Myocyte Enhancer Factor-2 Expression Research Articles

Enhanced expression of the myogenic factor Myocyte enhancer factor-2 in imaginal disc myoblasts activates a partial, but incomplete, muscle development program

The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process.

Gene Structure, Expression and Function Analysis of MEF2 in the Pacific White Shrimp Litopenaeus vannamei

The Pacific white shrimp Litopenaeus vannamei is the most economically important crustacean in the world. The growth and development of shrimp muscle has always been the focus of attention. Myocyte Enhancer Factor 2 (MEF2), a member of MADS transcription factor, has an essential influence on various growth and development programs, including myogenesis. In this study, based on the genome and transcriptome data of L. vannamei, the gene structure and expression profiles of MEF2 were characterized. We found that the LvMEF2 was widely expressed in various tissues, mainly in the Oka organ, brain, intestine, heart, and muscle. Moreover, LvMEF2 has a large number of splice variants, and the main forms are the mutually exclusive exon and alternative 5' splice site. The expression profiles of the LvMEF2 splice variants varied under different conditions. Interestingly, some splice variants have tissue or developmental expression specificity. After RNA interference into LvMEF2, the increment in the body length and weight decreased significantly and even caused death, suggesting that LvMEF2 can affect the growth and survival of L. vannamei. Transcriptome analysis showed that after LvMEF2 was knocked down, the protein synthesis and immune-related pathways were affected, and the associated muscle protein synthesis decreased, indicating that LvMEF2 affected muscle formation and the immune system. The results provide an important basis for future studies of the MEF2 gene and the mechanism of muscle growth and development in shrimp.

MEF2 intervened LPS-induced acute lung injury by binding to KLF2 promoter and modulating macrophage phenotype

Transcription factor myocyte enhancer factor 2 (MEF2) exerts anti-inflammatory activities in endothelial cells, yet its role in acute lung injury (ALI) remains unclear. Homeostasis dysfunction in macrophage polarization is essential for the pathogenesis of ALI. Krüppel-like factor 2 (KLF2) and microRNA-33 (miR-33), important regulators in macrophage polarization, have been predicted as the downstream and upstream modulator of MEF2, respectively. This study aimed to investigate the function of MEF2 in ALI and its regulatory mechanism on macrophage polarization. Murine ALI models were established by intratracheal instillation of 5 mg/kg lipopolysaccharide (LPS) for 24 h. M1-type macrophage polarization was induced with LPS and interferon γ, while the M2-type one was induced with interleukin-4 for 24 h in vitro. In ALI murine models, pulmonary MEF2 and KLF2 expressions were decreased. MEF2 and KLF2 expressions were higher in M2 macrophages than those in M1 macrophages in vitro. Conversely, microRNA-33 level was higher in M1 macrophages. Our study firstly demonstrated that MEF2 could increase M2 polarization but inhibit M1 polarization of macrophages in vitro and its overexpression mitigated LPS-induced ALI, increased pulmonary KLF2 expression and drove alveolar macrophage polarization from an M1 to an M2 phenotype in vivo. MEF2 was confirmed to bind to KLF2 promoter. Moreover, MEF2 regulated macrophage phenotypes via KLF2. In addition, miR-33 could bind to MEF2 and inhibit MEF2 expression in macrophages. It was speculated that miR-33 might be implicated in macrophage polarization through inhibiting MEF2 expression. Taken together, MEF2 may be a novel target for treating ALI.

Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation.

Arginine-vasopressin (AVP) promotes muscle differentiation, hypertrophy, and regeneration through the combined activation of the calcineurin and Calcium/Calmodulin-dependent Protein Kinase (CaMK) pathways. The AVP system is impaired in several neuromuscular diseases, suggesting that AVP may act as a physiological factor in skeletal muscle. Since the Phosphoinositide 3-kinases/Protein Kinase B/mammalian Target Of Rapamycin (PI3K/Akt/mTOR) signaling plays a significant role in regulating muscle mass, we evaluated its role in the AVP myogenic effect. In L6 cells AKT1 expression was knocked down, and the AVP-dependent expression of mTOR and Forkhead box O3 (FoxO) was analyzed by Western blotting. The effect of the PI3K inhibitor LY294002 was evaluated by cellular and molecular techniques. Akt knockdown hampered the AVP-dependent mTOR expression while increased the levels of FoxO transcription factor. LY294002 treatment inhibited the AVP-dependent expression of Myocyte Enhancer Factor-2 (MEF2) and myogenin and prevented the nuclear translocation of MEF2. LY294002 also repressed the AVP-dependent nuclear export of histone deacetylase 4 (HDAC4) interfering with the formation of multifactorial complexes on the myogenin promoter. We demonstrate that the PI3K/Akt pathway is essential for the full myogenic effect of AVP and that, by targeting this pathway, one may highlight novel strategies to counteract muscle wasting in aging or neuromuscular disorders.

Salvianolic Acid B Inhibits Activation of Human Primary Hepatic Stellate Cells Through Downregulation of the Myocyte Enhancer Factor 2 Signaling Pathway.

Various isoforms of myocyte enhancer factor 2 (MEF2) have been shown to play a role in the activation of rat hepatic stellate cells (HSCs) in culture. The signals that regulate MEF2 in HSCs are unknown. In addition, whether MEF2s regulate the activation of human HSCs (H-HSCs) is unclear. Here, we studied the expression and function of MEF2s in H-HSCs. Our data showed that the levels of MEF2A, C, and D proteins were high in liver tissues from patients with cirrhosis and increased during culture-induced activation of primary H-HSCs. Exposure of H-HSCs to transforming growth factor beta 1 (TGF-β1) led to a significant increase in MEF2A and C protein levels and enhanced MEF2 activity. Interestingly, TGF-β1 did not further enhance MEF2D levels. Furthermore, TGF-β1 activated p38 mitogen-activated protein kinase (MAPK) and led to increased phosphorylation of MEF2C at its p38 recognition site. Inhibition of p38 MAPK inhibited both TGF-β1- and culture-induced activation of MEF2. The activity of collagen I reporter in H-HSCs was significantly reduced when MEF2A and MEF2C were blocked with overexpression of dominant negative MEF2 mutants. Salvianolic-acid B (SA-B), a water-soluble element of Salvia miltiorrhiza known to have anti-fibrosis effects, attenuated both basal and TGF-β1-induced increased levels of MEF2A and C mRNA and protein. In addition, SA-B inhibited MEF2 activity, which correlated with reduced expression of the HSC activation markers, α-smooth muscle actin (α-SMA), and collagen I. Administration of SA-B reduced MEF2A in vivo, which was accompanied by reduced levels of α-SMA in a model of dimethylnitrosamine-induced rat liver fibrosis. We concluded that the MEF2 transcription factor was stimulated by TGF-β1 in H-HSCs. Antagonizing TGF-β1-induced activation of the MEF2 signaling pathway may account in part for the anti-fibrosis effects of SA-B.

Drosophila mef2 is essential for normal mushroom body and wing development.

ABSTRACTMEF2 (myocyte enhancer factor 2) transcription factors are found in the brain and muscle of insects and vertebrates and are essential for the differentiation of multiple cell types. We show that in the fruit fly Drosophila, MEF2 is essential for the formation of mushroom bodies in the embryonic brain and for the normal development of wings in the adult. In embryos mutant for mef2, there is a striking reduction in the number of mushroom body neurons and their axon bundles are not detectable. The onset of MEF2 expression in neurons of the mushroom bodies coincides with their formation in the embryo and, in larvae, expression is restricted to post-mitotic neurons. In flies with a mef2 point mutation that disrupts nuclear localization, we find that MEF2 is restricted to a subset of Kenyon cells that project to the α/β, and γ axonal lobes of the mushroom bodies, but not to those forming the α’/β’ lobes.

MEF2 Transcription Factors Regulate Human Trophoblast Invasion and Differentiation [33P

INTRODUCTION: Adverse pregnancy outcomes, including preeclampsia and preterm birth, two significant causes of maternal and fetal morbidity and mortality, are associated with placental trophoblast dysfunction. Myocyte enhancer factor 2 (MEF2) transcription factors play an important role in cell differentiation and invasion in various cell types and tissues. The objective of our study is to explore the role of MEF2 in the invasion/differentiation of trophoblasts in normal placental development and preeclampsia. METHODS: Human cytotrophoblasts (CTBs) were isolated from term placentas and used to analyze the expression of MEF2 transcription factors and MEF2-dependent cell signaling. The effect of MEF2 on human trophoblast invasion/differentiation and its underlying mechanisms were examined in two trophoblast cell lines: HTR8/SVneo (first trimester human villous-derived) and BeWo (choriocarcinoma CTB-like) cells. RESULTS: MEF2 isoforms are differentially expressed in the CTBs of first and third trimester human placentas. MEF2D and 2B are highly expressed in first trimester trophoblasts. Introduction of MEF2D into HTR8/SVneo promotes cell invasion and migration. The invasive capacity is impeded by exogenous expression of dominant negative MEF2. MEF2A is a major isoform of MEF2 in term CTBs and is required for proper CTB differentiation into syncytiotrophoblasts. During in vitro CTB differentiation, p38 MAPK activity was detected for days 1-4, and diminished for days 5-6 suggesting that p38 MAPK signaling is essential for CTB differentiation but not for the maintenance of syncytiotrophoblasts. CONCLUSION: Our studies suggest individual MEF2 family members differentially regulate trophoblast invasion/differentiation. Dysregulation of MEF2 signaling may be associated with placenta-related pregnancy disorders, including preeclampsia.

MEF2 Transcription Factor Regulates Osteogenic Differentiation of Dental Pulp Stem Cells

The myocyte enhancer factor-2 (MEF2) is a member of the MADS-box family. It controls the expression of genes that are critical for biological processes such as proliferation, cell death, and differentiation. Some studies have shown that MEF2 expression is enhanced in osteogenic progenitor cells established from bone marrow stromal cells with other types of mesenchymal progenitor cells. However, the effect of MEF2 on dental pulp stem cells (DPSCs) is unclear. In this study, we investigate the effect of MEF2 on regulating osteogenic differentiation and proliferation of DPSCs. We find that MEF2 is stably expressed in DPSCs, and the expression is increased time-dependently along with cell osteogenic differentiation. MEF2 expression also increases the alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) activity, and enhances mineralization in DPSCs. SB202190, inhibitor of p38, blocks the p38/MEF2 pathway and osteogenic differentiation. In addition, MEF2 overexpression inhibits DPSC proliferation. In summary, our data indicate that MEF2 not only regulates DPSCs as an inhibitor of cell proliferation but is also a promoter of osteogenic differentiation through the p38/MEF2 signaling pathway.

Astaxanthin Prevented and Reversed the Activation of Mouse Primary Hepatic Stellate Cells by Inhibiting the Myocyte Enhancer Factor 2‐Dependent Expression of Histone Deacetylase 9

Dysregulated activation of hepatic stellate cells (HSCs) is a critical event in the development of liver fibrosis/cirrhosis. We previously reported that astaxanthin (ASTX), a xanthophyll carotenoid, prevents the activation of HSCs and facilitates the reversal of activated HSCs to a quiescent phenotype using primary HSCs from C57BL/6J mice. We further evaluated the underlying mechanismsand their human relevance. As histone deacetylases (HDACs) are known to play important roles in HSC activation, we examined changes in the expression of 11 classical HDAC isoforms and the effect of ASTX on their expression during HSC activation. HDAC9 mRNA and protein were significantly higher in activated HSCs than quiescent HSCs. The presence of ASTX during HSC activation attenuated the induction by ~70-80%. The expression of myocyte enhancer factor 2 (MEF2), a known transcriptional activator of HDAC9, was induced by HSC activation, but diminished by ASTX. Activated HSCs incubated with ASTX displayed a quiescent phenotype with concomitant decreases in HDAC9 and MEF2 expression. In the livers of patients with primary biliary cirrhosis, HDAC9 expression was also significantly higher than in normal livers. Taken together, the inhibitory effect of ASTX on HSC activation is attributable at least partly to the repression of MEF2-mediated expression of HDAC9. The findings suggest that ASTX may be developed as a potential preventive/therapeutic agent for liver fibrosis/cirrhosis. (Funded by USDA AFRI 2012-67018-19290 and Multi-state Hatch)

The molecular characterization and temporal–spatial expression of myocyte enhancer factor 2 genes in the goat and their association with myofiber traits

The myocyte enhancer factor-2 (MEF2) gene family in vertebrates includes MEF2A, MEF2B, MEF2C, and MEF2D, which have important functions in the regulation of muscular growth and development. To investigate their temporal–spatial expression and functions in the goat, these genes were cloned (accession nos. JN967621–24) and their expression patterns characterized at five postnatal stages (3, 30, 60, 90, and 120days). Association analysis was then applied regarding MEF2 expression levels and myofiber diameter and density. MEF2B was shown to be weakly homologous with other species, the distant branches with other members and the lowest expression levels, suggesting that it is distinct from other family members. Expression of the other three MEF2 genes was widely distributed, but this was largely accumulated in the skeletal muscle and myocardium compared with the viscera at all developmental stages. MEF2A and MEF2D expression levels were higher overall than MEF2B and MEF2C in six tissues, and were significantly positively correlated with the myofiber diameter of the longissimus dorsi. These findings suggest that goat MEF2 genes mainly function in the skeletal muscle and myocardium, and that MEF2A and MEF2D are likely to effectively promote muscular growth and development during postnatal stages. MEF2A expression was highest in the myocardium, where MEF2C expression increased with age, implying that both gene products are related to the growth and development of postnatal myocardium.

Abstract 4609: MEF2 plays a critical role in RENCA macrobead-induced tumor cell growth inhibition

Abstract Regulation of cell proliferation and cell death is often dependent on the coupling of extracellular signals to the activation or repression of specific intracellular signaling pathways. We have previously demonstrated the capacity of mouse renal adenocarcinoma (RENCA) cells encapsulated in agarose macrobeads to form numerous small tumor colonies within the macrobeads over a period of several months. As the encapsulated tumor colonies mature and reach a maximum size, the macrobeads themselves, or conditioned media from cultured macrobeads, markedly inhibit the in vitro and in vivo growth of epithelial-derived tumor cells outside the macrobead microenvironment. Myocyte enhancer factor 2 (MEF2) is a family of four transcription factors that have been shown to play a central role in linking signaling pathways to the genes responsible for cell division, differentiation and death. In this study, we evaluated the expression and role of MEF2 in regulating cell proliferation in mouse (RENCA) and human cell lines (J82, MCF7) in response to RENCA macrobeads. Freely growing RENCA target cells transiently transfected with a MEF2 transcriptional response element and exposed to conditioned media from variously aged macrobeads demonstrated increased response element activation in parallel with the increasing age of the macrobeads. Similarly, growth inhibition of the freely growing RENCA cells increased with older macrobeads. Suppression of the expressed MEF2 isoforms in target RENCA cells, either individually (MEF2a, MEF2b, and MEF2d) or in combination (MEF2pool) using synthetic small interfering RNA (siRNA) markedly reduced the growth inhibitory effects of RENCA macrobeads. In J82 and MCF7 cell lines, MEF2D expression was responsive to replete media from RENCA macrobeads, increasing 2.0-fold and 2.7-fold respectively. Degradation of MEF2D mRNA transcripts in the human cell lines studied resulted in abatement of the growth inhibitory effect of the RENCA macrobeads. These findings reveal an essential role for MEF2 in RENCA macrobead-induced cancer cell growth inhibition and raise interesting questions about the molecular basis of this response. Citation Format: Prithy C. Martis, Atira Dudley, Melissa A. Laramore, Barry H. Smith, Lawrence S. Gazda. MEF2 plays a critical role in RENCA macrobead-induced tumor cell growth inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4609. doi:10.1158/1538-7445.AM2014-4609

Caffeine induces cardiomyocyte hypertrophy via p300 and CaMKII pathways

Caffeine is commonly utilized to trigger intracellular calcium in cardiomyocyte. It is well accepted that caffeine could induce cardiac arrhythmia, but it is not clear with regard of its impacts on the cardiac function. This article presents a recent study concerning the effects of caffeine on the cardiomyocyte hypertrophy and the associated signal pathway. The experimental results showed that the total protein contents, the surface area of cardiomyocyte and β-myosin heavy chain (β-MHC) expression increased in ventricular myocytes of neonatal Sprague–Dawley (SD) rats after 24h caffeine incubation. It is also observed that the basal intracellular calcium (Ca2+) level has increased, while the amplitude of Ca2+ oscillation and Ca2+ content have decreased in sarcoplasmic reticulum (SR). The caffeine-induced myocyte enhancer factor-2 (MEF2) expression and hypertrophy can be completely abolished by the inhibition of cardiac ryanodine receptor (RyR2), as well as KN93 and curcumin treatments. Meanwhile, the amplitude of Ca2+ oscillation and the Ca2+ content of SR in the completely-inhibited group have reached the physiological level. These results suggest that the caffeine-induced cardiomyocyte hypertrophy established the connection between Ca2+ release from SR and cytosol that activates CaMKII and p300, which in turn enhances the expression of MEF2 that promotes cardiomyocyte hypertrophy.

Estrogen alone or in combination with parathyroid hormone can decrease vertebral MEF2 and sclerostin expression and increase vertebral bone mass in ovariectomized rats.

The study is about the regulatory effects of estrogen and parathyroid hormone (PTH) on sclerostin, a protein that inhibits the Wnt/β-catenin pathway. The results indicate that estrogen may down-regulate sclerostin expression and that estrogen displays synergistic action with PTH. These results provide a new perspective on the relationship between estrogen and bone. To investigate whether estrogen can down-regulate SOST and MEF2 (myocyte enhancer factor 2) expression and whether co-treatment with estrogen and PTH has a stronger effect on suppressing SOST than PTH applied alone in ovariectomized rats. Forty-three-month-old virgin female Sprague-Dawley (SD) rats were ovariectomized and divided into four groups (n = 10). Another ten age-matched rats received sham operations as controls. After allowing 8 weeks for the development of vertebral osteopenia, the rats were administered the drug intervention. For this intervention, the estrogen group was subcutaneously injected with 17β-estradiol at 25 μg/kg body weight, the PTH group was injected with 80 μg/kg synthetic human PTH (1-34), and the co-treatment group was concurrently treated with PTH and estrogen at the above dosage. The OVX group and sham group were treated with vehicle. The drug treatment was conducted for 12 weeks. After the lumbar spine bone mineral density (BMD) was measured, the rats were sacrificed, and the lumbar spine and blood were collected for qPCR, Western blot, immunohistochemistry and other tests. Estrogen can down-regulate MEF2 and sclerostin expression, and co-treatment with estrogen and PTH has a stronger effect on suppressing MEF2 and SOST mRNA than PTH alone. The co-treatment group displayed slightly higher bone mass and biomechanical properties than the PTH group, but the differences were not significant. Estrogen appears to be a regulator of sclerostin, and the effect may involve suppressing MEF2s. Combined treatment with PTH and estrogen is not more beneficial for vertebral bone mass and strength than treatment with PTH alone in ovariectomized rats.

Myocyte enhancer factor-2 plays critical roles in HTLV-1 infection and transformation of CD4+ T cells associated with adult T cell leukemia (ATL) by stabilizing complex between Tax and CREB (HUM8P.335)

Abstract The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4+ T cell transformation have yet to be fully delineated. Herein, utilizing virus infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the role of myocyte enhancer factor-2 (MEF-2) in Tax-mediated transactivation of the LTR. Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and HTLV-mediated T cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was shown to be recruited to the HTLV-1 LTR followed by an assessment of MEF-2 binding site(s). A novel promoter-binding assay confirmed that MEF-2 constitutes the transcriptional complex that assembles on the LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (the known Tax-interacting transcription factor) and HATs - p300, CBP, and p/CAF. Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. A number of MEF-2-integrated signaling pathways were activated during HTLV-1 infection of primary CD4+ T cells, including PI3K/Akt, NF-κB, MAPK, JAK/STAT and TGF-β possibly regulating MEF-2 activity. Overall, these studies are the first to describe the involvement and regulation of a novel transcription factor during the course of a retroviral infection and associated disease syndrome.

Perturbation of Transcription Factor Nur77 Expression Mediated by Myocyte Enhancer Factor 2D (MEF2D) Regulates Dopaminergic Neuron Loss in Response to 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

We have earlier reported the critical nature of calpain-CDK5-MEF2 signaling in governing dopaminergic neuronal loss in vivo. CDK5 mediates phosphorylation of the neuronal survival factor myocyte enhancer factor 2 (MEF2) leading to its inactivation and loss. However, the downstream factors that mediate MEF2-regulated survival are unknown. Presently, we define Nur77 as one such critical downstream survival effector. Following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo, Nur77 expression in the nigrostriatal region is dramatically reduced. This loss is attenuated by expression of MEF2. Importantly, MEF2 constitutively binds to the Nur77 promoter in neurons under basal conditions. This binding is lost following 1-methyl-4-phenylpyridinium treatment. Nur77 deficiency results in significant sensitization to dopaminergic loss following 1-methyl-4-phenylpyridinium/MPTP treatment, in vitro and in vivo. Furthermore, Nur77-deficient MPTP-treated mice displayed significantly reduced levels of dopamine and 3,4-Dihydroxyphenylacetic acid in the striatum as well as elevated post synaptic FosB activity, indicative of increased nigrostriatal damage when compared with WT MPTP-treated controls. Importantly, this sensitization in Nur77-deficient mice was rescued with ectopic Nur77 expression in the nigrostriatal system. These results indicate that the inactivation of Nur77, induced by loss of MEF2 activity, plays a critical role in nigrostriatal degeneration in vivo.

Postsynaptic FMRP bidirectionally regulates excitatory synapses as a function of developmental age and MEF2 activity

Rates of synapse formation and elimination change over the course of postnatal development, but little is known of molecular mechanisms that mediate this developmental switch. Here, we report that the dendritic RNA-binding protein fragile X mental retardation protein (FMRP) bidirectionally and cell autonomously regulates excitatory synaptic function, which depends on developmental age as well as function of the activity-dependent transcription factor myocyte enhancer factor 2 (MEF2). The acute postsynaptic expression of FMRP in CA1 neurons of hippocampal slice cultures (during the first postnatal week, P6-P7) promotes synapse function and maturation. In contrast, the acute expression of FMRP or endogenous FMRP in more mature neurons (during the second postnatal week; P13-P16) suppresses synapse number. The ability of neuronal depolarization to stimulate MEF2 transcriptional activity increases over this same developmental period. Knockout of endogenous MEF2 isoforms causes acute postsynaptic FMRP expression to promote, instead of eliminate, synapses onto 2-week-old neurons. Conversely, the expression of active MEF2 in neonatal neurons results in a precocious FMRP-dependent synapse elimination. Our findings suggest that FMRP and MEF2 function together to fine tune synapse formation and elimination rates in response to neuronal activity levels over the course of postnatal development.

During post-natal human myogenesis, normal myotube size requires TRPC1 and TRPC4 mediated Ca2+ entry

Myogenesis involves expression of muscle-specific transcription factors such as myogenin and myocyte enhancer factor 2 (MEF2), and is essentially regulated by fluctuations of cytosolic Ca(2+) concentration. Recently we demonstrated that molecular players of store-operated Ca(2+) entry (SOCE), stromal interacting molecule (STIM) and Orai, were fundamental in the differentiation process of post-natal human myoblasts. Besides STIM and Orai proteins, the family of transient receptor potential canonical (TRPC) channels was shown to be part of SOCE in several cellular systems. In the present study, we investigated the role of TRPC channels in the human myogenesis process. We demonstrate, using an siRNA strategy or dominant negative TRPC overexpression, that TRPC1 and TRPC4 participate in SOCE, are necessary for MEF2 expression, and allow the fusion process to generate myotubes of normal size. Conversely, the overexpression of STIM1 with TRPC4 or TRPC1 increased SOCE, accelerated myoblast fusion, and produced hypertrophic myotubes. Interestingly, in cells depleted of TRPC1 or TRPC4, the normalization of SOCE by increasing the extracellular calcium concentration or by overexpressing STIM1 or Orai1 was not sufficient to restore normal fusion process. A normal differentiation occurred only when TRPC channel was re-expressed. These findings indicate that Ca(2+) entry mediated specifically by TRPC1 and TRPC4 allow the formation of normal-sized myotubes.

Expression of myocyte enhancer factor-2 and downstream genes in ground squirrel skeletal muscle during hibernation

Myocyte enhancer factor-2 (MEF2) transcription factors regulate the expression of a variety of genes encoding contractile proteins and other proteins associated with muscle performance. We proposed that changes in MEF2 levels and expression of selected downstream targets would aid the skeletal muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) in meeting metabolic challenges associated with winter hibernation; e.g., cycles of torpor-arousal, body temperature that can fall to near 0°C, long periods of inactivity that could lead to atrophy. MEF2A protein levels were significantly elevated when animals were in torpor (maximally 2.8-fold higher than in active squirrels) and the amount of phosphorylated active MEF2A Thr312 increased during entrance into torpor. MEF2C levels also rose significantly during entrance and torpor as did the amount of phosphorylated MEF2C Ser387. Furthermore, both MEF2 members showed elevated amounts in the nuclear fraction during torpor as well as enhanced binding to DNA indicating that MEF2-mediated gene expression was up-regulated in torpid animals. Indeed, the protein products of two MEF2 downstream gene targets increased in muscle during torpor (glucose transporter isoforms 4; GLUT4) or early arousal (myogenic differentiation; MyoD). Significant increases in Glut4 and MyoD mRNA transcript levels correlated with the rise in protein product levels and provided further support for the activation of MEF2-mediated gene expression in the hibernator. Transcript levels of Mef2a and Mef2c also showed time-dependent patterns with levels of both being highest during arousal from torpor. The data suggest a significant role for MEF2-mediated gene transcription in the selective adjustment of muscle protein complement over the course of torpor-arousal cycles.

Fluid shear stress stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS

AbstractFluid shear stress generated by steady laminar blood flow protects vessels from atherosclerosis. Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) are fluid shear stress–responsive genes and key mediators in flow anti-inflammatory and antiatherosclerotic actions. However, the molecular mechanisms underlying flow induction of KLF2 and eNOS remain largely unknown. Here, we show a novel role of histone deacetylase 5 (HDAC5) in flow-mediated KLF2 and eNOS expression. We found for the first time that fluid shear stress stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a calcium/calmodulin-dependent pathway. Consequently, flow induced the dissociation of HDAC5 and myocyte enhancer factor-2 (MEF2) and enhanced MEF2 transcriptional activity, which leads to expression of KLF2 and eNOS. Adenoviral overexpression of a HDAC5 phosphorylation–defective mutant (Ser259/Ser498 were replaced by Ala259/Ala498, HDAC5-S/A), which shows resistance to flow-induced nuclear export, suppressed flow-mediated MEF2 transcriptional activity and expression of KLF2 and eNOS. Importantly, HDAC5-S/A attenuated the flow-inhibitory effect on monocyte adhesion to endothelial cells. Taken together, our results reveal that phosphorylation-dependent derepression of HDAC5 mediates flow-induced KLF2 and eNOS expression as well as flow anti-inflammation, and suggest that HDAC5 could be a potential therapeutic target for the prevention of atherosclerosis.

Chronic ethanol feeding impairs AMPK and MEF2 expression and is associated with GLUT4 decrease in rat myocardium

Chronic and heavy alcohol consumption is one of the causes of heart diseases. However, the effects of ethanol on insulin sensitivity in myocardium has been unclear. To investigate the effects of ethanol on the expression of AMP-activated protein kinase (AMPK), myocyte enhancer factor 2 (MEF2) and glucose transporter 4 (GLUT4), all of which are involved in the regulation of insulin sensitivity, in the myocardium, we performed three parts of experiments in vivo and in vitro. I: Rats were injected with 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR, 0.8 mg.kg(-l)) for 2 h. II: Rats received different dose (0.5, 2.5 or 5 g.kg(-l).d(-l)) of ethanol for 22-week. III: Primary neonatal rat cardiomyocytes were isolated and treated with or without 100 mM ethanol or 1 mM AICAR for 4 h. The cardiac protein and mRNA expression of AMPKalpha subunits, MEF2 and GLUT4 were observed by western-blotting and RT-PCR, respectively. Serum TNFalpha levels were assessed by ELISA. The results showed chronic ethanol exposure induced insulin resistance. Ethanol decreased the mRNA levels of AMPKalpha1 and alpha2, the protein levels of total- and phospho-AMPKalpha in cardiomyocytes. Similarly, ethanol showed inhibitory effects on both the mRNA and protein levels of MEF2A and 2D, and GLUT4 in a dose-response-like fashion. Correlation analysis implied an association between phospho-AMPKalpha and MEF2A or MEF2D, and between the levels of MEF2 protein and GLUT4 transcription. In addition, ethanol elevated serum TNFalpha level. Taken together, chronic ethanol exposure decreases the expression of AMPKalpha and MEF2, and is associated with GLUT4 decline in rat myocardium.