Glycogen Synthase Kinase-3β
A Novel Regulator of Cardiac Hypertrophy and Development
- GSK-3β Is a Proline-Directed Serine/Threonine Protein Kinase Whose Activity Is Negatively Regulated by Multiple Mechanisms
- GSK-3β Inhibits Cardiac Hypertrophy In Vivo and In Vitro
- Regulation of Wnt Signaling Pathway by GSK-3β and Its Role in Cardiac Development and Postnatal Hearts
- Unanswered Questions: Future Perspectives
- Figures & Tables
- Info & Metrics
Glycogen synthase kinase-3β (GSK-3β) is a ubiquitously expressed constitutively active serine/threonine kinase that phosphorylates cellular substrates and thereby regulates a wide variety of cellular functions, including development, metabolism, gene transcription, protein translation, cytoskeletal organization, cell cycle regulation, and apoptosis. The activity of GSK-3β is negatively regulated by protein kinase B/Akt and by the Wnt signaling pathway. Increasing lines of evidence show that GSK-3β is an essential negative regulator of cardiac hypertrophy and that the inhibition of GSK-3β by hypertrophic stimuli is an important mechanism contributing to the development of cardiac hypertrophy. GSK-3β also plays an important role in regulating cardiac development. In this review, the role of GSK-3β in cardiac hypertrophy and development and the potential underlying mechanisms are discussed.
The signaling mechanisms leading to cardiac hypertrophy have been intensively investigated over the past decade.1–4⇓⇓⇓ Identification of the signaling molecules necessary for the development of cardiac hypertrophy might lead to the development of therapeutic strategies to prevent or reverse the disease. During the past few years, mitogen-activated protein kinases (MAPKs), calcineurin (a Ca2+/calmodulin-dependent protein phosphatase), and Ca2+/calmodulin-dependent protein kinases have been intensively investigated because they positively regulate cardiac hypertrophy (see reviews5,6⇓). Equally important in exploring the signaling mechanisms promoting hypertrophy is the dissection of pathways counteracting these prohypertrophic signaling mechanisms, which thereby alleviates the hypertrophic responses. Accumulating evidence suggests that glycogen synthase kinase-3β (GSK-3β) negatively regulates cardiac hypertrophy and that the inhibition of GSK-3β by hypertrophic stimuli is an important mechanism in the stimulation of cardiac hypertrophy.7–12⇓⇓⇓⇓⇓ GSK-3β also regulates cardiac development through its effect on Wnt signaling. In the present article, it is our goal to review recent findings regarding the role of GSK-3β in cardiac hypertrophy and development and to discuss the potential underlying mechanisms.
GSK-3β Is a Proline-Directed Serine/Threonine Protein Kinase Whose Activity Is Negatively Regulated by Multiple Mechanisms
GSK-3 belongs to a family of conserved serine/threonine kinases present in all eukaryotic groups. GSK-3 consists of two isoforms in humans, namely, GSK-3α (51 kDa) and GSK-3β (47 kDa) (see reviews13–15⇓⇓). GSK-3α and GSK-3β have 97% sequence homology within their kinase domains, whereas GSK-3α has an extended N-terminal glycine-rich tail.16 Although both isoforms share substrates, their expression patterns, substrate preferences, and cellular functions are not identical.15,17⇓ Kinase activities of GSK-3α and GSK-3β are regulated similarly in some cases18 but differently in other cases.19,20⇓ Although both isoforms are expressed in the heart,21 most studies that have been conducted thus far have examined the role of GSK-3β, possibly because of the general lack of specific reagents for GSK-3α.
Although GSK-3β was initially described for its function to inhibit glycogen synthesis through phosphorylation of glycogen synthase,22,23⇓ it has been revealed that GSK-3β regulates a wide range of cellular functions, including metabolism, gene expression, and cytoskeletal integrity13 (Figure 1). GSK-3β is also involved in a variety of disease processes, such as tumorigenesis and the development of Alzheimer’s disease.24–26⇓⇓ The minimal recognition determinant for phosphorylation by GSK-3β has been identified as -S-P-X-X-S-,27 where the first serine is phosphorylated by GSK-3β. GSK-3β tends to phosphorylate serine/threonine residues situated N-terminally next to a proline and is thus considered to be a proline-directed kinase.27,28⇓ The affinity of GSK-3β to some substrates is enhanced if the substrate is prephosphorylated at +4 serine/threonine by other kinases (referred to as “priming kinases”), including protein kinase A, casein kinases (Iα and II), and DYRK (which indicates dual-specificity tyrosine-phosphorylated and -regulated kinase).29–31⇓⇓ When sequential overlapping GSK-3β sites are present, GSK-3β itself can act as its own priming kinase.32 GSK-3–catalyzed phosphorylation of some substrates, such as the axin–adenomatous polyposis coli (APC)–β-catenin complex, may not require priming phosphorylation.33
GSK-3β is localized predominantly in the cytoplasm but is also found in the nucleus. Its subcellular localization is changed in response to stimuli.9,10,34,35⇓⇓⇓ For example, endothelin-1 stimulates nuclear translocation,10 whereas isoproterenol causes the nuclear exit of GSK-3β in cardiac myocytes.9 Serum withdrawal causes a marked increase in nuclear GSK-3β in neuronal cells.35
An important characteristic of GSK-3 is the fact that it is catalytically active in cells even under unstimulated conditions. Thus, total cellular activities of GSK-3 may be predominantly regulated at the level of protein expression. Interestingly, however, phosphorylation of GSK-3 at Tyr216 can further increase its kinase activity.36,37⇓ In Dictyostelium, stimulation of the cAMP receptor (CAR3) activates ZAK1, a non–receptor-type tyrosine kinase, which in turn phosphorylates and activates GskA, a homologue of mammalian GSK-3.38 Tyrosine kinases homologous to ZAK1 may exist in higher eukaryotic organisms.38
Because GSK-3β negatively regulates downstream signaling mechanisms, inactivation of GSK-3β in fact stimulates many cellular functions by removing the negative constraint imposed by GSK-3β. The activity of GSK-3β is regulated by multiple mechanisms (Figure 1). Most importantly, inactivation of GSK-3β is induced by phosphorylation via upstream protein kinases (see review33). The phosphorylation sites that lead to the inactivation of GSK-3 have been identified as Ser21 for GSK-3α and as Ser9 for GSK-3β.39–41⇓⇓ Protein kinase B (PKB)/Akt is one of the most thoroughly studied of the kinases that have been identified as upstream regulators of GSK-3β. Because PKB/Akt is a major kinase downstream from phosphatidylinositol 3-kinase (PI3K), many stimuli that activate PI3K inhibit GSK-3β through PKB/Akt. Morisco et al8 have recently demonstrated that β-adrenergic stimulation activates PKB/Akt and inactivates GSK-3β in cardiac myocytes. Other cardiac hypertrophic stimuli, including endothelin-1, Fas, and pressure overload, also inhibit GSK-3β, possibly through activation of the PI3K-PKB/Akt pathway.10,11⇓ Other upstream protein kinases that inhibit GSK-3β include integrin-linked kinase (ILK),42 protein kinase A,43 protein kinase Cδ,44 protein kinase Cζ,45 p90 ribosomal S6 kinase, and p70 S6 kinase-146 (Figure 1). Interestingly, ILK induces phosphorylation of GSK-3β at an amino acid residue distinct from Ser9.42
Activation of the Wnt signaling pathway (see below) is another important mechanism to inhibit activity of GSK-3β. In the presence of Wnt, Dishevelled (Dvl) and FRAT (frequently rearranged in advanced T-cell lymphomas) disrupt interaction between GSK-3 and axin, thereby leading to inactivation of GSK-3 via mechanisms distinct from the phosphorylation of GSK-3 at the residue targeted by insulin.47,48⇓
Although both insulin and Wnt pathways inactivate GSK-3β, they regulate distinct targets: Insulin induces an increased activity of glycogen synthase but has no influence on the protein level of β-catenin. In contrast, Wnt increases the cytosolic pool of β-catenin but not glycogen synthase activity.49 It has been suggested that GSK-3β phosphorylates β-catenin only when it is sequestered by the axin-APC complex.15,33⇓
Besides endogenous regulators of GSK-3β, several compounds directly inhibit kinase activities of GSK-3β. LiCl is the most commonly used inhibitor of GSK-3β.50 SB-216763 and SB-415286, structurally distinct maleimides, are potent and selective cell-permeable inhibitors of GSK-3β.51
GSK-3β Inhibits Cardiac Hypertrophy In Vivo and In Vitro
Recent evidence shows that GSK-3β functions as a negative regulator of cardiac hypertrophy.7–11⇓⇓⇓⇓ Increased cellular activities of GSK-3β by overexpression inhibit many aspects of cardiac hypertrophy, including increases in the rate of protein synthesis and hypertrophic gene expression.7–11⇓⇓⇓⇓ Activation of the hypertrophic program by stimulation of β-adrenergic receptors, Gq-coupled receptors, and Fas receptors leads to inactivation of endogenous GSK-3β via phosphorylation of Ser9, predominantly through the PI3K/Akt pathway.7–11⇓⇓⇓⇓ This prevents endogenous GSK-3β from executing its inhibitory effects on hypertrophy, thereby stimulating hypertrophy. Cardiac-specific overexpression of a mutant form of GSK-3β (GSK-3βS9A), which cannot be phosphorylated at Ser9, in transgenic mice inhibits hypertrophy in response to aortic banding and isoproterenol stimulation and partially prevents cardiac hypertrophy caused by an activated form of calcineurin.7 Lack of Fas receptor–induced inhibition of GSK-3β by pressure overload in lpr mice, which do not have functional Fas receptors, causes rapid-onset left ventricular dilatation and heart failure that are due to the absence of compensatory hypertrophy.11 Insulin-induced phosphorylation/inactivation of GSK-3β is impaired in diabetes. Resistance of GSK-3β phosphorylation by insulin may contribute not only to impaired glycogen synthesis but also to reduced protein synthesis, leading to the development of diabetic cardiomyopathy.52 Because GSK-3β regulates nuclear transcription, protein translation, and cytoskeletal organization in other cell types, it is likely that GSK-3β affects cardiac hypertrophy and the development of cardiomyopathy through multiple mechanisms, as proposed in Figure 2.
Regulation of Cardiac Transcription Factors
GSK-3β phosphorylates a wide variety of transcription factors, thereby regulating the nuclear transcription process. The Table lists the transcription factors known to be phosphorylated by GSK-3β and summarizes how GSK-3β–induced phosphorylation affects them. In general, phosphorylation of transcription factors by GSK-3β causes ubiquitination, nuclear exit, or decreases in the DNA binding, leading to decreases in nuclear transcription (Table). However, in some transcription factors, such as C/EBPα, CREB, and nuclear factor-κB, phosphorylation by GSK-3β stimulates transcription.17,53,54⇓⇓
Recent data from our laboratory show that GSK-3β directly phosphorylates GATA4 and negatively regulates transcription through GATA4 by stimulating Crm-1–mediated nuclear export of GATA4.9 Members of the GATA family zinc finger transcription factors play an important role in mediating cardiac development55,56⇓ and cardiac hypertrophy57–61⇓⇓⇓⇓ (see review62). A number of genes whose expression is altered during cardiac hypertrophy, including atrial natriuretic factor (ANF), brain natriuretic peptide, α- and β-myosin heavy chain, cardiac troponin I, platelet-derived growth factor receptor β, angiotensin II type 1a receptor, and Na+-Ca2+ exchanger, are critically regulated by GATA4 and other GATA family transcription factors.57–60,63,64⇓⇓⇓⇓⇓ Thus, the regulation of GATA4 by GSK-3β should widely affect phenotypic changes of cardiac myocytes during cardiac hypertrophy. Morisco et al8 have demonstrated that overexpression of either wild-type or Akt-insensitive GSK-3β negatively regulates the transcription of ANF in neonatal rat cardiac myocytes, whereas stimulation of the β-adrenergic receptors increases transcription of ANF through the inactivation of GSK-3β. The induction of ANF expression by endothelin was markedly inhibited by overexpression of GSK-3βS9A in cardiac myocytes.10
Haq et al10 have demonstrated that GSK-3β inhibits endothelin-1–induced hypertrophy in neonatal rat cardiac myocytes. GSK-3β maintains nuclear factors of activated T cells (NF-AT, an important positive mediator of cardiac hypertrophy65) in the cytosol or delays endothelin-1–induced nuclear import of NF-AT, thereby preventing NF-AT–mediated nuclear transcription. Because nuclear localization of NF-AT is positively regulated through its dephosphorylation by calcineurin,65 phosphorylation by GSK-3β acts as a counterregulatory mechanism against the calcineurin pathway. As with the stimulation of calcineurin, inhibition of GSK-3β by endothelin-1 causes nuclear translocation of NF-AT, thereby stimulating cardiac hypertrophy. Additional overexpression of GSK-3βS9A in transgenic mice overexpressing an activated form of calcineurin significantly decreased the nuclear localization of NF-AT.7
Whether stimulation of calcineurin or inhibition of GSK-3β predominates in the induction of cardiac hypertrophy seems to depend on the character of the hypertrophic stimulus. For example, inhibition of GSK-3β seems to predominate over the stimulation of calcineurin in the induction of β-adrenergic cardiac hypertrophy, inasmuch as inhibition of the calcineurin pathway with cyclosporin A only partially blocks β-adrenergic ANF transcription, whereas overexpression of GSK-3β completely blocks it in neonatal rat cardiac myocytes.8 In contrast, both mechanisms may be equally important for endothelin-1–induced cardiac hypertrophy.10 What is the importance of GSK-3β among the many signaling mechanisms known to promote cardiac hypertrophy? We speculate that the PI3K/PKB/Akt/GSK-3β pathway may be as important as the MAPK signaling mechanisms in some types of cardiac hypertrophy, such as Gq-mediated hypertrophy.10 However, the PI-3K/PKB/Akt/GSK-3β pathway can be the predominant mechanism when activation of the MAPK pathway is less prominent, such as in the case of β-adrenergic cardiac hypertrophy.7,8⇓
Regulation of Protein Synthesis
Protein synthesis is a complex process involving three essential steps: initiation, elongation, and termination.66 The concerted actions of multiple signaling molecules are required for the regulation of protein synthesis (see reviews66,67⇓). One of the critical steps controlling the initiation of protein translation is the binding of eukaryotic translation initiation factor 2 (eIF2) to the activated initiator tRNA (met-tRNAmet) and subsequent formation of a ternary complex that binds to the 40S ribosomal subunit. eIF2Bε is the largest of the five subunits of eIF2B and is required for the GDP/GTP exchange reaction of eIF2. GSK-3β phosphorylates eIF2Bε at Ser540 and inactivates it.68 Inactivation of GSK-3β by cell growth stimuli leads to decreases in the phosphorylation and activation of eIF2Bε, which promotes the initiation of protein synthesis.
Haq et al10 have demonstrated that overexpression of GSK-3βS9A in neonatal rat cardiac myocytes blocks development of the key features of cardiac hypertrophy, including increases in the rate of protein synthesis, on Gαq-coupled receptor stimulation. Overexpression of wild-type GSK-3β also inhibits increases in cell size as well as the total cellular protein content in cardiac myocytes in response to β-adrenergic stimulation in vitro (authors’ unpublished data, 2002) and in response to pressure overload and β-adrenergic stimulation in vivo.7 By contrast, inhibition of GSK-3β with LiCl increases protein synthesis.10 The downstream signaling mechanism regarding how GSK-3β negatively affects protein synthesis in cardiac myocytes remains to be investigated. Inhibition of protein translation initiation through the phosphorylation of eIF2Bε may be one of the mechanisms in mediating the antihypertrophic effects of GSK-3β. Alternatively, GSK-3β may transcriptionally regulate expression of the critical regulators for protein translation or autocrine/paracrine factors. Inhibition of GSK-3β by hypertrophic stimuli is, in many cases, accompanied by the activation of PI3K and Akt, which also positively regulate other regulators of protein synthesis via the FK506-binding protein/rapamycin–associated protein/mammalian target of rapamycin pathway, including eukaryotic initiation factor 4E–binding protein, p70 ribosomal S6 kinase-1, and eukaryotic elongation factor 2.66,67,69⇓⇓ Therefore, it is likely that GSK-3β works in concert with other molecules downstream from PI3K/Akt to regulate protein synthesis during cardiac hypertrophy.
Regulation of Cytoskeletal Organization
GSK-3β has been shown to phosphorylate microtubule-associated proteins (MAPs), including tau, MAP2c, and MAP1B, thereby contributing to cytoskeletal remodeling events in many cell types.70–72⇓⇓ Neurofibrillary tangles of paired helical filaments are neuropathological hallmarks of Alzheimer’s disease, and abnormally phosphorylated tau is the major subunit of paired helical filaments.73 Phosphorylation of tau inhibits microtubule assembly and reduces its ability to stabilize microtubules. Inhibition of GSK-3β by hypertrophic stimuli potentially affects microtubules and other cytoskeletal structures of cardiac myocytes through its effects on β-catenin and microtubule-associated proteins. Overexpression of GSK-3βS9A inhibits endothelin-1–induced actin reorganization in neonatal rat cardiac myocytes.10
Regulation of Cell Cycle
Temporal and spatial expression and activation of the cell cycle regulators, including cyclins and cyclin-dependent kinases (cdks), are tightly regulated to control the cell-cycle transition.74 Among many cell-cycle regulators, cyclin D1, a regulator of G1- to S-phase transition, is a critical downstream target of the Wnt1 signaling.75 It is expected that GSK-3βnegatively regulates cyclin D1 expression by inhibiting the Wnt1 signaling. In addition, activity and nuclear localization of GSK-3β are regulated in a cell-cycle–dependent manner, which, in turn, controls subcellular localization and expression of cyclin D1 through phosphorylation of Thr286.34 Phosphorylation of cyclin D1 by GSK-3β causes increased association of cyclin D1 with a nuclear exportin (Crm-1) and promotes nuclear exit and subsequent proteasomal degradation of cyclin D1.76 Because the cyclin-cdk complex has recently been implicated in various cellular functions in cardiac myocytes not restricted to DNA synthesis77,78⇓ (see reviews79,80⇓) GSK-3β–induced regulation of cyclin D1 may be involved in cell growth/death responses in the heart. GSK-3β phosphorylates p21Cip1, thereby stimulating its degradation in human umbilical vein endothelial cells.81 Interestingly, inhibition of GSK-3β by LiCl causes accumulation of p21Cip1 and hypertrophy of endothelial cells.81
Regulation of Apoptosis
GSK-3β plays an important role in the regulation of apoptosis/cell survival. GSK-3β promotes apoptosis in neuronal cells82,83⇓ and vascular smooth muscle cells.84 Increased cAMP levels promote survival of neuronal cells by inactivating GSK-3β via a protein kinase A–dependent mechanism.83 Overexpression of a mutant form of eIF2Bε, which cannot be phosphorylated by GSK-3β, inhibits cytochrome c release in PC12 cells,85 suggesting that apoptosis by GSK-3β is mediated by the phosphorylation of eIF2Bε. GSK-3β inhibits antiapoptotic molecules, including heat shock factor-1 and the associated expression of heat shock protein-70,86,87⇓ which may, in turn, stimulate apoptosis.
By contrast, findings in other cell types suggest that GSK-3β mediates cell survival. For example, GSK-3β knockout mice die in utero with increased apoptosis in the liver caused by excessive production of tumor necrosis factor, suggesting that GSK-3β mediates cell survival mechanisms, such as activation of nuclear factor-κB.17 Furthermore, β-catenin promotes apoptosis in some mammalian cell lines.88 In this case, GSK-3β should inhibit apoptosis through the degradation of β-catenin.
Collectively, whether GSK-3β promotes apoptosis or cell survival depends on the cell types. It has been shown that PKB/Akt promotes cell survival in cardiac myocytes.89 The cell survival effect of Akt may be partially mediated by phosphorylation/inhibition of GSK-3β. It has been suggested that calcineurin promotes cell survival in cardiac myocytes.90 It is of great interest to determine whether GSK-3β counteracts the antiapoptotic effect of calcineurin and promotes apoptosis in cardiac myocytes.
Regulation of Wnt Signaling Pathway by GSK-3β and Its Role in Cardiac Development and Postnatal Hearts
Wnt/Wingless proteins are a family of cysteine-rich glycoproteins that regulate cell fate decisions, embryonic development,91 and tumorigenesis.92 Association of Wnt and the Frizzled (Fz) family of transmembrane receptors leads to accumulation of β-catenin, a key downstream target of Wnt signaling93 (Figure 3). GSK-3β is a critical regulator of this signaling mechanism. GSK-3β forms a complex with axin (and its close relative conductin) and the tumor suppressor gene product APC. This complex, called the destruction complex, controls the stability of β-catenin. Axin is a scaffold molecule and sequesters GSK-3 β in this complex.15 When GSK-3β is active, it phosphorylates APC and β-catenin and stimulates interaction between β-catenin and β-Trcp, a regulator of E3 ubiquitin ligase, and subsequent degradation of β-catenin in proteasomes.94,95⇓ Binding of the Wnt molecules to Fz receptors activates Dvl, which inhibits the activity of GSK-3β, 96 thereby stabilizing β-catenin.97 Casein kinase Iε also makes a complex with axin, GSK-3β, and Dvl and works as a positive regulator of the Wnt signaling.98 Stabilization of β-catenin is associated with its translocation to the nucleus, where it interacts with members of the lymphoid enhancer factor (LEF)/T-cell factor (TCF) and activates specific target genes.99,100⇓ It should be noted that inhibition of GSK-3β alone is not sufficient to activate LEF/TCF and that an additional factor is required to facilitate dissociation of GSK-3β from axin.101
GSK/Wnt Signaling in Cardiac Development
Accumulating evidence shows that the components of the Wnt pathway play a significant role in heart development. Several recent studies have shown that the Wnt signaling pathway negatively regulates cardiogenesis.102–105⇓⇓⇓ For example, in Xenopus, Wnt3a and Wnt8 inhibit heart induction, and ectopic expression of GSK-3β induces cardiogenesis in ventral mesoderm.103 The secreted Wnt antagonists crescent and Dickkopf-1 are expressed in the anterior mesoderm and promote cardiogenesis by interfering with the Wnt signaling pathway.104 Similarly, inhibition of Wnt activity induces heart formation from the posterior mesoderm in chick embryos.105 In contrast to these findings, other reports suggest that the Wnt signaling positively regulates cardiac development. For example, wingless, zeste-white3/shaggy–encoded kinase, and armadillo, the Drosophila homologues of Wnt, GSK-3β, and β-catenin, respectively, have been shown to play a crucial role in specifying the heart progenitors in Drosophila.106,107⇓ In chicks, the ectopic expression of Wnt11 has been shown to promote cardiac development within noncardiac tissue.108 It should be noted that the effect of Wnt11 may be mediated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, not via GSK-3β and β-catenin.109 β-Catenin is involved in the initial differentiation of the heart-forming mesoderm in the chick embryo.110,111⇓ Oral treatment with lithium, a mood-stabilizing drug that is inhibitory for GSK-3, in pregnant women showed a higher incidence of congenital heart defects in babies.112 Collectively, GSK-3β is involved in heart development via the Wnt signaling pathway, yet the exact mechanisms of action remain to be elucidated.
Wnt Signaling in Postnatal Hearts
The Wnt signaling pathway may play a role in postnatal hearts as well. Wnt proteins, including Wnt10B, are expressed in the adult heart.113 The Fz class of cell surface receptors for Wnt proteins, including Fz1 and Fz2, are expressed in human myocardium.114 In myofibroblasts of infarcted hearts, Fz2 expression is considerably enhanced.115 In failing ventricles of humans, mRNA levels of secreted Fz-related proteins 3 and 4, which are endogenous Wnt antagonists, are elevated, and the Wnt/β-catenin pathway is attenuated.116 However, the functional role of Wnt signaling in the normal and pathological hearts remains to be elucidated.
When the Wnt signaling is activated and/or the kinase activity of GSK-3β is inhibited, β-catenin is stabilized and translocated into the nucleus, and it participates in the transcription process.99,117,118⇓⇓ Stabilized β-catenin markedly stimulates the transcription of connexin43, a major component of cardiac gap junction channels.119 Although β-catenin stimulates a reporter gene containing the consensus TCF binding sequence,26 the nuclear transcription factor that β-catenin binds, presumably a member of the LEF/TCF family, has not been identified in cardiac myocytes.
Recent evidence suggests that β-catenin is upregulated in human hypertrophy.120 Preliminary results from our laboratory also indicate that β-adrenergic stimulation of neonatal rat cardiac myocytes upregulates the expression of β-catenin.121 Because the β-catenin–LEF/TCF complex controls the activities of many genes mediating cell proliferation, it will be interesting to elucidate how β-catenin affects cardiac hypertrophy. The Wnt signaling pathway also activates β-catenin–independent signaling mechanisms. For example, Dvl activates c-Jun NH2-terminal kinases,122 which may stimulate cardiac hypertrophy.
Because β-catenin is a central component of the cadherin–cell adhesion complex, it is speculated that β-catenin also directly affects the formation of cell-cell junctions independently of its role as a transcriptional regulator. In fact, the Wnt-Fz2 signaling pathway is involved in the stabilization of the cadherin–β-catenin complex in neonatal rat cardiac myocytes.123 Chronic aortic stenosis in guinea pigs causes translocation of β-catenin and vinculin away from intercalated disks in failing myocytes, thereby impairing the mechanical linkage between N-cadherin and thin filaments and adversely affecting myocyte morphology.124 It is of great interest to determine whether the activity of GSK-3β is altered in this animal model. Collectively, these results suggest that GSK-3β may affect cadherin-mediated cellular responses of cardiac myocytes through the regulation of β-catenin.
Unanswered Questions: Future Perspectives
As we have summarized in this review, GSK-3β regulates various cellular phenomena relevant for the growth and death of cardiac myocytes. Because overexpression of constitutively active GSK-3β suppresses the development of hypertrophy in vivo7 and in vitro8–11⇓⇓⇓ and because the activity of GSK-3β is negatively regulated by cardiac hypertrophic stimuli,7–12⇓⇓⇓⇓⇓ inhibition of GSK-3β by hypertrophic stimuli represents an important signaling mechanism of cardiac hypertrophy. This signaling mechanism is unique among major hypertrophic signaling pathways because “disinhibition” mediates the positive mechanism. Although several downstream signaling molecules, including GATA4, NF-AT, eIF2Bε, and β-catenin, are likely to be regulated by GSK-3β in cardiac myocytes, the precise downstream signaling mechanisms regarding how GSK-3β negatively affects cardiac hypertrophy remain to be determined.
It has been shown that GSK-3β is phosphorylated and that its kinase activities are downregulated in patients with heart failure.12 Although overexpression of GSK-3 β (S9A) prevents pressure overload–induced cardiac hypertrophy in transgenic mice,7 recent evidence suggests that the inhibition of GSK-3 β is cardioprotective against ischemia.125 Whether or not restoring GSK-3β activity in patients with heart failure is salutary remains to be elucidated. Other important unanswered questions include the following: What is the unique function of GSK-3α in the heart? Does GSK-3β play an important role in mediating physiological forms of cardiac hypertrophy, such as exercise-induced and pathophysiological changes of the heart during aging? Do calcineurin and GSK-3β always counterregulate the phosphorylation status of the same target molecules? Does GSK-3β promote apoptosis in cardiac myocytes? What are the other functions of GSK-3β in the heart?
Some useful tools to address these issues, including adenoviral vectors harboring wild types and mutants of GSK-3β as well as transgenic mice with cardiac specific overexpression of GSK-3β (S9A), are available.7,9,10⇓⇓ Disruption of the murine GSK-3β gene results in embryonic lethality by severe liver degeneration.17 To obtain genetic evidence regarding whether GSK-3β is required for postnatal cardiac disease, cardiac-specific knockout models will be required. To test the therapeutic potential of modulating GSK-3β in patients with heart failure, the effect of conditional expression or deletion of GSK-3β needs to be evaluated in animal models. It is expected that more evidence will become available in the near future to further dissect the underlying mechanisms by which GSK-3β regulates the growth and death of cardiac myocytes.
Note Added in Proof
Recently, Liu et al136 have shown that priming phosphorylation of β-catenin by casein kinase Iα is required for its subsequent phosphorylation by GSK-3β and degradation.
This review was supported by grants from the NIH and the American Heart Association, National Headquarters. We thank Drs S.F. Vatner, D.E. Vatner, and H. Tomita for critical reading of this manuscript.
Original received April 4, 2001; resubmission received March 7, 2002; revised resubmission received April 5, 2002; accepted April 9, 2002.
- ↵Sugden PH. Signaling in myocardial hypertrophy: life after calcineurin? Circ Res. 1999; 84: 633–646.
- ↵Olson EN, Molkentin JD. Prevention of cardiac hypertrophy by calcineurin inhibition: hope or hype? Circ Res. 1999; 84: 623–632.
- ↵Molkentin JD. Calcineurin and beyond: cardiac hypertrophic signaling. Circ Res. 2000; 87: 731–738.
- ↵Antos CL, McKinsey TA, Frey N, Kutschke W, McAnally J, Shelton JM, Richardson JA, Hill JA, Olson EN. Activated glycogen synthase-3β suppresses cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2002; 99: 907–912.
- ↵Morisco C, Zebrowski D, Condorelli G, Tsichlis P, Vatner SF, Sadoshima J. The Akt-glycogen synthase kinase 3β pathway regulates transcription of atrial natriuretic factor induced by β-adrenergic receptor stimulation in cardiac myocytes. J Biol Chem. 2000; 275: 14466–14475.
- ↵Morisco C, Seta K, Hardt SE, Lee Y, Vatner SF, Sadoshima J. Glycogen synthase kinase 3β regulates GATA4 in cardiac myocytes. J Biol Chem. 2001; 276: 28586–28597.
- ↵Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A, Molkentin JD, Alessandrini A, Woodgett J, Hajjar R, Michael A, Force T. Glycogen synthase kinase-3β is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol. 2000; 151: 117–130.
- ↵Haq S, Choukroun G, Lim H, Tymitz KM, del Monte FF, Gwathmey J, Grazette L, Michael A, Hajjar R, Force T, Molkentin JD. Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation. 2001; 103: 670–677.
- ↵Woodgett JR. Judging a protein by more than its name: GSK-3. Sci STKE. September 18, 2001; 100: RE12.
- ↵Saito Y, Vandenheede JR, Cohen P. The mechanism by which epidermal growth factor inhibits glycogen synthase kinase 3 in A431 cells. Biochem J. 1994; 303: 27–31.
- ↵Wojtazsewski JF, Nielsen P, Kiens B, Richter EA. Regulation of glycogen synthase kinase-3 in human skeletal muscle: effects of food intake and bicycle exercise. Diabetes. 2001; 50: 265–269.
- ↵Fiol CJ, Mahrenholz AM, Wang Y, Roeske RW, Roach PJ. Formation of protein kinase recognition sites by covalent modification of the substrate: molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J Biol Chem. 1987; 262: 14042–14048.
- ↵Porter CM, Havens MA, Clipstone NA. Identification of amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization. J Biol Chem. 2000; 275: 3543–3551.
- ↵Woods YL, Cohen P, Becker W, Jakes R, Goedert M, Wang X, Proud CG. The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bε at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase. Biochem J. 2001; 355: 609–615.
- ↵Ginger RS, Dalton EC, Ryves WJ, Fukuzawa M, Williams JG, Harwood AJ. Glycogen synthase kinase-3 enhances nuclear export of a dictyostelium STAT protein. EMBO J. 2000; 19: 5483–5491.
- ↵Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 1998; 12: 3499–3511.
- ↵Bijur GN, Jope RS. Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3β. J Biol Chem. 2001; 276: 37436–37442.
- ↵Wang QM, Fiol CJ, DePaoli-Roach AA, Roach PJ. Glycogen synthase kinase-3β is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. J Biol Chem. 1994; 269: 14566–14574.
- ↵Sutherland C, Leighton IA, Cohen P. Inactivation of glycogen synthase kinase-3β by phosphorylation: new kinase connections in insulin and growth-factor signalling. Biochem J. 1993; 296: 15–19.
- ↵Stambolic V, Woodgett JR. Mitogen inactivation of glycogen synthase kinase-3β in intact cells via serine 9 phosphorylation. Biochem J. 1994; 303: 701–704.
- ↵Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A. 1998; 95: 11211–11216.
- ↵Fang X, Yu SX, Lu Y, Bast RCJr, Woodgett JR, Mills GB. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A. Proc Natl Acad Sci U S A. 2000; 97: 11960–11965.
- ↵Ballou LM, Tian PY, Lin HY, Jiang YP, Lin RZ. Dual regulation of glycogen synthase kinase-3β by the α1A-adrenergic receptor. J Biol Chem. 2001; 276: 40910–40916.
- ↵Cross DA, Alessi DR, Vandenheede JR, McDowell HE, Hundal HS, Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J. 1994; 303: 21–26.
- ↵Li L, Yuan H, Weaver CD, Mao J, Farr GH, Sussman DJ, Jonkers J, Kimelman D, Wu D. Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt- mediated regulation of LEF-1. EMBO J. 1999; 18: 4233–4240.
- ↵Ding VW, Chen RH, McCormick F. Differential regulation of glycogen synthase kinase 3β by insulin and Wnt signaling. J Biol Chem. 2000; 275: 32475–32481.
- ↵Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GJ, Carter PS, Roxbee Cox L, Mills D, Brown MJ, Haigh D, Ward RW, Smith DG, Murray KJ, Reith AD, Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000; 7: 793–803.
- ↵Laviola L, Belsanti G, Davalli AM, Napoli R, Perrini S, Weir GC, Giorgino R, Giorgino F. Effects of streptozocin diabetes and diabetes treatment by islet transplantation on in vivo insulin signaling in rat heart. Diabetes. 2001; 50: 2709–2720.
- ↵Fiol CJ, Williams JS, Chou CH, Wang QM, Roach PJ, Andrisani OM. A secondary phosphorylation of CREB341 at Ser129 is required for the cAMP-mediated control of gene expression: a role for glycogen synthase kinase-3 in the control of gene expression. J Biol Chem. 1994; 269: 32187–32193.
- ↵Ross SE, Erickson RL, Hemati N, MacDougald OA. Glycogen synthase kinase 3 is an insulin-regulated C/EBPα kinase. Mol Cell Biol. 1999; 19: 8433–8441.
- ↵Molkentin JD, Lin Q, Duncan SA, Olson EN. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 1997; 11: 1061–1072.
- ↵Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek MS, Soudais C, Leiden JM. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 1997; 11: 1048–1060.
- ↵Grepin C, Dagnino L, Robitaille L, Haberstroh L, Antakly T, Nemer M. A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription. Mol Cell Biol. 1994; 14: 3115–3129.
- ↵Hasegawa K, Lee SJ, Jobe SM, Markham BE, Kitsis RN. cis-Acting sequences that mediate induction of β-myosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction. Circulation. 1997; 96: 3943–3953.
- ↵Herzig TC, Jobe SM, Aoki H, Molkentin JD, Cowley AWJ, Izumo S, Markham BE. Angiotensin II type 1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload. Proc Natl Acad Sci U S A. 1997; 94: 7543–7548.
- ↵Cheng G, Hagen TP, Dawson ML, Barnes KV, Menick DR. The role of GATA, CArG, E-box, and a novel element in the regulation of cardiac expression of the Na+-Ca2+ exchanger gene. J Biol Chem. 1999; 274: 12819–12826.
- ↵Xia Y, McMillin JB, Lewis A, Moore M, Zhu WG, Williams RS, Kellems RE. Electrical stimulation of neonatal cardiac myocytes activates the NFAT3 and GATA4 pathways and up-regulates the adenylosuccinate synthetase 1 gene. J Biol Chem. 2000; 275: 1855–1863.
- ↵Molkentin JD. The zinc finger-containing transcription factors GATA-4, -5, and -6: ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem. 2000; 275: 38949–38952.
- ↵Morimoto T, Hasegawa K, Kaburagi S, Kakita T, Masutani H, Kitsis RN, Matsumori A, Sasayama S. GATA-5 is involved in leukemia inhibitory factor-responsive transcription of the β-myosin heavy chain gene in cardiac myocytes. J Biol Chem. 1999; 274: 12811–12818.
- ↵Charron F, Paradis P, Bronchain O, Nemer G, Nemer M. Cooperative interaction between GATA-4 and GATA-6 regulates myocardial gene expression. Mol Cell Biol. 1999; 19: 4355–4365.
- ↵Rhoads RE. Signal transduction pathways that regulate eukaryotic protein synthesis. J Biol Chem. 1999; 274: 30337–30340.
- ↵Wang L, Wang X, Proud CG. Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps. Am J Physiol. 2000; 278: H1056–H1068.
- ↵Lucas FR, Goold RG, Gordon-Weeks PR, Salinas PC. Inhibition of GSK-3β leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium. J Cell Sci. 1998; 111: 1351–1361.
- ↵Rimerman RA, Gellert-Randleman A, Diehl JA. Wnt1 and MEK1 cooperate to promote cyclin D1 accumulation and cellular transformation. J Biol Chem. 2000; 275: 14736–14742.
- ↵Alt JR, Cleveland JL, Hannink M, Diehl JA. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev. 2000; 14: 3102–3114.
- ↵Adachi S, Ito H, Tamamori-Adachi M, Ono Y, Nozato T, Abe S, Ikeda M-a, Marumo F, Hiroe M. Cyclin A/cdk2 activation is involved in hypoxia-induced apoptosis in cardiomyocytes. Circ Res. 2001; 88: 408–414.
- ↵Schneider MD, MacLellan WR. Cyclin-dependent kinase-2 in the birth and death of cardiac muscle cells. Circ Res. 2001; 88: 367–369.
- ↵Rossig L, Badorff C, Holzmann Y, Zeiher AM, Dimmeler S. GSK-3 couples AKT-dependent signaling to the regulation of p21Cip1 degradation. J Biol Chem. 2002; 277: 9684–9689.
- ↵Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem. 1998; 273: 19929–19932.
- ↵Li M, Wang X, Meintzer MK, Laessig T, Birnbaum MJ, Heidenreich KA. Cyclic AMP promotes neuronal survival by phosphorylation of glycogen synthase kinase 3β. Mol Cell Biol. 2000; 20: 9356–9363.
- ↵Hall JL, Chatham JC, Eldar-Finkelman H, Gibbons GH. Upregulation of glucose metabolism during intimal lesion formation is coupled to the inhibition of vascular smooth muscle cell apoptosis: role of GSK3β. Diabetes. 2001; 50: 1171–1179.
- ↵Pap M, Cooper GM. Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3β signaling pathway. Mol Cell Biol. 2002; 22: 578–586.
- ↵He B, Meng YH, Mivechi NF. Glycogen synthase kinase 3β and extracellular signal-regulated kinase inactivate heat shock transcription factor 1 by facilitating the disappearance of transcriptionally active granules after heat shock. Mol Cell Biol. 1998; 18: 6624–6633.
- ↵Xavier IJ, Mercier PA, McLoughlin CM, Ali A, Woodgett JR, Ovsenek N. Glycogen synthase kinase 3β negatively regulates both DNA-binding and transcriptional activities of heat shock factor 1. J Biol Chem. 2000; 275: 29147–29152.
- ↵Kim K, Pang KM, Evans M, Hay ED. Overexpression of β-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators. Mol Biol Cell. 2000; 11: 3509–3523.
- ↵Matsui T, Li L, del Monte F, Fukui Y, Franke TF, Hajjar RJ, Rosenzweig A. Adenoviral gene transfer of activated phosphatidylinositol 3′-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation. 1999; 100: 2373–2379.
- ↵De Windt LJ, Lim HW, Taigen T, Wencker D, Condorelli G, Dorn GW, Kitsis RN, Molkentin JD. Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: an apoptosis-independent model of dilated heart failure. Circ Res. 2000; 86: 255–263.
- ↵Parr BA, Shea MJ, Vassileva G, McMahon AP. Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds. Development. 1993; 119: 247–261.
- ↵Wang Y, Macke JP, Abella BS, Andreasson K, Worley P, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J. A large family of putative transmembrane receptors homologous to the product of theDrosophila tissue polarity gene frizzled. J Biol Chem. 1996; 271: 4468–4476.
- ↵Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. β-Catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 1997; 16: 3797–3804.
- ↵Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D, Birchmeier W. Functional interaction of an axin homolog, conductin, with β-catenin, APC, and GSK3β. Science. 1998; 280: 596–599.
- ↵Miller JR, Moon RT. Signal transduction through β-catenin and specification of cell fate during embryogenesis. Genes Dev. 1996; 10: 2527–2539.
- ↵Sakanaka C, Leong P, Xu L, Harrison SD, Williams LT. Casein kinase Iε in the wnt pathway: regulation of β-catenin function. Proc Natl Acad Sci U S A. 1999; 96: 12548–12552.
- ↵Yuan H, Mao J, Li L, Wu D. Suppression of glycogen synthase kinase activity is not sufficient for leukemia enhancer factor-1 activation. J Biol Chem. 1999; 274: 30419–30423.
- ↵Olson EN. Development: the path to the heart and the road not taken. Science. 2001; 291: 2327–2328.
- ↵Schneider VA, Mercola M. Wnt antagonism initiates cardiogenesis inXenopus laevis. Genes Dev. 2001; 15: 304–315.
- ↵Tzahor E, Lassar AB. Wnt signals from the neural tube block ectopic cardiogenesis. Genes Dev. 2001; 15: 255–260.
- ↵Marvin MJ, Di Rocco G, Gardiner A, Bush SM, Lassar AB. Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Dev. 2001; 15: 316–327.
- ↵Kuhl M, Sheldahl LC, Malbon CC, Moon RT. Ca2+/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J Biol Chem. 2000; 275: 12701–12711.
- ↵Schumann H, Holtz J, Zerkowski HR, Hatzfeld M. Expression of secreted frizzled related proteins 3 and 4 in human ventricular myocardium correlates with apoptosis related gene expression. Cardiovasc Res. 2000; 45: 720–728.
- ↵Rezvani M, Liew CC. Role of the adenomatous polyposis coli gene product in human cardiac development and disease. J Biol Chem. 2000; 275: 18470–18475.
- ↵Gaussin V, Sadoshima J. β-Adrenergic receptor stimulation activates β-catenin. Circulation. 2000; 102 (suppl II): II-196.Abstract.
- ↵Toyofuku T, Hong Z, Kuzuya T, Tada M, Hori M. Wnt/frizzled-2 signaling induces aggregation and adhesion among cardiac myocytes by increased cadherin-β-catenin complex. J Cell Biol. 2000; 150: 225–241.
- ↵Tong H, Imahashi K, Steenbergen C, Murphy E. Phosphorylation of glycogen synthase kinase-3β during preconditioning through a phosphatidylinositol-3-kinase–dependent pathway is cardioprotective. Circ Res. 2002; 90: 377–379.
- Beals CR, Sheridan CM, Turck CW, Gardner P, Crabtree GR. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science. 1997; 275: 1930–1934.
- GSK-3β Inhibits Cardiac Hypertrophy In Vivo and In Vitro
- Unanswered Questions: Future Perspectives
- Figures & Tables
- Info & Metrics