Knockdown Of MEF2C Research Articles

METTL3 promotes microglial inflammation via MEF2C in spinal cord injury.

Spinal cord injury (SCI) is a significant contributor to disability in contemporary society, resulting in substantial psychological and economic burdens for patients and their family. Microglia-mediated inflammation is an important factor affecting the nerve repair of SCI patients. N6-methyladenosine (m6A) is a prevalent epigenetic modification in mammals, which shows a strong association with inflammation. However, the mechanism of m6A modification regulating microglia-mediated inflammation is still unclear. Here, we observed that METTL3, a m6A methylase, was increased in SCI mice and lipopolysaccharide (LPS)-exposed BV2 cells. Knockdown of METTL3 inhibited the increased expression of iNOS and IL-1β induced by LPS in vitro. Subsequently, MEF2C, myocyte-specific enhancer factor 2C, was decreased in SCI mice and LPS-exposed BV2 cells. Knockdown of MEF2C promoted the expression of iNOS and IL-1β. Sequence analysis showed that there were multiple highly confident m6A modification sites on the MEF2C mRNA. METTL3 antibody could pull down a higher level of MEF2C mRNA than the IgG in RNA binding protein immunoprecipitation assay. Knockdown of METTL3 promoted MEF2C protein expression and MEF2C mRNA expression, accompanied by a reduced m6A modification level on the MEF2C mRNA. Knockdown of MEF2C inhibited the anti-inflammatory effect of METTL3 siRNA. Our results suggest that METTL3 promotes microglia inflammation via regulating MEF2C mRNA m6A modification induced by SCI and LPS treatment.

LncRNA UCA1 Suppresses the Inflammation Via Modulating miR-203-Mediated Regulation of MEF2C/NF-κB Signaling Pathway in Epilepsy.

Although many advances have been made in the pathogenesis of epilepsy recently, the pathological mechanisms of epilepsy are still largely unknown. Exploring the pathological mechanisms and developing novel therapeutic strategies for epilepsy are urgently needed. A SD rat model of epilepsy was established with lithium chloride-pilocarpine. Astrocytes were isolated, cultured from 8 to 12week rats and identified by flow cytometry and immunofluorescence. Immunohistochemical staining was usedfor MEF2C and NF-κB in paraffin-embedded sections. RT-qPCR and western blot were used to analyze gene expression. ELISA was used to analyze the concentration of IL-6, TNF-α and Cox-2. Cells were transfected with pcDNA-MEFC2, sh-MEFC2, pcDNA-UCA1, sh-UCA1, miR-203 mimic or miR-203 inhibitor. Cell viability was assessed by MTT assay. Dual luciferase assay was used to determine the direct interaction of lncRNA UCA1/miR-203 and miR-203/MEF2C. MEF2C was down-regulated and inhibited NF-κB expression and the secretion of IL-6 and TNF-α in epilepsy. LncRNA UCA1 was also down-regulated in epilepsy. LncRNA UCA1 over-expression increased the expression of MEF2C and its knock-down decreased MEF2C expression. Luciferase activity showed lncRNAUCA1 directly targeted miR-203 and miR-203 directly targeted MEF2C. MiR-203 suppressed the expression of MEF2C, and promoted NF-κB, phosphorylated IκB/IKK and inflammatory effectors, which was reversed by MEF2C knock-down. Moreover, lncRNA UCA1 could increase the expression of MEF2C to inhibit NF-κB, phosphorylated IκB/IKK and inflammatory effectors, which was also reversed by miR-203 mimic transfection. LncRNA UCA1 inhibited the inflammation via regulating miR-203 mediated regulation of MEF2C/NF-κB signaling in epilepsy. Our investigation elucidated novel pathological mechanisms and provided potential therapeutic targets for epilepsy.

MEF2C Interacts With c-FOS in PTH-Stimulated Mmp13 Gene Expression in Osteoblastic Cells.

Parathyroid hormone (PTH) regulates the transcription of many genes in the osteoblast. One of these genes is Mmp13, which is involved in bone remodeling and early stages of endochondral bone formation. Previously, we reported that PTH induces Mmp13 transcription by regulating the dissociation of histone deacetylase 4 (HDAC4) from runt-related transcription factor 2 (Runx2), and the association of the HATs, p300, and p300/CREB binding protein (CBP)-associated factor. It is known that, in addition to Runx2, HDAC4 binds to the transcription factor, myocyte-specific enhancer factor 2c (MEF2C), and represses its activity. In this work, we investigated whether MEF2C participates in PTH-stimulated Mmp13 gene expression in osteoblastic cells and how it does so. Knockdown of Mef2c in UMR 106-01 cells repressed Mmp13 messenger RNA expression and promoter activity with or without PTH treatment. Chromatin immunoprecipitation (ChIP) assays showed that MEF2C associated with the Mmp13 promoter; this increased after 4 hours of PTH treatment. ChIP-reChIP results indicate that endogenous MEF2C associates with HDAC4 on the Mmp13 promoter; after PTH treatment, this association decreased. From gel shift, ChIP, and promoter-reporter assays, MEF2C was found to associate with the activator protein-1 (AP-1) site without directly binding to DNA and had its stimulatory effect through interaction with c-FOS. In conclusion, MEF2C is necessary for Mmp13 gene expression at the transcriptional level and participates in PTH-stimulated Mmp13 gene expression by increased binding to c-FOS at the AP-1 site in the Mmp13 promoter. The observation of MEF2C interacting with a member of the AP-1 transcription factor family provides knowledge of the functions of HDAC4, c-FOS, and MEF2C.

Retinal expression and localization of Mef2c support its important role in photoreceptor gene expression

Photoreceptor-specific gene expression is controlled by a hierarchical network of transcription factors, including the master regulators cone-rod homeobox (Crx) and neural retina leucine zipper (Nrl). Myocyte-enhancer factor 2c (Mef2c) is an ubiquitously expressed transcription factor with important functions in the cardiovascular system. Here, we performed a detailed analysis of Mef2c expression, localization and function in the retina to further elucidate its potential role for photoreceptor gene regulation. We showed that murine retinal Mef2c mRNA expression was high at birth and peaked at late postnatal developmental stages. Using immunohistochemistry and Western blot, Mef2c protein was detected in the outer nuclear layer of adult mouse and human retinas and localized to the nucleus of 661W photoreceptor-like cells. Mef2c knock-down in 661W cells reduced the expression of arrestin 3 (Arr3) and medium-wave-sensitive cone opsin (Opn1mw) but increased transcript levels of mitogen-activated protein kinase 15 (Mapk15) and phosphodiesterase 6h (Pde6h). In conclusion, Mef2c is highly expressed in the retina where it modulates photoreceptor-specific gene expression.

1,25-Dihydroxyvitamin D3 induces monocytic differentiation of human myeloid leukemia cells by regulating C/EBPβ expression through MEF2C

Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27Kip1 and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (activating transcription factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, are mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression.

HDAC5 Controls MEF2C-Driven Sclerostin Expression in Osteocytes

Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here, we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin-positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of corepressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.

Abstract 4233: Mef2C enhances 1,25-dihydroxyvitamin D3-induced monocytic differentiation of human myeloid leukemia cells by regulating C/EBPβ expression

Abstract Myogenic enhancer factors 2 (Mef2) are members of the family of MADS (MCM1-agamous-deficiens-serum response factor)-box transcription factors known to have multiple roles in morphogenesis of skeletal, cardiac and smooth muscle cells. Recently, Mef2C was found to be also involved in hematopoiesis. Bone marrow cells isolated from Mef2C knockout mice demonstrated decreased monocytic differentiation in response to cytokine stimulation, whereas constitutive expression of Mef2C promoted monopoiesis. The above findings led us to examine the role of Mef2C in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation. Human acute myeloid leukemia (AML) cell lines, HL60 and U937, were used in this study. We found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreased the expression of the monocytic marker CD14, as well as of nonspecific esterase (NSE), suggesting that Mef2C is required for 1,25D-induced monocytic differentiation in AML cells. Our laboratory has previously demonstrated that optimal monocytic differentiation induced by 1,25D requires ERK5, and that ERK5 expression shows positive correlation with Mef2C expression. Here we show that Mef2C knockdown demonstrates similar inhibition of CD14 expression as ERK5 knockdown. CCAAT-enhancer-binding proteins (C/EBPs) α and β are two major transcription factors that can directly regulate CD14 expression. Since 1,25D has been shown to increase both C/EBPα and β expression, and these two transcription factors form heterodimers to enhance CD14 expression, we used siRNA to knock down Mef2C and found decreased expression of 1,25D-induced C/EBPβ, but not C/EBPα. Similar results were obtained using an alternative method to titrate down Mef2C with dsDNA containing its DNA binding sequence. We conclude that Mef2C is a functional downstream target of ERK5, and that 1,25D-induced CD14 and NSE expression is mediated through ERK5/Mef2C activation. (Supported by T32 CA134268 training grant and R01 CA044722 both from the National Cancer Institute, NIH, and by the American Institute for Cancer Research grant 10A049) Citation Format: Ruifang Zheng, Xuening Wang, George P. Studzinski. Mef2C enhances 1,25-dihydroxyvitamin D3-induced monocytic differentiation of human myeloid leukemia cells by regulating C/EBPβ expression. [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 4233. doi:10.1158/1538-7445.AM2014-4233

The transient expression of miR-203 and its inhibiting effects on skeletal muscle cell proliferation and differentiation.

Previous studies have shown that miR-203 is a skin-specific microRNA (miRNA) with a profound role in skin cell differentiation. However, emerging microarray and deep sequencing data revealed that miR-203 is also expressed in embryonic skeletal muscle and myoblasts. In this study, we found that miR-203 was transiently upregulated in chicken embryos on days 10 to 16 (E10–E16) and was sharply downregulated and even not expressed after E16 in chicken embryonic skeletal muscle. Histological profiles and weight variations of embryo skeletal muscle revealed that miR-203 expression is correlated with muscle development. In vitro experiments showed that miR-203 exhibited downregulated expression during myoblast differentiation into myotubes. miR-203 overexpression inhibited myoblast proliferation and differentiation, whereas its loss-of-function increased myoblast proliferation and differentiation. During myogenesis, miR-203 can target and inhibit the expression of c-JUN and MEF2C, which were important for cell proliferation and muscle development, respectively. The overexpression of c-JUN significantly promoted myoblast proliferation. Conversely, knockdown of c-JUN by siRNA suppressed myoblast proliferation. In addition, the knockdown of MEF2C by siRNA significantly inhibited myoblast differentiation. Altogether, these data not only suggested that the expression of miR-203 is transitory during chicken skeletal muscle development but also showed a novel role of miR-203 in inhibiting skeletal muscle cell proliferation and differentiation by repressing c-JUN and MEF2C, respectively.

Members of the Myocyte Enhancer Factor 2 Transcription Factor Family Differentially RegulateBdnfTranscription in Response to Neuronal Depolarization

Transcription of the gene encoding brain-derived neurotropic factor (BDNF) is induced in response to a wide variety of extracellular stimuli via the activation of a complex array of transcription factors. However, to what degree individual transcription factors confer specificity upon the regulation of Bdnf is poorly understood. Previous studies have shown that members of the myocyte enhancer factor 2 (MEF2) transcription factor family bind a regulatory element upstream of Bdnf promoter I and associate with an unknown binding site in Bdnf promoter IV. Here we identify calcium-response element 1 as the MEF2 binding site in promoter IV of the Bdnf gene and determine the requirements for individual MEF2 family members in Bdnf regulation. MEF2A, MEF2C, and MEF2D are all highly expressed in embryonic rat cortical neurons; however, only the Mef2c gene encodes an MEF2 splice variant that lacks the γ repressor-domain. We find that MEF2C variants lacking the γ-domain are particularly sensitive to activation by membrane depolarization, raising the possibility that the MEF2s may differentially contribute to activity-regulated gene expression. We find that only knockdown of MEF2C significantly impairs membrane depolarization-induced expression of Bdnf exon IV. By contrast, knockdown of MEF2D significantly enhanced depolarization-induced expression of Bdnf exon I. Together, these data show that individual members of the MEF2 family of transcription factors differentially regulate the expression of Bdnf, revealing a new mechanism that may confer specificity on the induction of this biologically important gene.

MEF2C Ablation in Endothelial Cells Reduces Retinal Vessel Loss and Suppresses Pathologic Retinal Neovascularization in Oxygen-Induced Retinopathy

Ischemic retinopathies, including retinopathy of prematurity and diabetic retinopathy, are major causes of blindness. Both have two phases, vessel loss and consequent hypoxia-driven pathologic retinal neovascularization, yet relatively little is known about the transcription factors regulating these processes. Myocyte enhancer factor 2 (MEF2) C, a member of the MEF2 family of transcription factors that plays an important role in multiple developmental programs, including the cardiovascular system, seems to have a significant functional role in the vasculature. We, therefore, generated endothelial cell (EC)-specific MEF2C-deficient mice and explored the role of MEF2C in retinal vascularization during normal development and in a mouse model of oxygen-induced retinopathy. Ablation of MEF2C did not cause appreciable defects in normal retinal vascular development. However, MEF2C ablation in ECs suppressed vessel loss in oxygen-induced retinopathy and strongly promoted vascular regrowth, consequently reducing retinal avascularity. This finding was associated with suppression of pathologic retinal angiogenesis and blood-retinal barrier dysfunction. MEF2C knockdown in cultured retinal ECs using small-interfering RNAs rescued ECs from death and stimulated tube formation under stress conditions, confirming the endothelial-autonomous and antiangiogenic roles of MEF2C. HO-1 was induced by MEF2C knockdown in vitro and may play a role in the proangiogenic effect of MEF2C knockdown on retinal EC tube formation. Thus, MEF2C may play an antiangiogenic role in retinal ECs under stress conditions, and modulation of MEF2C may prevent pathologic retinal neovascularization.

VEGF Promotes the Transcription of the Human PRL-3 Gene in HUVEC through Transcription Factor MEF2C

Phosphatase of regenerating liver 3 (PRL-3) is known to be overexpressed in many tumors, and its transcript level is high in the vasculature and endothelial cells of malignant tumor tissue. However, the mechanism(s) underlying its enhanced expression and its function in endothelial cells remain unknown. Here, we report that vascular endothelial growth factor (VEGF) can induce PRL-3 transcription in human umbilical vein endothelial cells (HUVEC). An analysis of its 5′UTR revealed that PRL-3 transcription is initiated from two distinct sites, which results in the formation of the two transcripts, PRL-3-iso1 and PRL-3-iso2, but only the latter is up-regulated in HUVEC by VEGF. The PRL-3-iso2 promoter region includes two functional MEF2 (myocyte enhancer factor2) binding sites. The over-expression of the constitutively active form of MEF2C promotes the abundance of the PRL-3-iso2 transcript in a number of human cell lines. The siRNA-induced knockdown of MEF2C abolished the stimulative effect of VEGF on PRL-3 transcript in HUVEC, indicating that the VEGF-induced promotion of PRL-3 expression requires the presence of MEF2C. Finally, blocking PRL-3 activity or expression suppresses tube formation by HUVEC. We suggest that PRL-3 functions downstream of the VEGF/MEF2C pathway in endothelial cells and may play an important role in tumor angiogenesis.

Myocyte Enhancer Factor 2C, an Osteoblast Transcription Factor Identified by Dimethyl Sulfoxide (DMSO)-enhanced Mineralization

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

The Myocyte Enhancer Factor 2C (MEF2C) Is a Direct Transcriptional Target of HOXA9 and Mediates Leukemia Survival in Human Mixed-Lineage Leukemias.

Leukemias that harbor rearrangements of the mixed-lineage leukemia gene (MLL) possess unique clinical and biological characteristics. Aberrant high-level expression of select Homeobox (HOX) genes including HOXA9 is a consistent feature of MLL-rearranged leukemias, implicating an important role of dysregulated HOX gene expression in this entity. Applying HOXA9-directed RNAi, we have previously shown that aberrant HOXA9 expression is a prerequisite for leukemia survival in human MLL-rearranged leukemias and murine Mll-leukemia stem cells as HOXA9 suppression leads to a progressive induction of apoptosis in vitro and in vivo. In this study we aimed to identify potential direct HOXA9 target genes in human MLL-rearranged leukemias utilizing gene expression profiling after shRNA mediated HOXA9 knockdown and subsequent chromatin immunoprecipitation (ChIP) analysis. To establish efficient HOXA9 knockdown, 2 previously validated shRNA constructs targeting human HOXA9 with high efficiency (>80% mRNA suppression) were transduced into t(9;11) MOLM-14 AML cells. 72h after transduction, RNA was hybridized to Affymetrix HU 133A2.0 expression arrays. Supervised analysis identified a large group of genes concomitantly downregulated after HOXA9 suppression. Gene set enrichment analysis demonstrated significant enrichment of the top 300 genes in the HOXA9 suppression signature and genes more highly expressed in human MLL-AML as compared to other genetically defined AML subtypes (p=0.02). Interestingly, genes previously implicated in the pathogenesis of MLL-rearranged leukemias were among the most highly enriched (HOXA5, MEIS1, PBX3, MEF2C). To analyze if any of this genes might be a direct physiological HOXA9 target, we performed ChIP assays from FLAG-tagged HOXA9 expressing MOLM-14 cells. Whereas the promotor regions of HOXA5, MEIS1 and PBX3 did not show HOXA9 binding, the MEF2C promotor region DNA was highly enriched by ChIP. Furthermore HOXA9 binding was confined to the MEF2C promotor region as the regions -5kb 5'-upstream, +5kb and +10kb 3'-downstream of the MEF2C promotor region did not show any enrichment. Motive search analysis of the MEF2C promoter further revealed a perfect consensus binding site (TGATTTAT) for HOXA9 and its DNA binding partner PBX located at −721 to −714 from the transcriptional start site. We then analyzed if direct shRNA mediated targeting of MEF2C also abrogates leukemia survival in human MLL-rearranged leukemias similar to upstream HOXA9 suppression. Analysis of viability after MEF2C knockdown with two different shRNA constructs (>80% mRNA knockdown) in a panel of human 8 AML/ALL cell lines (4 MLL-rearranged; 4 MLL-wildtype) and primary AML patient cells (5 MLL-rearranged; 5 MLL-wildtype) revealed progressive induction of apoptosis which was significantly correlated with the presence of the MLL-fusion oncogene in the cell lines (mean viability at day 7: MLL-rearranged: 14.5% MLL-wildtype: 86.75%: p<0.001) and in the primary AML cells (p=0.006). Our data implicates MEF2C as a direct downstream target of HOXA9 and aberrant MEF2C expression is necessary for survival of MLL-rearranged leukemias. Targeting the HOXA9-MEF2C program may be a novel therapeutic approach.