Myocyte Enhancer Factor-2 Transcription Factors Research Articles

The deacetylase HDAC4 controls myocyte enhancing factor‐2‐dependent structural gene expression in response to neural activity

Histone deacetylase 4 (HDAC4) binds and inhibits activation of the critical muscle transcription factor myocyte enhancer factor-2 (MEF2). However, the physiological significance of the HDAC4-MEF2 complex in skeletal muscle has not been established. Here we show that in skeletal muscle, HDAC4 is a critical modulator of MEF2-dependent structural and contractile gene expression in response to neural activity. We present evidence that loss of neural input leads to concomitant nuclear accumulation of HDAC4 and transcriptional reduction of MEF2-regulated gene expression. Cell-based assays show that HDAC4 represses structural gene expression via direct binding to AT-rich MEF2 response elements. Notably, using both surgical denervation and the neuromuscular disease amyotrophic lateral sclerosis (ALS) model, we found that elevated levels of HDAC4 are required for efficient repression of MEF2-dependent structural gene expression, indicating a link between the pathological induction of HDAC4 and subsequent MEF2 target gene suppression. Supporting this supposition, we show that ectopic expression of HDAC4 in muscle fibers is sufficient to induce muscle damage in mice. Our study identifies HDAC4 as an activity-dependent regulator of MEF2 function and suggests that activation of HDAC4 in response to chronically reduced neural activity suppresses MEF2-dependent gene expression and contributes to progressive muscle dysfunction observed in neuromuscular diseases.

Cocaine Regulates MEF2 to Control Synaptic and Behavioral Plasticity

Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.

Signal-dependent regulation of transcription by histone deacetylase 7 involves recruitment to promyelocytic leukemia protein nuclear bodies.

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes, including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase (HDAC) 7 is a potent corepressor that inhibits transcription by myocyte enhancer factor 2 (MEF2) transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor (TNF)-alpha treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells. Consequently, TNF-alpha promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase (MMP)-10, leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, small interfering RNA knockdown of PML decreases basal and TNF-alpha-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression.

MEF2C Transcription Factor Controls Chondrocyte Hypertrophy and Bone Development

Chondrocyte hypertrophy is essential for endochondral bone development. Unexpectedly, we discovered that MEF2C, a transcription factor that regulates muscle and cardiovascular development, controls bone development by activating the gene program for chondrocyte hypertrophy. Genetic deletion of Mef2c or expression of a dominant-negative MEF2C mutant in endochondral cartilage impairs hypertrophy, cartilage angiogenesis, ossification, and longitudinal bone growth in mice. Conversely, a superactivating form of MEF2C causes precocious chondrocyte hypertrophy, ossification of growth plates, and dwarfism. Endochondral bone formation is exquisitely sensitive to the balance between MEF2C and the corepressor histone deacetylase 4 (HDAC4), such that bone deficiency of Mef2c mutant mice can be rescued by an Hdac4 mutation, and ectopic ossification in Hdac4 null mice can be diminished by a heterozygous Mef2c mutation. These findings reveal unexpected commonalities in the mechanisms governing muscle, cardiovascular, and bone development with respect to their regulation by MEF2 and class II HDACs.

Mechanisms underlying myogenesis: complex and likely to become more so!

in this issue, Geiger and her colleagues ([7][1]) provide an excellent and comprehensive database of mRNA abundance and protein expression of myosin heavy chain (MHC) isoforms in the rat diaphragm during a period of rapid growth [i.e., postnatal days ( P ) 0 , 14 , 28 , and 84 (adult)]. Although the

MEF2 Activates a Genetic Program Promoting Chamber Dilation and Contractile Dysfunction in Calcineurin-Induced Heart Failure

Hypertrophic growth, a risk factor for mortality in heart disease, is driven by reprogramming of cardiac gene expression. Although the transcription factor myocyte enhancer factor-2 (MEF2) is a common end point for several hypertrophic pathways, its precise cardiac gene targets and function in cardiac remodeling remain to be elucidated. We report the existence of synergistic interactions between the nuclear factor of activated T cells and MEF2 transcription factors triggered by calcineurin signaling. To circumvent the embryonic lethality and mitochondrial deficiency associated with germ-line null mutations for MEF2C and MEF2A respectively, we used conditional transgenesis to express a dominant-negative form of MEF2 in the murine postnatal heart and combined this with magnetic resonance imaging to assess MEF2 transcriptional function in Ca2+/calcineurin-induced cardiac remodeling. Surprisingly, end-diastolic and end-systolic ventricular dimensions and contractility were normalized in the presence of severely hypertrophied left ventricular walls on MEF2 inhibition in calcineurin transgenic mice. In line, we generated lines of transgenic mice expressing MEF2A in the heart, which displayed primarily chamber dilation. Microarray profiling indicated that MEF2 promotes a gene profile functioning primarily to or at the nucleus, cytoskeletal and microtubular networks, and mitochondria. These findings assign a novel function to MEF2 transcription factors in the postnatal heart, where they activate a genetic program that minimally affects cardiac growth yet promotes chamber dilation, mechanical dysfunction, and dilated cardiomyopathy.

Myocyte Enhancer Factors 2A and 2C Induce Dilated Cardiomyopathy in Transgenic Mice

Cardiac hypertrophy and dilation are mediated by neuroendocrine factors and/or mitogens as well as through internal stretch- and stress-sensitive signaling pathways, which in turn transduce alterations in cardiac gene expression through specific signaling pathways. The transcription factor family known as myocyte enhancer factor 2 (MEF2) has been implicated as a signal-responsive mediator of the cardiac transcriptional program. For example, known hypertrophic signaling pathways that utilize calcineurin, calmodulin-dependent protein kinase, and MAPKs can each affect MEF2 activity. Here we demonstrate that MEF2 transcription factors induced dilated cardiomyopathy and lengthening of myocytes. Specifically, multiple transgenic mouse lines with cardiac-specific overexpression of MEF2A or MEF2C presented with cardiomyopathy at base line or were predisposed to more fulminant disease following pressure overload stimulation. The cardiomyopathic response associated with MEF2A and MEF2C was not further altered by activated calcineurin, suggesting that MEF2 functions independently of calcineurin in this response. In cultured cardiomyocytes, MEF2A, MEF2C, and MEF2-VP16 overexpression induced sarcomeric disorganization and focal elongation. Mechanistically, MEF2A and MEF2C each programmed similar profiles of altered gene expression in the heart that included extracellular matrix remodeling, ion handling, and metabolic genes. Indeed, adenoviral transfection of cultured cardiomyocytes with MEF2A or of myocytes from the hearts of MEF2A transgenic adult mice showed reduced transient outward K(+) currents, consistent with the alterations in gene expression observed in transgenic mice and partially suggesting a proximal mechanism underlying MEF2-dependent cardiomyopathy.

Myospryn Is a Direct Transcriptional Target for MEF2A That Encodes a Striated Muscle, α-Actinin-interacting, Costamere-localized Protein

The full repertoire of proteins that comprise the striated muscle Z-disc and peripheral structures, such as the costamere, have yet to be discovered. Recent studies suggest that this elaborate protein network, which acts as a structural and signaling center for striated muscle, harbors factors that function as mechanosensors to ensure coordinated contractile activity. Mutations in genes whose products reside in this region often result in skeletal and cardio myopathies, demonstrating the importance of this macromolecular complex in muscle structure and function. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the costamere in striated muscle. This gene, called myospryn, was identified by microarray analysis as a transcript down-regulated in MEF2A knock-out mice. MEF2A knock-out mice develop cardiac failure during the perinatal period with mutant hearts exhibiting several cardiac abnormalities including myofibrillar disarray. Myospryn is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy-associated gene 5 (CMYA5). Myospryn is expressed as a single, large transcript of approximately 12 kilobases in adult heart and skeletal muscle with an open reading frame of 3739 amino acids. This protein, belonging to the tripartite motif superfamily of proteins, contains a B-box coiled-coil (BBC), two fibronectin type III (FN3) repeats, and SPRY domains and interacts with the sarcomeric Z-disc protein, alpha-actinin-2. Our findings demonstrate that myospryn functions directly downstream of MEF2A at the costamere in striated muscle potentially playing a role in myofibrillogenesis.

Transcriptional Regulation of Synaptic Change

Changes in synaptic activity of neurons can result in stable changes in neuronal function. Many such long-term changes are thought to be mediated by altered transcription of key target genes. Two studies have identified a role for the transcriptional regulator MEF2 in the control of synapse number (see the Perspective by Beg and Scheiffele). In cultured rat embryonic neurons, Flavell et al. found that the calcium-dependent signaling that accompanied neuronal activity led to activation of myocyte enhancer factor 2 (MEF2) and decreased numbers of synapses. Depletion of MEF2A and MEF2D caused increased synapse formation, and MEF2 activated transcription of genes known to reduce synapse formation. Shalizi et al. also found regulation of synapse number by MEF2 in rat cerebellum and cerebellar slices. MEF2A appeared to act by repressing transcription of the gene encoding the transcription factor Nur77. MEF2A showed this repression activity when sumoylated (modified by covalent linking of small ubiquitin-related modifier protein, or SUMO). Sumoylation was, in turn, dependent on calcium-dependent dephosphorylation of MEF2A. Phosphorylation of MEF2A appeared to switch MEF2A between an acetylated and a sumoylated state. A. A. Beg, P. Scheiffele, SUMO wrestles the synapse. Science 311 , 962-963 (2006). [Summary] [Full Text] S. W. Flavell, C. W. Cowan, T.-K. Kim, P. L. Greer, Y. Lin, S. Paradis, E. C. Griffith, L. S. Hu, C. Chen, M. E. Greenberg, Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science 311 , 1008-1012 (2006). [Abstract] [Full Text] A. Shalizi, B. Gaudillière, Z. Yuan, J. Stegmüller, T. Shirogane, Q. Ge, Y. Tan, B. Schulman, J. W. Harper, A. Bonni, A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation. Science 311 , 1012-1017 (2006). [Abstract] [Full Text]

PI3K/Akt‐dependent regulation of the transcription factor myocyte enhancer factor‐2 in insulin‐like growth factor‐1‐ and membrane depolarization‐mediated survival of cerebellar granule neurons

Survival signals such as insulin-like growth factor-1 (IGF-1) or membrane depolarization convey their neuronal protective effects through the activation of signaling networks and nuclear factors. In cerebellar granule neurons, IGF-1 mediates survival primarily through the PI3K/Akt pathway. The function of the transcription factor myocyte enhancer factor-2 (MEF2) is required for mediating membrane depolarization-dependent neuronal survival. However, whether PI3K/Akt regulates MEF2 and the role of MEF2 in IGF-1-mediated survival of neurons are unknown. In addition, the contribution of the PI3K/Akt pathway in membrane depolarization-induced neuronal survival remains undefined. We show here that the PI3K/Akt pathway promotes the survival of cerebellar granule neurons derived from Long-Evans rats following IGF-1 stimulation or membrane depolarization through regulation of MEF2 activity. IGF-1 stimulated the gene transactivation activity of MEF2 and its DNA binding potential. Moreover, regulation of MEF2 function by IGF-1 was dependent on the activity of the PI3K/Akt signaling pathway. Blocking MEF2 function reduced IGF-1-induced survival of cerebellar granule neurons. Membrane depolarization stimulated phosphorylation of Akt in cerebellar granule neurons. Blocking of the PI3K/Akt pathway with either a pharmacological inhibitor of PI3K, LY294002, or dominant negative mutants of PI3K and Akt inhibited the membrane depolarization-induced increase in MEF2 transactivation as well as its DNA binding activity and reduced neuronal survival. Together, these findings provide clear evidence to support an important role of the PI3K/Akt pathway in the regulation of nuclear survival factor MEF2 upon either IGF-1 stimulation or membrane depolarization, thus placing MEF2 as a novel downstream effector of the PI3K/Akt pathway in neurons.

Regulation of Neuroprotective Activity of Myocyte-enhancer Factor 2 by cAMP-Protein Kinase A Signaling Pathway in Neuronal Survival

The transcription factor myocyte-enhancer factor 2 (MEF2) has been shown to be required for the survival of different types of neurons. However, the death- or survival-inducing second messenger pathways that regulate MEF2 activity remain to be fully elucidated. Membrane depolarization by KCl induces neuronal survival that is dependent upon MEF2-mediated gene transactivation. Here we report that membrane depolarizationinduced activation of MEF2 requires the cAMP-protein kinase A (PKA) pathway. Inhibition of the activity of cAMP-PKA pathway attenuates membrane depolarization-induced activation of MEF2 activity and neuronal survival, whereas enhancing the activity of this pathway prevents KCl withdrawal-induced inhibition of MEF2 and neuronal apoptosis. Moreover, PKA directly phosphorylates MEF2 at Thr-20 in vitro to increase MEF2 DNA binding activity. A mutation of Thr-20 to Ala renders MEF2 resistant to PKA phosphorylation in vitro and reduces its DNA binding activity. Transfection of this T20A mutant blocks survival and induces apoptosis in cultured cortical and cerebellar granule neurons. This study identifies the transcription factor MEF2 as a target of cAMP-PKA pathway and demonstrates that PKA phosphorylation of MEF2 is a key step in modulating its DNA binding activity and ability to promote neuronal survival.

Myocyte Enhancer Factor 2 Activates Promoter Sequences of the Human AβH-J-J Locus, Encoding Aspartyl-β-Hydroxylase, Junctin, and Junctate

Alternative splicing of the locus AbetaH-J-J generates three functionally distinct proteins: an enzyme, AbetaH (aspartyl-beta-hydroxylase), a structural protein of the sarcoplasmic reticulum membrane (junctin), and an integral membrane calcium binding protein (junctate). Junctin and junctate are two important proteins involved in calcium regulation in eukaryotic cells. To understand the regulation of these two proteins, we identified and functionally characterized one of the two promoter sequences of the AbetaH-J-J locus. We demonstrate that the P2 promoter of the AbetaH-J-J locus contains (i) a minimal sequence localized within a region -159 bp from the transcription initiation site, which is sufficient to activate transcription of both mRNAs; (ii) sequences which bind known transcriptional factors such as those belonging to the myocyte enhancer factor 2 (MEF-2), MEF-3, and NF-kappaB protein families; and (iii) sequences bound by unknown proteins. The functional characterization of the minimal promoter in C2C12 cells and in the rat soleus muscle in vivo model indicates the existence of cis elements having positive and negative effects on transcription. In addition, our data demonstrate that in striated muscle cells the calcium-dependent transcription factor MEF-2 is crucial for the transcription activity directed by the P2 promoter. The transcription directed by the AbetaH-J-J P2 promoter is induced by high expression of MEF-2, further stimulated by calcineurin and Ca2+/calmodulin-dependent protein kinase I, and inhibited by histone deacetylase 4.

Association with Class IIa Histone Deacetylases Upregulates the Sumoylation of MEF2 Transcription Factors

The myocyte enhancer factor-2 (MEF2) family of transcription factors plays an important role in regulating cellular programs like muscle differentiation, neuronal survival, and T-cell apoptosis. Multisite phosphorylation is known to control the transcriptional activity of MEF2 proteins, but it is unclear whether other modifications are involved. Here, we report that human MEF2D, as well as MEF2C, is modified by SUMO2 and SUMO3 at a motif highly conserved among MEF2 proteins from diverse organisms. This motif is located within the C-terminal transcriptional activation domain, and its sumoylation inhibits transcription. As a transcriptional corepressor of MEF2, histone deacetylase 4 (HDAC4) potentiates sumoylation. This potentiation is dependent on the N-terminal region but not the C-terminal deacetylase domain of HDAC4 and is inhibited by the sumoylation of HDAC4 itself. Moreover, HDAC5, HDAC7, and an HDAC9 isoform also stimulate sumoylation of MEF2. Opposing the action of class IIa deacetylases, the SUMO protease SENP3 reverses the sumoylation to augment the transcriptional and myogenic activities of MEF2. Similarly, the calcium/calmodulin-dependent kinases [corrected] and extracellular signal-regulated kinase 5 signaling pathways negatively regulate the sumoylation. These results thus identify sumoylation as a novel regulatory mechanism for MEF2 and suggest that this modification interplays with phosphorylation to promote intramolecular signaling for coordinated regulation in vivo.

Innervation-dependent and fiber type-specific transcriptional regulation of the slow myosin heavy chain 2 promoter in avian skeletal muscle fibers.

Skeletal muscle fiber type is regulated, in part, by innervation leading to transcriptional regulation of fiber type-specific genes. Here, we report the initial characterization of the transcriptional regulation of the slow myosin heavy chain 2 (MyHC2) promoter in innervated and noninnervated slow medial adductor (MA) and fast pectoralis major (PM) muscle fibers in cell culture. The proximal 1358 bp of slow MyHC2 upstream DNA contains a functional E-box and binding sites for myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT). Mutagenesis studies indicated that both MEF2 and NFAT binding sites are required for innervation-induced slow MyHC2 promoter activity in MA muscle fibers. However, MEF2 transcription factor activity was unaffected by innervation and did not demonstrate fiber type-specific interactions with the slow MyHC2 MEF2 binding site. NFAT transcription factor activity did increase in innervated MA muscle fibers and not in PM muscle fibers, indicating innervation and muscle fiber type-specific regulation. However, transfection of constitutively active NFAT indicated that NFAT is insufficient to induce slow MyHC2 gene expression in either fast PM or slow MA muscle fibers without innervation. These results indicate the requirement for MEF2 and NFAT in innervation-induced slow MyHC2 gene expression and suggest that additional innervation-dependent and fiber type-specific control of slow MyHC2 gene expression resides in MA and PM muscle fibers, respectively.

Membrane Hyperpolarization Triggers Myogenin and Myocyte Enhancer Factor-2 Expression during Human Myoblast Differentiation

It is widely thought that myogenin is one of the earliest detectable markers of skeletal muscle differentiation. Here we show that, during human myoblast differentiation, an inward rectifier K(+) channel (Kir2.1) and its associated hyperpolarization trigger expression and activity of the myogenic transcription factors, myogenin and myocyte enhancer factor-2 (MEF2). Furthermore, Kir2.1 current precedes and is required for the developmental increase in expression/activity of myogenin and MEF2. Drugs or antisense reducing Kir2.1 current diminished or suppressed fusion as well as expression/activity of myogenin and MEF2. In contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase (a pathway controlling initiation of the myogenic program) that inhibited both myogenin/MEF2 expression and fusion, did not affect Kir2.1 current. This non-blockade by LY294002 indicates that Kir2.1 acts upstream of myogenin and MEF2. We propose that Kir2.1 channel activation is a required key early event that initiates myogenesis by turning on myogenin and MEF2 transcription factors via a hyperpolarization-activated Ca(2+)-dependent pathway.

Functional interaction between class II histone deacetylases and ICP0 of herpes simplex virus type 1.

This study describes the physical and functional interactions between ICP0 of herpes simplex virus type 1 and class II histone deacetylases (HDACs) 4, 5, and 7. Class II HDACs are mainly known for their participation in the control of cell differentiation through the regulation of the activity of the transcription factor MEF2 (myocyte enhancer factor 2), implicated in muscle development and neuronal survival. Immunofluorescence experiments performed on transfected cells showed that ICP0 colocalizes with and reorganizes the nuclear distribution of ectopically expressed class I and II HDACs. In addition, endogenous HDAC4 and at least one of its binding partners, the corepressor protein SMRT (for silencing mediator of retinoid and thyroid receptor), undergo changes in their nuclear distribution in ICP0-transfected cells. As a result, during infection endogenous HDAC4 colocalizes with ICP0. Coimmunoprecipitation and glutathione S-transferase pull-down assays confirmed that class II but not class I HDACs specifically interacted with ICP0 through their amino-terminal regions. This region, which is not conserved in class I HDACs but homologous to the MITR (MEF2-interacting transcription repressor) protein, is responsible for the repression, in a deacetylase-independent manner, of MEF2 by sequestering it under an inactive form in the nucleus. Consequently, we show that ICP0 is able to overcome the HDAC5 amino-terminal- and MITR-induced MEF2A repression in gene reporter assays. This is the first report of a viral protein interacting with and controlling the repressor activity of class II HDACs. We discuss the putative consequences of such an interaction for the biology of the virus both during lytic infection and reactivation from latency.

Myocyte enhancer factor-2 transcription factors in neuronal differentiation and survival.

Myocyte enhancer factor-2 (MEF2) transcription factors regulate genes that control critical cellular processes including proliferation, differentiation, and survival. Although MEF2 proteins were first identified as transcription factors that bound A/T rich DNA sequences and controlled muscle-specific genes during myogenic development, it is now apparent that MEF2 transcription factors are also highly expressed in neurons and are critical determinants of neuronal differentiation and fate. Here we focus our discussion on the role of MEF2 proteins in nervous tissue and the regulation of these transcription factors by calcium and phosphorylation signaling pathways.

TEF-1 and MEF2 transcription factors interact to regulate muscle-specific promoters

Many muscle-specific genes are regulated by transcriptional enhancer factor-1 (TEF-1), serum response factor (SRF), and myocyte enhancer factor-2 (MEF2) transcription factors. TEF-1 interacts with the MADS domain of SRF and together SRF and TEF-1 co-activate the skeletal α-actin promoter. MEF2 factors also contain a MADS domain with 50% amino acid identity to the SRF MADS domain. Because of this sequence divergence, some SRF co-factors do not interact with MEF2. To demonstrate that TEF-1 factors could also interact with MEF2 through its MADS domain, we used co-immunoprecipitation and GST pull-down assays in vitro and a mammalian two-hybrid assay in vivo. The MADS domain was not sufficient for MEF2 interaction with TEF-1, because additional sequences in the activation domains of both proteins were required for in vivo association. The physiological significance of this interaction was also demonstrated by transient transfection assays using muscle-specific promoters. Our results suggest that by their interaction with MEF2 factors, TEF-1 factors can control MEF2-dependent muscle-specific gene expression.

Vascular smooth muscle cell proliferation requires both p38 and BMK1 MAP kinases

Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of atherosclerosis. Induction of both c-fos (through the transcription factor Elk-1) and c-jun, both immediate early genes, is important for the stimulation of VSMC proliferation and migration. It was earlier found that p38 mitogen-activated protein (MAP) kinase upregulates c-jun gene transcription through phosphorylation of two myocyte enhancer factor 2 (MEF2) family transcription factors, MEF2A and MEF2C, while big MAP kinase 1 (BMK1) may upregulate c-jun gene transcription through MEF2A, MEF2C, and also MEF2D. Here, we report that inhibition of BMK1 by a dominant negative form of MEK5 or pharmacologic inhibition of p38 by SB 203580 additively suppress serum-induced VSMC proliferation. This additive effect of p38 and BMK1 inhibition implies that these two kinases coordinately regulate MEF2 transcription factors. The exclusive activation of MEF2D by BMK1 appears required for this cooperative upregulation of c-jun in VSMC, and coactivation of p38 and BMK1 also has additive effects on the activation of a reporter gene linked to the c-jun promoter in our experimental system. Thus, coordinate activity of both the p38 and BMK1 pathways appears necessary for optimal transcription of c-jun and, pari pasu, VSMC proliferation. These results may have implications for the future design of pharmacologic agents for inhibition of VSMC growth.

Association of COOH-terminal-binding Protein (CtBP) and MEF2-interacting Transcription Repressor (MITR) Contributes to Transcriptional Repression of the MEF2 Transcription Factor

The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. We show that the amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs.