Myocyte Enhancer Factor 2D Research Articles

Sodium Tungstate Promotes Neurite Outgrowth and Confers Neuroprotection in Neuro2a and SH-SY5Y Cells.

Sodium tungstate (Na2WO4) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na2WO4 in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na2WO4 promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na2WO4 increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na2WO4 increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na2WO4 on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na2WO4 were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na2WO4. These findings support the role of Na2WO4 in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound.

Oral Piwi-Interacting RNA Delivery Mediated by Green Tea-Derived Exosome-Like Nanovesicles for the Treatment of Aortic Dissection.

Aortic dissection (AD) is a severe cardiovascular disease necessitating active therapeutic strategies for early intervention and prevention. Nucleic acid drugs, known for their potent molecule-targeting therapeutic properties, offer potential for genetic suppression of AD. Piwi-interacting RNAs, a class of small RNAs, hold promise for managing cardiovascular diseases. Limited research on these RNAs and AD exists. This study demonstrates that an antagomir targeting heart-apoptosis-associated piRNA (HAAPIR) effectively regulates vascular remodeling, mitigating AD occurrence and progression through the myocyte enhancer factor 2D (Mef2D) and matrix metallopeptidase 9 (MMP9) pathways. Green tea-derived plant exosome-like nanovesicles (PELNs) are used for oral administration of antagomir. The antagomir-HAAPIR-nanovesicle complex, after purification and optimization, exhibits a high packing rate, while the antagomir is resistant to enzyme digestion. Administered to mice, the complex targets the aortic lesion, reducing AD incidence and improving survival. Moreover, MMP9 and Mef2D expression decrease significantly, inhibiting the phenotypic conversion of human aortic smooth muscle cells. PELNs encapsulate the antagomir-HAAPIR complex, maintaining stability, mediating transport into the bloodstream, and delivering Piwi-interacting RNAs to AD sites. Thus, HAAPIR is a potential target for persistent clinical AD prevention and treatment, and nanovesicle-encapsulated nucleic acids offer a promising cardiovascular disease treatment, providing insights for other therapeutic targets.

Mef2d regulates mutually exclusive expression of IgZ and IgM isotypes through epigenetic modulation in a zebrafish model

The discovery of IgZ, or its counterpart IgT, represents a novel immunoglobulin isotype (named ζ or τ) found in teleosts, introducing a new member to the existing Ig classes (µ, δ, γ, α, and ε) among vertebrates. The distinctive intrachromosomal organization of ighz and ighm loci implies the necessity of a distinct, mutually exclusive recombination process for the independent generation of IgZ and IgM isotypes. However, the molecular mechanisms governing this process remain elusive. In this study, we unveil the regulatory function of myocyte enhancer factor 2D (Mef2d) in the assembly of ighz genes through epigenetic modulation in a zebrafish model. Mechanistically, Mef2d selectively hinders the recombination of ighm locus in IgZ+ B cells by binding to the 3′Eµ site of the ighm locus and helping establish a repressive modification pattern of H3K4me0/H3K9me2/H3K27me2 in Dµ/Jµ regions through recruiting the co-repressive Sin3/Hdac1 complex with the assistance of cohesin complex and Setdb1/Ezh2 methyltransferases. Consequently, this renders the Dµ/Jµ regions inaccessible to Rag1/2, thus preventing ighm rearrangement. As a pivotal regulator for IgZ isotype production, Mef2d exhibits differential expression in committed IgZ+ B cells, a process regulated by the Il-7/Il-7r-mediated p38 Mapk signaling pathway. These results indicate the existence of a unique isotypic exclusion mechanism underlying recombination between ighz and ighm locus in teleosts. This mechanism highlights an unrecognized strategy for generating diverse isotypes in vertebrates, distinct from the well-established class switch recombination process. This study significantly contributes to our understanding of paradigms, diversifications, and the evolutionary history of vertebrate adaptive immunity.

MEF2D facilitates liver metastasis of gastric cancer cells through directly inducing H1X under IL-13 stimulation

Liver metastasis is the most common metastatic occurrence in gastric cancer patients, although the precise mechanism behind it remains unclear. Through a combination of proteomics and quantitative RT-PCR, our study has revealed a significant correlation between the upregulation of myocyte enhancer factor-2D (MEF2D) and both distant metastasis and poor prognosis in gastric cancer patients. In mouse models, we observed that overexpressing or knocking down MEF2D in gastric cancer cells respectively promoted or inhibited liver metastasis. Furthermore, our research has demonstrated that MEF2D regulates the transcriptional activation of H1X by binding to the H1X promoter. This regulation leads to the upregulation of H1X, which, in turn, promotes the in vivo metastasis of gastric cancer cells along with the upregulation of the downstream gene β-CATENIN. Additionally, we found that the expression of MEF2D and H1X at both mRNA and protein levels can be induced by the inflammatory factor IL-13, and this induction exhibits a time gradient dependence. In human gastric cancer tissues, the expression of IL13RA1, the receptor for IL-13, positively correlates with the expression of MEF2D and H1X. IL13RA1 has been identified as an intermediate receptor through which IL-13 regulates MEF2D. In conclusion, our findings suggest that MEF2D plays a crucial role in promoting liver metastasis of gastric cancer by upregulating H1X and downstream target β-CATENIN in response to IL-13 stimulation. Targeting MEF2D could therefore be a promising therapeutic strategy for the clinical management of gastric cancer. Statement of significanceMEF2D promotes its transcriptional activation in gastric cancer cells by binding to the H1X promoter and is upregulated by IL-13-IL13RA1, thereby promoting distant metastasis of gastric cancer.

Abstract 5629: MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas

Abstract Background: Immune escape remains a challenge in the treatment of hepatocellular carcinoma (HCC), one of the most common and deadly cancers in the world. The transcription factor, myocyte enhancer factor 2D (MEF2D), is important in the regulation of differentiation and adaptive responses in many cancers, such that we tested its role in HCC. Methods: We knocked down MEF2D in HCC cell lines and analyzed HCC tissues and cell lines by RNA sequencing, Western blot and immunohistochemistry. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation assay. Chromatin immunoprecipitation was used to analyze the regulation of CD70 transcription by MEF2D. H22 cells, with MEF2D knockout or without (controls), were injected into the livers of syngeneic BALB/c mice. Flow cytometry was used to analyze the function of T cells in tumors, spleens, and lymph nodes. Results: Through database searches, we noted that MEF2D and CD70 were upregulated in HCC and associated with shorter patient survival times. Compared to WT tumors, injection of MEF2D-knockdown HCC cells into immunocompetent mice led to smaller tumors with increased T cell infiltration, activation, and impaired T-regulatory (Treg) cell suppressive function. Mechanistically, we found that MEF2D binds to the promoter region of CD70 gene and activates its transcription. Moreover, acetylation of MEF2D enhances the binding of MEF2D to the CD70 promoter and further promotes its transcription. CD70 blocking antibody inhibited activation of the CD70-CD27 signaling axis in murine HCC tumors, leading to impaired immunosuppressive function of Tregs and enhanced T cell-mediated anti-tumor immune responses. Thus, regulation of the CD70-CD27 signaling axis by MEF2D affects the numbers and functions of Treg cells and thereby controls T cell infiltration. Conclusions: MEF2D in HCC cells increases the expression of CD70 and blocks T cell-mediated anti-tumor immunity via the CD70-CD27 signaling axis. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC. Citation Format: Fanhua Kong, Liqing Wang, Qifa Ye, Yan Xiong, Wayne W. Hancock. MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5629.

The Dual Functions of Andrographolide in the Epstein–Barr Virus-Positive Head-and-Neck Cancer Cells: The Inhibition of Lytic Reactivation of the Epstein–Barr Virus and the Induction of Cell Death

Andrographolide, a medicinal compound, exhibits several pharmacological activities, including antiviral and anticancer properties. Previously, we reported that andrographolide inhibits Epstein–Barr virus (EBV) lytic reactivation, which is associated with viral transmission and oncogenesis in epithelial cancers, including head-and-neck cancer (HNC) cells. However, the underlying mechanism through which andrographolide inhibits EBV lytic reactivation and affects HNC cells is poorly understood. Therefore, we investigated these mechanisms using EBV-positive HNC cells and the molecular modeling and docking simulation of protein. Based on the results, the expression of EBV lytic genes and viral production were significantly inhibited in andrographolide-treated EBV-positive HNC cells. Concurrently, there was a reduction in transcription factors (TFs), myocyte enhancer factor-2D (MEF2D), specificity protein (SP) 1, and SP3, which was significantly associated with a combination of andrographolide and sodium butyrate (NaB) treatment. Surprisingly, andrographolide treatment also significantly induced the expression of DNA Methyltransferase (DNMT) 1, DNMT3B, and histone deacetylase (HDAC) 5 in EBV-positive cells. Molecular modeling and docking simulation suggested that HDAC5 could directly interact with MEF2D, SP1, and SP3. In our in vitro study, andrographolide exhibited a stronger cytotoxic effect on EBV-positive cells than EBV-negative cells by inducing cell death. Interestingly, the proteome analysis revealed that the expression of RIPK1, RIPK3, and MLKL, the key molecules for necroptosis, was significantly greater in andrographolide-treated cells. Taken together, it seems that andrographolide exhibits concurrent activities in HNC cells; it inhibits EBV lytic reactivation by interrupting the expression of TFs and induces cell death, probably via necroptosis.

A Ubiquitin-Dependent Switch on MEF2D Senses Pro-Metastatic Niche Signals to Facilitate Intrahepatic Metastasis of Liver Cancer.

Effective treatment for metastasis, a leading cause of cancer-associated death, is still lacking. To seed on a distal organ, disseminated cancer cells (DCCs) must adapt to the local tissue microenvironment. However, it remains elusive how DCCs respond the pro-metastatic niche signals. Here, systemic motif-enrichment identified myocyte enhancer factor 2D (MEF2D) as a critical sensor of niche signals to regulate DCCs adhesion and colonization, leading to intrahepatic metastasis and recurrence of liver cancer. In this context, MEF2D transactivates Itgb1 (coding β1-integrin) and Itgb4 (coding β4-integrin) to execute temporally unique functions, where ITGB1 recognizes extracellular matrix for early seeding, and ITGB4 acts as a novel sensor of neutrophil extracellular traps-DNA (NETs-DNA) for subsequent chemotaxis and colonization. In turn, an integrin-FAK circuit promotes a phosphorylation-dependent USP14-orchastrated deubiquitination switch to stabilize MEF2D via circumventing degradation by the E3-ubiquitin-ligase MDM2. Clinically, the USP14(pS432)-MEF2D-ITGB1/4 feedback loop is often hyper-active and indicative of inferior outcomes in human malignancies, while its blockade abrogated intrahepatic metastasis of DCCs. Together, DCCs exploit a deubiquitination-dependent switch on MEF2D to integrate niche signals in the liver mesenchyme, thereby amplifying the pro-metastatic integrin-FAK signaling. Disruption of this feedback loop is clinically applicable with fast-track potential to block microenvironmental cues driving metastasis.

Expression of MEF2D in Lung Adenocarcinoma and Its Correlation with Prognosis

Myocyte enhancer factor 2D (MEF2D) can participate in the process of tumor lesions by regulating the transcription of oncogenes. In a previous study, MEF2D was demonstrated to enhance the proliferation and metastasis of lung adenocarcinoma cells A549 and H1299 by promoting the transcription of NUSAP1. The research aimed to explore the expression level and clinical significance of MEF2D in lung adenocarcinoma. A total of 199 patients with lung adenocarcinoma were collected. Immunohistochemical staining was used to detect MEF2D expression levels in cancer and adjacent tissues. After the clinical and follow-up data were collated, the correlation between MEF2D expression level and clinical characteristics and prognosis of the patients was analyzed. In the lung adenocarcinoma, the high expression rate of MEF2D in cancer tissues was significantly higher than that in adjacent tissues (P<0.05). According to immunohistochemical score, MEF2D expression level in lung adenocarcinoma tissues was correlated with tumor differentiation, N stage, M stage and intrapulmonary metastasis (P<0.05). Kaplan-Meier analysis showed that patients with low MEF2D expression had significantly better prognosis than patients with high MEF2D expression (P<0.05). Cox multivariate analysis showed that MEF2D expression level, M stage, N stage and bone metastasis of lung cancer were independent risk factors for prognosis of lung adenocarcinoma patients. MEF2D expression level is closely related to the metastasis of lung adenocarcinoma and other clinical characteristics, and can be used as an independent risk factor for the prognosis of patients with lung adenocarcinoma, which has the potential to be developed as a clinical diagnosis and treatment target of lung adenocarcinoma.

P38γ and p38δ modulate innate immune response by regulating MEF2D activation.

Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using Mapk12/Mapk13-deficient (p38γ/δKO) mice, which show low levels of TPL2, the kinase upstream of MKK1-ERK1/2 in myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here, we generated a Mapk12D171A/D171A/Mapk13-/- (p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to lipopolysaccharide (LPS)-induced septic shock and Candida albicans infection than wild-type (WT) mice. Gene expression analyses in LPS-activated wild-type and p38γ/δKIKO macrophages revealed that p38γ/p38δ-regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and in vitro kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of Nos2 and Il1b mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.

Knockdown of MEF2D inhibits the development and progression of B-cell acute lymphoblastic leukemia.

Myocyte enhancer factor 2D (MEF2D) is involved in the progression of various malignant tumors. However, its impact on B-cell acute lymphoblastic leukemia (B-ALL) has not been elucidated. In this study, the expression level of MEF2D in B-ALL patients was validated through the Gene Expression Omnibus (GEO) database and clinical specimens. MEF2D-knockdown B-ALL cell lines were constructed by lentivirus transfection, and the effects of MEF2D on the viability, apoptosis, cycle progression, and drug sensitivity of B-ALL cells were verified by Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM). The effect of MEF2D on the proliferation of B-ALL cells in vivo was verified via the construction of a xenograft mouse model. The mechanism of MEF2D regulating B-ALL cells was explored by RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemical (IHC). In this study, overexpression of MEF2D was observed in B-ALL patients and was remarkably correlated to disease progression in ALL patients. The knockdown of MEF2D expression suppressed cell viability, induced cell apoptosis, blockaded cell cycle progression, enhanced drug sensitivity of B-ALL cells in vitro, and reduced the tumor load in vivo. Furthermore, mechanistic studies revealed that MEF2D knockdown downregulated the expression of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Our research demonstrated that MEF2D was markedly expressed in B-ALL. MEF2D knockdown inhibited cancer progression of B-ALL both in vitro and in vivo, which may be related to the downregulation of the PI3K-AKT signaling pathway. The data suggest that MEF2D plays a vital role in the process of tumorigenesis and may be a potential novel target for B-ALL therapy.

Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes.

Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease.

MiR-18 inhibitor promotes the differentiation of bovine skeletal muscle-derived satellite cells by increasing MEF2D expression.

Skeletal muscle is composed of muscle fibers formed from myoblast differentiation. Recently, numerous researchers have demonstrated that microRNAs (miRNAs) play an essential role in modulating the proliferation and differentiation of myoblasts. Our previous study has shown that among the miR-17-92 cluster members, miR-17 and miR-20a together with miR-19b can efficiently promote the differentiation of murine C2C12 and bovine primary myoblasts. However, the role of miR-18 in this process remains elusive. In this study, we revealed that miR-18 inhibited the differentiation of bovine skeletal muscle-derived satellite cells (bMDSCs), whereas an miR-18 inhibitor significantly promoted cell differentiation (p < 0.001). Then, a target gene of miR-18 was found to be myocyte enhancer factor 2D (MEF2D), which is critical for myoblast differentiation. Furthermore, we found that the combination of the miR-18 inhibitor and miR-19 significantly improved the formation of bMDSCs-derived muscle fibers (p < 0.001). This study revealed the role of miR-18 in bovine skeletal muscle differentiation and contributed to the understanding of the regulatory mechanism of mammalian myogenic differentiation.

Smad8 Is Increased in Duchenne Muscular Dystrophy and Suppresses miR-1, miR-133a, and miR-133b.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor β (TGFβ) signaling. In this report, we investigated the major transducers of TGFβ signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.

Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy

BackgroundThe development of diabetes vascular calcification (VC) is tightly associated with the inhibition of vascular smooth muscle cell (VSMC) autophagy. Previously, our team found that miR-32-5p (miR-32) promotes macrophage activation, and miR-32 is expressed at higher level in the plasma of patients with coronary calcification. However, whether miR-32 mediates the function of macrophages in type 2 diabetes (T2D) VC is still unclear.MethodsWild-type (WT) and miR-32−/− mice were used in this study. qRT-PCR and western blotting were used to analyze gene expression. Flow cytometry was used to analyze the influence of glucose concentration on macrophage polarization. Nanoparticle tracking analysis (NTA), transmission electron microscopy, and confocal microscopy were used to identify macrophage extracellular vehicles (EVs). Immunofluorescence, in situ hybridization (ISH), immunohistochemistry, and alizarin red staining were used to analyze the influence of macrophage EVs on autophagy and calcification of the aorta of miR-32−/− mice. A luciferase assay was used to analyze the effect of miR-32 on myocyte enhancer factor 2D (Mef2d) expression. Co-IP combined with mass spectrometry (MS) and transcriptome sequencing was used to analyze the signalling pathway by which Mef2d acts in VSMC autophagy.ResultsWe found that high glucose conditions upregulate miR-32 expression in macrophages and their EVs. Importantly, macrophages and their EVs promote VSMC osteogenic differentiation and upregulate miR-32 expression in VSMCs. Moreover, miR-32 mimics transfection promoted osteogenic differentiation and inhibited autophagy in VSMCs. In vitro and in vivo experiments showed that Mef2d is the key target gene of miR-32 that inhibits VSMC autophagy. Furthermore, MS and transcriptome sequencing found that cGMP-PKG is an important signalling pathway by which Mef2d regulates VSMC autophagy. In addition, after T2D miR-32−/− mice were injected with macrophage EVs via the caudal vein, miR-32 was detected in aortic VSMCs of miR-32−/− mice. Moreover, autophagy was significantly inhibited, and calcification was significantly enhanced in aorta cells.ConclusionsThese results reveal that EVs are the key pathway by which macrophages promote T2D VC, and that EVs miR-32 is a key cause of autophagy inhibition in VSMCs.

SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers.

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7–associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

O-GlcNAcylation of myocyte-specific enhancer factor 2D negatively regulates insulin secretion from pancreatic β-cells

Patients with type 2 diabetes often exhibit impairments in both glucose-induced insulin secretion (GIIS) and incretin-induced insulin secretion (IIIS). These phenotypes are associated with altered glucose metabolism in pancreatic β-cells, although the molecular mechanisms remain unclear. Here, we used MIN6-K8 pancreatic β-cell lines as a model to examine the effect of O-linked N-acetylglucosamine glycosylation (O-GlcNAcylation), a glucose-induced protein posttranslational modification, on insulin secretion. O-GlcNAcylation was enhanced in high-glucose-treated MIN6-K8 cells, and high levels of O-GlcNAcylation attenuated PKA-dependent phosphorylation, suggesting that the two protein modifications may compete with each other. Immunoprecipitation proteomic analysis identified six candidate proteins that were O-GlcNAcylated by high-glucose treatment, whereas the O-GlcNAcylations were removed by treatment with an incretin mimetic, exendin-4. Among these proteins, knockdown of myocyte enhancer factor 2D (Mef2d) enhanced insulin secretion, and high-glucose treatment increased the level of O-GlcNAcylation of Mef2d in MIN6-K8 cells. Furthermore, knockout of Mef2d promoted GIIS in MIN6-K8 cells, whereas adenovirus-mediated rescue of Mef2d decreased GIIS in the knockout cells. These results suggest that Mef2d negatively regulates insulin secretion through O-GlcNAcylation.

Acidic Tumor Microenvironment Promotes Pancreatic Cancer through miR-451a/MEF2D Axis.

Pancreatic cancer (PC), as a highly malignant and aggressive solid tumor, is common in the digestive system. The acidic microenvironment is one of the critical markers of cancer. Nonetheless, there are few studies on how the acidic microenvironment affects the development of PC. This study focused on investigating the specific molecular mechanisms of the acidic microenvironment in PC. In our study, qRT-PCR was conducted for examining microRNA (miR)-451a and myocyte enhancer factor 2D (MEF2D) expressions in PANC-1 cells. Then, detailed functional effects of an acidic environment on miR-451a and MEF2D in PANC-1 cells were detected by CCK-8, colony formation, flow cytometry, wound healing, transwell, mitochondrial functionality measurement, JC-1 staining, DCFH-DA staining, and sphere formation assays. The relationship between miR-451a and MEF2D was confirmed by luciferase reporter analysis. Under acidic conditions, the increase of proliferation, migration, and invasion of PANC-1 cells was observed. Moreover, the mitochondrial oxidative respiration-related gene miR-451a was reduced in acidic conditions. In addition, we found that, in PANC-1 cells under an acidic environment, miR-451a overexpression enhanced oxygen consumption, mitochondrial membrane potential (MMP) loss, and ROS generation and inhibited proliferation, migration, invasion, and stemness via sponging MEF2D. In a word, our results revealed that the acidic microenvironment regulated PC progression by affecting the miR-451a/MEF2D axis, indicating a novel avenue for the future treatment of PC.

RETRACTED ARTICLE: FAM83H-AS1/miR-485-5p/MEF2D axis facilitates proliferation, migration and invasion of hepatocellular carcinoma cells

BackgroundAbundant evidence has manifested that long noncoding RNAs (lncRNAs) are closely implicated in human cancers, including hepatocellular carcinoma (HCC). Remarkably, lncRNA FAM83H antisense RNA 1 (FAM83H-AS1) has been reported to be a tumor-propeller in multiple cancers. However, its effect on HCC progression remains unknown.MethodsFAM83H-AS1 expression was analyzed by RT-qPCR. Colony formation, EdU, and flow cytometry as well as transwell assays were implemented to analyze the biological functions of FAM83H-AS1 on HCC progression. Luciferase reporter, RIP and RNA pull-down assays were implemented to detect the interaction among FAM83H-AS1, microRNA-485-5p (miR-485-5p), and myocyte enhancer factor 2D (MEF2D) in HCC cells.ResultsFAM83H-AS1 expression in HCC cells was markedly elevated. FAM83H-AS1 accelerated cell proliferation, migration and invasion whereas inhibiting cell apoptosis in HCC. Besides, we confirmed that FAM83H-AS1 acts as a miR-485-5p sponge in HCC cells. Additionally, MEF2D was verified to be a direct target of miR-485-5p. FAM83H-AS1 could upregulate MEF2D expression via sponging miR-485-5p. Further, rescue experiments testified that MEF2D upregulation or miR-485-5p downregulation offset the repressive effect of FAM83H-AS1 depletion on HCC cell progression.ConclusionsFAM83H-AS1 facilitates HCC malignant progression via targeting miR-485-5p/MEF2D axis, suggesting that FAM83H-AS1 may be a promising biomarker for HCC treatment in the future.

Coupling HDAC4 with transcriptional factor MEF2D abrogates SPRY4-mediated suppression of ERK activation and elicits hepatocellular carcinoma drug resistance.

Hepatocellular carcinoma (HCC) lacks effective treatment, and the patients rapidly develop the acquired resistance to sorafenib with less defined mechanisms. Here, we demonstrate that transcriptional factor myocyte enhancer factor 2D (MEF2D) overexpression is detected in sorafenib-resistant HCC specimens and HCC cell lines and predicts poor prognosis of sorafenib-treated HCC patients. Mechanistically, MEF2D in complex with histone deacetylase HDAC4 directly binds to the SPRY4 promoter regions and suppresses the transcriptional expression of SPRY4, which is a negative regulator of MAPK/ERK signaling pathway. Inhibition of HDAC4 with its clinically used inhibitor induces SPRY4 expression and inhibition of ERK activity, resulting in sensitization of HCC cells to sorafenib-induced apoptosis and greatly improved inhibition of liver tumor growth in mice with sorafenib treatment. These findings highlight the critical role of coupling HDAC4 with MEF2D in activation of ERK by suppressing SPRY4 and underscore the great potential to improve HCC treatment by combined administration of sorafenib with HDAC4 inhibitors.

MEF2D-NR4A1-FAM134B2-mediated reticulophagy contributes to amino acid homeostasis

ABSTRACT We recently identified FAM134B2, which is an N-terminal truncated reticulophagy receptor highly induced by starvation such as fasting of mice and treatment of mammalian cells with a starvation medium that does not contain amino acids, glucose and growth factors. However, which starvation signal mediates the induction of FAM134B2 is still obscure. In this study, we found that amino acid deficiency (AAD) could mimic the starvation condition to induce FAM134B2 expression. Unexpectedly, EIF2AK4/GCN2-mediated integrated signal response (ISR) and MTOR (mechanistic target of rapamycin kinase) signals, which constitute two major signaling pathways that respond to AAD, did not contribute to AAD-induced FAM134B2 induction. mRNA-seq and siRNA screenings identified two ISR-independent transcription factors, MEF2D (myocyte enhancer factor 2D) and NR4A1 (nuclear receptor subfamily 4 group A member 1), involved in AAD-induced FAM134B2 expression. AAD induces MEF2D, resulting in the induction of NR4A1, which in turn induces FAM134B2-mediated reticulophagy to maintain intracellular amino acid levels. In conclusion, the MEF2D-NR4A1-FAM134B2 cascade is a critical signal in amino acid homeostasis. Abbreviations AAD: amino acid deficiency; APOC3: apolipoprotein C3; BACH1: BTB domain and CNC homolog 1; CEBP: CCAAT enhancer binding protein; DDIT3/CHOP: DNA damage inducible transcript 3; EBSS: Earle’s Balanced Salt Solution; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; HisOH: histidinol; ISR: integrated stress response; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF2D: myocyte enhancer factor 2D; MTOR: mechanistic target of rapamycin kinase; NR4A1: nuclear receptor subfamily 4 group A member 1; RETREG1/FAM134B: reticulophagy regulator 1; RTN2: reticulon 2, TF: transcription factor; TFEB: transcription factor EB; ZBTB10: zinc finger and BTB domain containing 10