and ALK4, 5, or 7 and result in the activation of SMAD 2 and 3. BMP, which binds to BMPR-II, and ALK2, 3, or 6 activates SMADs 1, 5, or 8. As discussed before, the TGFβ and BMP family members exert distinct effects in the heart: while TGFβ family members have prevailing detrimental effects in the adult heart, BMP members are essential for heart development and promote differentiation of cardiomyocytes.


Figure 3
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  		Fig. 3 Important signaling pathways of TGFβ superfamily members in the cardiovascular system. Overview of TGFβ superfamily members coupling to their specific type I and II receptors, which results in distinct SMAD isoform activation.

 
The ability of receptors to activate SMAD proteins is not only determined by ligand binding but also by soluble blocking peptides, which bind to the ligand and thereby inhibit its access to the receptor. The proteoglycan decorin [36] and {alpha}2-macroglobulin [37] belong to this group of peptides. Interestingly, both peptides are found upregulated after myocardial infarction [6,38]. Further regulatory proteins are membrane-anchored proteins like SARA (SMAD anchor for receptor activation), which enhance recruitment of SMAD 2 or 3 to its receptor [39]. A factor important in the vascular system is endoglin, which is a TGF-β co-receptor that facilitates TGFβ binding to its receptor. Endoglin is expressed mainly on endothelial cells and is involved in cardiovascular development, angiogenesis, and vascular remodeling [5,40]. In endothelial cells, TGFβ activates two distinct type I receptors, the classical receptor ALK5 and the endothelial-specific ALK1. ALK1 is commonly activated by BMP family members. Thus, TGFβ signaling in endothelial cells is different from other cell types. Activation of either receptor results in opposing effects on endothelial cell proliferation, thereby demonstrating that the first divergence in SMAD signaling starts already at the receptor site.


   5. Transcriptional control by SMADs in the cardiovascular system
Top
 Abstract
 1. Introduction
 2. SMAD activation in...
 3. Structure and interaction...
 4. Receptor activation of...
 5. Transcriptional control by...
6. Cardiac effects of...
 7. Conclusions
 References
 
The consensus binding site of SMAD proteins comprises only a four base pair sequence (5'-GTCT-3') to which SMADs bind with a low affinity of 10–7 M [41]. Such a sequence can be found in nearly every promoter region of a gene and is therefore not sufficient to confer promoter selectivity. Consequently, recruitment of SMADs to DNA is regulated by cooperation with other transcription factors. These factors facilitate binding of SMADs to DNA and, depending on the factors that are simultaneously induced with SMADs, create a large spectrum of sequence-specific binding patterns. Since there is a large number of transcription factors able to interact with SMADs, we focus here on transcription factors that are known to be expressed in the heart (summarized in Figs. 3 and 4Go).


Figure 4
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  		Fig. 4 Factors influencing signaling of TGFβ family members in the heart. TGFβ family members couple to type I and II receptors. This results in activation of SMAD 2 and 3 and initiates binding of Co-SMAD4. This complex translocates to the nucleus and regulates gene transcription. Calmodulin, Akt, or ERK can inhibit, whereas p38 and JNK enhance SMAD activity. In the nucleus, interaction of the SMAD complex with other transcription factors or the co-activator CBP/p300 modulates SMAD binding activity to diverse promoter elements.

 
5.1 Fibrotic, apoptotic, and hypertrophic action of AP-1 in the heart
One of the first transcription factors detected to interact with SMADs is the activator protein AP-1. It binds to the TPA (12-O-tetradecanoylphorbol-13-acetate)-responsive element TRE, which is the AP-1 consensus binding site. Interestingly, Zhang et al. [42] detected that SMAD proteins are also able to bind to TRE sites and stimulate transcription. However, not all AP-1 binding sites have equal affinities to SMADs, which may depend on the sequences flanking the TRE sites. In the presence of AP-1, binding of SMADs to TRE sites and also to neighbouring SMAD binding sites is enhanced and results in elevated transcriptional activity. Physical interaction of SMAD with AP-1 is responsible for attraction of SMADs into the transcription complex [43,44].

All of these analyses of AP-1/SMAD binding and also the primary assays of transcriptional activation were performed in vitro. However, these findings have a great in vivo relevance. TGFβ is a cytokine that is released under several pathophysiologic conditions and that simultaneously activates SMAD and AP-1. Both AP-1 and SMAD mediate enhanced expression of TGFβ-responsive genes, such as collagen [42], c-Jun [45], endothelin-1 [46], or peroxisome proliferator activated receptor gamma (PPAR{gamma}) [47]. All of these genes have important functions in the cardiovascular system, e.g. in ventricular remodeling due to cardiac fibrosis or in vascular angiogenesis. Another functional consequence of the concerted action of AP-1/SMAD signaling is the induction of apoptosis in TGFβ-stimulated cells [44,48]: in isolated adult cardiomyocytes, involvement of AP-1/SMAD signaling in apoptosis induction by TGFβ or nitric oxide was demonstrated [4]. Although the functional involvement of both transcription factors in apoptosis induction are obvious, their pro-apoptotic target genes have not yet been identified.

In the heart, elevated levels of SMADs and AP-1 are found under pathophysiological conditions: activation of AP-1 in correlation with induction of apoptosis has been shown in some models of ischemia/reperfusion injury [49–51]. Additionally, there is increasing evidence for an enhanced expression and activation of SMAD 2, 3, and 4 proteins after myocardial infarction [2,6]. Therefore, the simultaneous activation of AP-1 and SMAD after myocardial infarction may be the trigger for fibrosis and apoptosis in the heart. This may enhance the unfavourable remodeling process which indicates a central role of AP-1/SMAD signaling in deteriorating processes in the heart.

It is noteworthy that AP-1 also has other effects on cardiomyocytes: it is a mediator of hypertrophic growth as a result of {alpha}-adrenergic stimulation [52]. The conflicting AP-1 actions–either stimulation of apoptosis or hypertrophy–in one and the same cell type cannot be explained by a different composition of the AP-1 dimer under both conditions. However, there is an obvious difference in the activation of SMAD proteins: they are induced only under apoptotic and not under hypertrophic conditions in isolated cardiomyocytes. Therefore, the additional activation of SMADs may shift AP-1 signaling from hypertrophy to apoptosis. This hypothesis suggests that activation of AP-1/SMAD signaling always overrides a sole AP-1 activation and results in apoptosis induction. But this may be a too simplistic view of things, because activation of {alpha}-adrenoceptors, which activates only AP-1, can protect cardiomyocytes against apoptosis induced by ischemic conditions [53]. Reasons for this may be the activation of extracellular signal-regulated kinase (ERK) or the protein kinase Akt during {alpha}-adrenoceptor stimulation. Both inhibit SMAD activity and may therefore provide protection against ischemic injury. Furthermore, other co-activating transcription factors stimulated via {alpha}-adrenoceptors may override AP-1/SMAD signaling.

5.2 Important target genes of SMAD/SP1 signaling in the heart
Another factor that can interact with SMAD and AP-1 is the transcription factor SP1. In the {alpha}2I-collagen promoter, SP1- and AP-1 sites are linked via a SMAD consensus binding site. Upon stimulation with TGFβ, collagen expression is increased via SP1 and SMAD3/4 signaling [54]. In an earlier study, involvement of AP-1 was also demonstrated. In this situation, SMADs are assumed to act as bridging molecules between SP1 and AP-1 in order to enhance collagen synthesis, which would enhance cardiac stiffness. Neighbouring SP1/AP-1 sites are also present in the connexin 43 promoter [55]. Whether SMAD proteins may also influence the transcription of connexins in the heart, thereby influencing their electrical coupling via gap junctions, is not yet known. Other genes that are regulated via SP-1/SMAD interaction are the cyclin-dependent kinase inhibitors p15INK4B and p21 [56,57], which influence growth and differentiation of cardiomyocytes. Expression of the cell adhesion receptor integrin is also modulated by SP1/SMAD [58]. Integrins act as cellular mechanoreceptors, thereby participating in the mechano-biochemical coupling of the heart. In recent years, their participation in cardiac hypertrophy has been demonstrated (reviewed in Ref. [59]).

5.3 NF{kappa}B/SMAD signaling in the cardiovascular system
Similar to AP-1 and SMADs the transcription factor NF{kappa}B (nuclear factor-kappaB) is activated upon myocardial infarction [49] and in failing hearts [60]. NF{kappa}B possesses diverse roles in cardiovascular pathology, aggravating ischemic injury, triggering preconditioning, and enhancing or inhibiting apoptosis. These different functions of NF{kappa}B may again be explained by cooperation of NF{kappa}B with other transcription factors in order to change the outcome of NF{kappa}B activation. Interaction between NF{kappa}B and SMADs has been shown to enhance collagen VIIA and JunB expression in TGFβ stimulated cells [61,62]. As discussed in the previous paragraph, this is a situation when AP-1 is also induced. Therefore, simultaneous induction of AP-1, NF{kappa}B, and SMADs may contribute to unfavourable remodeling by enlargement of fibrotic tissue.

5.4 HIF1{alpha}/SMAD signaling in angiogenesis
Another factor upregulated in ischemic hearts is the hypoxia-inducible transcription factor HIF1{alpha}. At the same time at which hypoxia induces HIF1{alpha}, elevated concentrations of SMAD are present in ischemic hearts [6]. This resembles the situation in a study performed by Sanchez-Elsner et al. [63] in which Hep3 B cells were subjected to hypoxia. Only in the presence of TGFβ did hypoxic conditions provoke VEGF (vascular endothelial growth factor) expression mediated by a synergistic interaction of HIF1{alpha} and SMAD2/3 and 4. VEGF is a major stimulus for angiogenesis, promoting formation of blood vessels by inducing proliferation, migration, elongation, and branching of endothelial cells. The effect of HIF1{alpha}/SMAD interaction on angiogenesis is additionally enhanced by upregulation of endoglin via this pathway [64]: under basal conditions endoglin expression is regulated by SP1. Hypoxia alone moderately enhances endoglin expression, but in the presence of TGFβ, a marked increase is found. This enhancement of endoglin expression is due to a multiprotein complex (SP1/SMAD3/HIF1{alpha}) on the endoglin promoter mediating the cooperation between hypoxia and TGFβ pathways. As already described for AP-1/SMAD/SP1 complexes, SMAD seems to act again as an adaptor molecule between HIF1{alpha} and SP1. Therefore, SMAD activation is necessary for the switch from basal to highly induced gene expression of endoglin. Since endoglin plays a critical role in angiogenesis [65], its upregulation during hypoxia can mediate adaptive responses in the ischemic heart to counteract low oxygen levels. In conclusion, HIF1{alpha}/SMAD cooperation has positive effects for neovascularisation of ischemic tissues due to endoglin and VEGF induction.

5.5 Growth inhibiting effects of p300/SMAD signaling
SMAD proteins utilize the co-activators CBP/p300 to exert their effects (Fig. 4). SMAD3 has a dramatically enhanced affinity toward CBP/p300 after phosphorylation by TGF type I kinase receptor at its SSxS motif or after amino- or carboxy-terminal truncation. Association of CBP/p300 with SMAD mediates full activation of gene transcription [66]. The major functional component of CBP/p300 is its histone acetyltransferase domain, which potentiates transcription by acetylation-dependent loosening of the chromatin structure [67]. The SMAD-dependent stimulation of type I collagen expression in TGFβ-stimulated fibroblasts depends on functional cooperation with p300/CBP. Interferon gamma (IFN{gamma}) exerts antagonistic effects on collagen expression in fibroblasts [68]. IFN{gamma} activates the transcription factor Stat1{alpha}, and this appears to induce competition between activated Stat1{alpha} and SMAD3 for interaction with limiting amounts of cellular p300/CBP. This reveals a mechanism for antagonistic interaction of SMAD and Jak-Stat pathways in regulation of target genes and illustrates that the relative cellular levels of p300/CBP compared to other available transcription factors determine gene expression.

Nuclear acetylation mediated by p300 plays a critical role in myocyte hypertrophy and represents a pathway that contributes to the development of heart failure [69]. Thus far, association of histone acetyltransferases with MEF2, GATA, and NFAT families of transcription factors, which all control fetal cardiac gene expression, has been shown. At least a part of the hypertrophic effects of p300 is explainable via this interaction. Additional activation of SMAD proteins may attract p300 away from pro-hypertrophic transcription factors, thereby ceasing its action and reducing hypertrophy. The hypothesis that activation of SMADs results rather in inhibition of hypertrophic growth is supported by other studies: (i) overexpression of Ski, a co-repressor of SMADs, results in selective growth of skeletal muscle in transgenic mice [70], (ii) myostatin, a member of the TGFβ/activin subfamily, is a negative regulator of skeletal muscle mass [71], (iii) under hypertrophy-provoking {alpha}-adrenoceptor stimulation, SMAD activation is not found [4].

5.6 GATA/SMAD interaction in cardiac development
The GATA transcription factor family is essential for cardiac development and can mediate hypertrophic effects in the heart. It has recently been recognized that GATA is able to cooperate with SMADs and that during stimulation with TGFβ, physical and functional interaction between GATA-3 and SMAD3 allows TGFβ to regulate GATA target genes [72]. Cooperative interaction of these factors regulates transcription from promoters of the cytokines IL-5 and IL-10 in a TGFβ-dependent manner in T helper cells. By this interaction, SMAD3 and GATA-3 are able to integrate a genetic program of cell differentiation with an extracellular signal, providing a molecular framework for the effects of TGFβ on cell development.

The transcription factor Nkx2-5 plays an early role in regulating cardiac gene expression during cardiac morphogenesis. BMP, which is a member of the TGFβ superfamily but activates a totally different pattern of SMAD isoforms (Fig. 3), is crucial in the regulation of Nkx2-5 expression. Recently, a novel upstream Nkx2-5 enhancer, composed of clustered repeats of SMAD and GATA binding sites, has been identified [21]. This composite Nkx2-5 enhancer is a direct target of BMP signaling via cooperative interactions between SMAD1/4 and GATA-4. Deletion of this enhancer significantly reduced Nkx2-5 expression. Therefore, synergism of GATA and SMAD signaling contributes to the specification of cardiac progenitors (Fig. 1). These findings on Nkx2-5 regulation are extended by the findings of Lee et al. [73], who identified at least three distinct cardiac activating regions in the chick Nkx2.5 promoter. They demonstrate that in addition to GATA4/5/6 and SMAD1/4, binding of YY1 is also necessary for BMP-mediated induction and heart-specific expression, thus resulting in an even more complex pattern for transcriptional co-activators of SMAD proteins.

In the adult heart, pressure overload and hypertrophic stimulation of cardiac myocytes in vitro provide adequate stimuli for activation of GATA (reviewed in Ref. [74]). However, SMAD activation has not yet been described under such conditions. Therefore, activation of other cofactors implicated in cardiac hypertrophy, like NFAT, MEF-2, and SRF, seems to converge with GATA to stimulate hypertrophic growth signaling without SMAD involvement in the adult heart.


   6. Cardiac effects of SMAD modulation by kinases
Top
 Abstract
 1. Introduction
 2. SMAD activation in...
 3. Structure and interaction...
 4. Receptor activation of...
 5. Transcriptional control by...
 6. Cardiac effects of...
7. Conclusions
 References
 
Besides the cooperative binding of SMADs with other transcription factors or co-activators in the heart, kinases have additional impact on SMAD signaling. In oncogenic cell lines, the Ras-MEK-ERK pathway can (i) decrease TGFβ receptor levels by controlling expression, (ii) increase the level of SMAD co-repressor TGIF by stabilizing this protein, and (iii) attenuate SMAD accumulation in the nucleus by phosphorylation of SMAD1, 2, or 3 in the linker region [75] (Fig. 4). This phosphorylation inhibits nuclear transport of SMADs and thereby attenuates transcription of TGFβ- or BMP-induced genes. Thus, activation of ERK can counterbalance BMP/TGFβ activity. In cardiomyocytes, ERK activation can be found after hypertrophy-inducing {alpha}-adrenoceptor stimulation. Whereas short-term activation is responsible for the hypertrophy-associated fetal gene shift [76], sustained ERK activation mediates hypertrophic growth in adult cardiomyocytes [77]. The inhibition of SMADs by ERK again supports the view that SMADs are not involved in the hypertrophic growth of cardiomyocytes. Having in mind that SMAD activation in the heart correlates with maladaptive responses like apoptosis or fibrosis, one might assume a protective role of {alpha}-adrenoceptor stimulation in the process of heart failure development because this would inhibit SMAD activity by stimulation of ERK (Fig. 1). This hypothesis is substantiated by findings in the ALLHAT study (Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial), where heart failure progression was still observed in patients treated with antihypertensive {alpha}-adrenoceptor blockers [78]. And in the V-HeFT study (Vasodilator Heart Failure Trial) [79], the {alpha}-adrenoceptor antagonist prazosin did not reduce mortality due to chronic heart failure, in spite of its hypertension-reducing effects.

Along the same lines as ERK, effects of calmodulin with regard to SMAD inhibition and hypertrophic growth can be mentioned. Calmodulin binds at the N-terminal MH1 region of SMAD2 (Fig. 4). This inhibits SMAD2 activity and transcriptional activation of TGFβ-stimulated genes [80]. On the cardiac level, calmodulin activation is considered to be a hypertrophic growth signal [81]. Therefore, this is another example in which activation of a hypertrophic pathway in the heart has inhibitory effects on SMAD signaling and may protect the heart against injury.

Another factor known to have beneficial effects on cardiac function is insulin. The signaling pathway of insulin is mediated via PKB/Akt kinase activation. As Matsui et al. [82] have shown, constitutively active Akt can prevent cardiac injury and apoptosis after transient ischemia. Interestingly, Akt acts, similarly to ERK, as an inhibitor of SMADs [83]. In contrast to ERK, inhibition by Akt is not mediated by phosphorylation of SMADs but via a direct interaction of both proteins. Akt binding at the MH2 and linker region sequesters SMAD3 outside the nucleus, thereby inhibiting the transcriptional capability of SMAD3. This inhibition specifically reduces TGFβ-mediated apoptosis in cells, but does not affect other functions of TGFβ. Thus, this represents another instance of kinase-mediated SMAD inhibition. Since this Akt-mediated SMAD inhibition has anti-apoptotic effects, one might assume that one effect of Akt in the heart may be reduction of apoptosis after myocardial infarction.

Kinases that modulate SMAD signaling in a positive way are the stress-related MAP kinases, p38 and JNK. JNK can phosphorylate SMADs, and this phosphorylation is needed for enhancement of SMAD–SMAD interaction and nuclear transport [84] (Fig. 4). JNK is activated after myocardial infarction and is a contributor to nitric oxide-induced apoptosis in cardiomyocytes [85]. Since apoptosis induction is also mediated by SMADs, activation of JNK may accelerate apoptosis induction in the ischemic heart via SMAD signaling. p38 acts via phosphorylation of MSK1, which phosphorylates CBP/p300 and enhances chromatin remodeling. This facilitates transcriptional activation by SMADs ([86], Fig. 4). Several p38 isoforms with different functions exist. Therefore, activation of p38 may either induce apoptosis or hypertrophy in cardiomyocytes, and different opinions about the outcome of p38 activation in the heart have been discussed [87].

An indirect influence of the MKK4/JNK and MKK3/p38 pathways results from their activation of Jun, which is a component of AP-1, and ATF2. Both of those transcription factors can cooperate with SMADs through direct physical contact. In certain cell types and conditions, the MKK4/JNK and MKK3/p38 pathways are activated by TGFβ itself and the proteins XIAP (xenopus inhibitor of apoptosis) or TAK-1 [31]. TAK1 activates MAP kinase kinase (MKK) in combination with an adaptor molecule, TAB1 [88].


   7. Conclusions
Top
 Abstract
 1. Introduction
 2. SMAD activation in...
 3. Structure and interaction...
 4. Receptor activation of...
 5. Transcriptional control by...
 6. Cardiac effects of...
 7. Conclusions
References
 
SMAD proteins have a great potential of integrating different signals in cells. Modulation of their activity by diverse kinases and cooperation with different transcription factors make them ideal candidates for fine tuning of signaling pathways. Although there is still a great potential of defining new pathways in SMAD signaling, from our present knowledge concerning SMAD function in the heart, it becomes obvious that absence or even inhibition of SMADs correlates with conditions found in hypertrophic growth processes, whereas the activation of SMAD2/3 and 4 by the TGFβ/activin subfamily contributes to cardiac fibrosis and apoptosis. Considering the fact that SMADs interact with normally pro-hypertrophic factors like AP-1, one might assume that the additional activation of SMADs during the adaptive process of hypertrophy may detract from the hypertrophic action of AP-1, thereby diverting this process in the detrimental direction of ventricular remodeling and progression to heart failure. There is some first evidence for this hypothesis showing that SMADs mediate apoptosis via cooperation with AP-1 in cardiomyocytes and that they are linked to fibrotic stiffness in the heart. Since many of the studies on modulation of SMAD activity and their functional effects were done in cell culture models, future work has to validate their deleterious cardiac effects and may definitively identify SMADs as molecular switches between adaptive and detrimental processes in the heart.


   Notes
 
* Sian E. Harding acted as guest editor for this manuscript.

Time for primary review 33 days


   References
Top
 Abstract
 1. Introduction
 2. SMAD activation in...
 3. Structure and interaction...
 4. Receptor activation of...
 5. Transcriptional control by...
 6. Cardiac effects of...
 7. Conclusions
 References
 

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