Myocyte Enhancer Factor 2B Research Articles

MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis

The myocyte enhancer factor 2B (MEF2B) transcription factor is frequently mutated in germinal center (GC)-derived B-cell lymphomas. Its ammino (N)-terminal mutations drive lymphomagenesis by escaping interaction with transcriptional repressors, while the function of carboxy (C)-terminal mutations remains to be elucidated. Here, we show that MEF2B C-tail is physiologically phosphorylated at specific residues and phosphorylation at serine (S)324 is impaired by lymphoma-associated mutations. Lack of phosphorylation at S324 enhances the interaction of MEF2B with the SWI/SNF chromatin remodeling complex, leading to higher transcriptional activity. In addition, these mutants show an increased protein stability due to impaired interaction with the CUL3/KLHL12 ubiquitin complex. Mice expressing a phosphorylation-deficient lymphoma-associated MEF2B mutant display GC enlargement and develop GC-derived lymphomas, when crossed with Bcl2 transgenic mice. These results unveil converging mechanisms of action for a diverse spectrum of MEF2B mutations, all leading to its dysregulation and GC B-cell lymphomagenesis.

The transcription factor ZEB2 drives the formation of age-associated B cells.

Age-associated B cells (ABCs) accumulate during infection, aging, and autoimmunity, contributing to lupus pathogenesis. In this study, we screened for transcription factors driving ABC formation and found that zinc finger E-box binding homeobox 2 (ZEB2) is required for human and mouse ABC differentiation in vitro. ABCs are reduced in ZEB2 haploinsufficient individuals and in mice lacking Zeb2 in B cells. In mice with toll-like receptor 7 (TLR7)-driven lupus, ZEB2 is essential for ABC formation and autoimmune pathology. ZEB2 binds to +20-kb myocyte enhancer factor 2b (Mef2b)'s intronic enhancer, repressing MEF2B-mediated germinal center B cell differentiation and promoting ABC formation. ZEB2 also targets genes important for ABC specification and function, including Itgax. ZEB2-driven ABC differentiation requires JAK-STAT (Janus kinase-signal transducer and activator of transcription), and treatment with JAK1/3 inhibitor reduces ABC accumulation in autoimmune mice and patients. Thus, ZEB2 emerges as a driver of B cell autoimmunity.

Identification of two novel SNPs in the myocyte enhancer factor 2B (MEF2B) gene and its association with growth traits in two breeds of Turkish sheep

Myocyte enhancer factor 2B (MEF2B) is a member of the transcription factor family that is thought to regulate the gene expression of variable regulators of embryonic development and cellular differentiation. The study aimed to detect the possible association of the MEF2B gene with growth traits in Awassi and Cukurova meat sheep (CMS). Sanger sequencing method was utilized to identify the possible polymorphism in four amplified fragments in the MEF2B gene. Two novel SNPs of g .14327 G>C and g .16706 T > A were detected in intron-2 and exon-8 respectively. The g .16706 T > A was only observed in CMS with three genotypes (TT, TA, and AA), while this locus did not show any polymorphism in Awassi. In contrast, two genotypes (GG and CC) were exerted from g .14327 G>C in Awassi without showing any variation in CMS. Association analysis showed that g .16706 T > A and g .14327 G>C showed significant variable associations with growth traits in CMS and Awassi breeds respectively. CMS individuals with the AA genotype showed higher BW, RW, CG, BL, and CW than those with TA and TT respectively. Whereas, Awassi individuals with the GG genotype showed higher BW, RH, CW, and ADG than those with CC genotype. The substitution of T to A at the g.16706 led to the change of amino acid from Leucine to Histidine in the 324th amino acid residue of MEF2B. Molecular docking showed that p.Leu342His induced a dramatic alteration in the complex formed between MEF2B and its corresponding DNA sequences with reduced numbers of interacted amino acids upon mutation. It can be stated that g .16706 T > A and g .14327 G>C are promising biomarkers for evaluating productive traits in CMS and Awassi respectively. The predicted mechanism by which g .16706 T > A influence DNA has validated its suggestive action. Large-scale association analysis is required to assess the extent of both SNPs on the growth traits in both breeds.

Epstein-Barr virus infection patterns in nodular lymphocyte-predominant Hodgkin lymphoma.

To investigate Epstein-Barr virus (EBV) latency types in 19 cases of EBV-positive nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), as such information is currently incomplete. Immunohistochemistry (IHC) for CD20, CD79a, PAX5, OCT2, CD30, CD15, CD3 and programmed cell death protein 1 was performed. For EBV detection, in-situ hybridisation (ISH) for EBV-encoded RNA (EBER) was employed combined with IHC for EBV-encoded latent membrane protein (LMP)-1, EBV-encoded nuclear antigen (EBNA)-2, and EBV-encoded BZLF1. In 95% of the cases, neoplastic cells with features of Hodgkin and Reed-Sternberg (HRS) cells were present, mostly showing expression of CD30. In all cases, the B-cell phenotype was largely intact, and delineation from classic Hodgkin lymphoma (CHL) was further supported by myocyte enhancer factor 2B (MEF2B) detection. All tumour cells were EBER-positive except in two cases. EBV latency type II was most frequent (89%) and type I was rare. Cases with latency type I were CD30-negative. Five cases contained some BZLF1-positive and/or EBNA-2-positive bystander lymphocytes. As HRS morphology of neoplastic cells and CD30 expression are frequent features of EBV-positive NLPHL, preservation of the B-cell transcription programme, MEF2B expression combined with NLPHL-typical architecture and background composition facilitate distinction from CHL. EBER ISH is the method of choice to identify these cases. The majority present with EBV latency type II, and only rare cases present with latency type I, which can be associated with missing CD30 expression. The presence of occasional bystander lymphocytes expressing BZLF1 and/or EBNA-2 and the partial EBV infection of neoplastic cells in some cases could indicate that EBV is either not primarily involved or is only a transient driver in the pathogenesis of EBV-positive NLPHL.

Systematic transition modeling analysis in the MEF2B-DNA binding interface due to Y69H and K4E variants

Transcriptional coactivator myocyte enhancer factor 2B (MEF2B) mutations are the most common cause of germinal center-derived B-cell non-Hodgkin lymphoma. Despite well-established contributions in lymphomagenesis, the structure-function paradigms of these mutations are largely unknown. Here through in silico approaches, we present structural evaluation of two reported missense variants (K4E and Y69H) in MEF2B to investigate their impact on DNA-binding through molecular dynamics simulation assays. Notably, MEF2B-specific MADs box domain (Lys23, Arg24 and Lys31) and N-terminal loop residues (Gly2, Arg3, Lys4, Lys5, Ile6 and Asn13) contribute in DNA binding, while in MEF2BK4E, DNA binding is facilitated by Gly2, Arg3 and Arg91 (α3) residues. Conversely, in MEF2BY69H, Arg3, Lys5, Ser78, Arg79 and Asn81 residues mediate DNA binding. DNA binding induces pronounced conformational readjustments in MEF2BWT-specific α1-N-terminal loop region, while MEF2BY69H and MEF2BK4E exhibit fluctuations in both α1 and α3. Hydrogen (H)-bond occupancy analysis reveals a similar DNA binding behavior for MEF2WT and MEF2BY69H, compared to MEF2BK4E structure. The Anisotropic Network Model analysis depicts α1 and α3 as more fluctuant regions in MEF2BK4E as compared to other systems. MEF2BWT and MEF2BK4E, Tyr69 residue is involved in p300 binding thus possible influence of Y69H variation in the functions other than DNA binding, such as p300 co-activator recruitment may explain the reduced transcriptional activation of MEF2BY69H. Thus, present study may provide a structural basis of DNA recognition by pinpointing the underlying conformational changes in the dynamics of MEF2BK4E, MEF2BY69H, and MEF2BWT structures that may contribute in the identification of novel therapeutic strategies for lymphomagenesis.

The molecular landscape and other distinctive features of primary cutaneous follicle center lymphoma

Primary cutaneous follicle center lymphoma (PCFCL) is distinguished from other follicular lymphomas (FLs) based on its clinicopathologic features including diminished CD10 and frequent lack of BCL2 rearrangements (R). Whether newer germinal center-associated markers would also be less commonly expressed and whether mutational studies would support its segregation from classic FL and FL subsets, including those which also typically lack BCL2R, are uncertain. To address these questions, 22 PCFCLs were stained for myocyte enhancer factor 2B (MEF2B) and human germinal center-associated lymphoma (HGAL), and targeted next-generation sequencing was performed with results compared to a meta-analysis of FL, pediatric-type FL (PTFL), low stage FL (LSFL) and other FL subsets. Selected fluorescence in situ hybridization studies were also performed. Although 27% of cases lacked CD10, all tested were MEF2B+ and HGAL+. The most common somatic mutations in the 12 to 19 analyzable PCFCL were TNFRSF14 (40%, plus 10% with 1p36 deletions), followed by CREBBP, TNFAIP3, KMT2D, SOCS1, EP300, STAT6, and FOXO1 (17-25%). Three of the most commonly mutated genes in FL (KMT2D, CREBBP, and BCL2) were significantly less commonly mutated in PCFCL than in FL, and TNFAIP was more commonly mutated with no difference for TNFRSF14 between PCFCL and FL or PTFL. CREBBP was also less frequently mutated than in LSFL but more frequently mutated than in PTFL. MAP2K1 mutations were much more common in PTFL (44% versus 0%). Two of 22 of the PCFCL had a BCL2 rearrangement and zero of 12 had a BCL6 rearrangement. These findings, while showing well-recognized and new shared features between PCFCL and other FL, highlight a distinctive mutational profile further supporting its recognition as a distinct entity.

Landscape of DNA binding signatures of myocyte enhancer factor-2B reveals a unique interplay of base and shape readout.

Myocyte enhancer factor-2B (MEF2B) has the unique capability of binding to its DNA target sites with a degenerate motif, while still functioning as a gene-specific transcriptional regulator. Identifying its DNA targets is crucial given regulatory roles exerted by members of the MEF2 family and MEF2B’s involvement in B-cell lymphoma. Analyzing structural data and SELEX-seq experimental results, we deduced the DNA sequence and shape determinants of MEF2B target sites on a high-throughput basis in vitro for wild-type and mutant proteins. Quantitative modeling of MEF2B binding affinities and computational simulations exposed the DNA readout mechanisms of MEF2B. The resulting binding signature of MEF2B revealed distinct intricacies of DNA recognition compared to other transcription factors. MEF2B uses base readout at its half-sites combined with shape readout at the center of its degenerate motif, where A-tract polarity dictates nuances of binding. The predominant role of shape readout at the center of the core motif, with most contacts formed in the minor groove, differs from previously observed protein–DNA readout modes. MEF2B, therefore, represents a unique protein for studies of the role of DNA shape in achieving binding specificity. MEF2B–DNA recognition mechanisms are likely representative for other members of the MEF2 family.

Expression of myocyte enhancer factor 2B in mantle cell lymphoma and its clinical significance

Objective: To investigate the expression of myocyte enhancer factor 2B (MEF2B) in mantle cell lymphomas (MCL), and to analyze the correlation between the expression of MEF2B and pathological subtypes, structural subtypes, SOX11 expression and its clinical significance. Methods: Paraffin-embedded tissues were stained with HE, immunohistochemistry (EnVision method) and fluorescence in situ hybridization (FISH) , in addition, the clinical and pathological data of 60 cases of MCL were collected at Sun Yat-sen University Foshan Hospital and Sun Yat-sen University Cancer Center from January,2002 to May, 2019 for analysis. Results: Of the 60 MCLs, males is predominant (M∶F=3∶1). Histologically, the typical MCL is the majority (classical MCL: variant type MCL=48 cases:12 cases) . Fifty cases were classified into non-complete FDC meshwork type MCL, and the remaining 10 cases were classified into the complete-FDC meshwork type MCL group. Patients with classical MCL were more than 60 years old. The coexistent lesion sites both node and extranode in pathological subtype or structural subtype was the most common lesion sites. SOX11(+) MCL was common in classical MCL (P=0.040) and tended to be complete-FDC meshwork type MCL (P=0.086). The expression rate of MEF2B in MCL was 60.0%(36/60). This rate of MEF2B in classical type, complete-FDC meshwork type and SOX11(+) MCL was significantly higher than that variant type, no complete-FDC meshwork type, SOX11(-)MCL (P<0.05), respectively. There was no difference in clinical characteristics of MCL between MEF2B positive and negative groups. Compared with SOX11(-)MCL, the percentage of MEF2B expressed in tumor cells of SOX11(+)MCL was significantly higher (P=0.027). The expression of MEF2B was not related to the proliferation of tumor cells (P=0.341). There was no significant difference in the survival rate between different expression groups of MEF2B and SOX11 (P=0.304 and P=0.819, respectively). Only the mortality of variant type (blastoid/pleomorphic) MCL within 2 years was significantly higher than that of classical type MCL (P<0.05). Conclusions: The expression of MEF2B in MCL is related to the pathological subtypes, structural subtypes and the expression of SOX11, but not to the proliferation and prognosis. The high mortality rate within 2 years is only found in variant MCL. However, the role of MEF2B in MCL needs to be further studied.

Akirin1 promotes myoblast differentiation by modulating multiple myoblast differentiation factors.

Akirin1 is found to be involved in myoblast differentiation. However, the mechanism by which the Akirin1 gene regulates myoblast differentiation still remains unclear. In the present study, we found that ectopic expression of Akirin1 promoted myoblast differentiation by increasing the expression of myogenic regulatory factor (MRF) 4 (MRF4) and myocyte enhancer factor 2B (MEF2B) mRNA. Additionally, we showed that ectopic Akirin1 induced cell cycle arrest by up-regulating p21 mRNA. To further uncover the mechanism by which Akirin1 promotes myoblast differentiation, we showed that the enhanced Akirin1 increased the mRNA expression of P38α. Importantly, the enhanced MRF4 expression by Akirin1 can be abrogated by treatment of SB203580, a p38 inhibitor. Similarly, we found that enhanced MEF2B expression by Akirin1 can be abrogated by treatment with LY294002, a PI3K inhibitor. Together, our results indicate that Akirin1 promotes myoblast differentiation by acting on the p38 and PI3K pathways and subsequently inducing the expression of myoblast differentiation factors.

Betaine improves the growth performance and muscle growth of partridge shank broiler chickens via altering myogenic gene expression and insulin-like growth factor-1 signaling pathway

This study was conducted to investigate the effect of betaine on growth performance, carcass characteristics, myogenic gene expression, and insulin-like growth factor-1 (IGF-1) signaling pathway in partridge shank broiler chickens. A total of 192 one-day-old partridge shank broiler chickens were randomly divided into 4 groups with 6 replicates of 8 chickens for a 52-d feeding trial. Broilers were fed a basal diet supplemented with 0 (control), 250 (B250), 500 (B500), or 1,000 (B1000) mg/kg betaine. Compared with the control group, the B500 and B1000 groups had higher (P < 0.05) body weight gain (BWG), and the B500 group had a lower (P < 0.05) feed/gain ratio (F:G) during the whole trial period. Moreover, the B1000 group increased (P < 0.05) the breast muscle yield and decreased (P < 0.05) relative abdominal fat weight. The mRNA expression of myocyte enhancer factor 2B (MEF2B) and mechanistic target of rapamycin (mTOR) and mTOR phosporylation were higher (P < 0.05) in both breast and thigh muscles in the B500 and B1000 groups than those in the control group. The higher (P < 0.05) concentration and mRNA expression of IGF-1 were also observed in breast muscle in the B500 and B1000 groups. Additionally, the B1000 group up-regulated (P < 0.05) the mRNA level of myogenic differentiation factor 1 (MyoD1) in breast muscle and myogenin (MyoG) in thigh muscle. In conclusion, diets supplemented with 500 or 1,000 mg/kg betaine improved the growth performance of partridge shank broiler chickens during the whole trial period, and the B1000 group significantly improved the breast muscle growth. These improvements might result from increased mRNA expression of MyoD1 and MEF2B in breast muscle and MyoG and MEF2B in thigh muscle, and through alterations in IGF-1/mTOR signaling pathway.

Direct Reprogramming of Fibroblasts Into Smooth Muscle-Like Cells With Defined Transcription Factors-Brief Report.

Objective- To identify the transcription factors that could contribute to direct reprogramming of fibroblasts toward smooth muscle cell fate. Approach and Results- We screened various combinations of transcription factors, including Myocd (myocardin), Mef2C (myocyte enhancer factor 2C), Mef2B (myocyte enhancer factor 2B), Mkl1 (MKL [megakaryoblastic leukemia]/Myocd-like 1), Gata4 (GATA-binding protein 4), Gata5 (GATA-binding protein 5), Gata6 (GATA-binding protein 6), Ets1 (E26 avian leukemia oncogene 1, 5' domain), and their corresponding carboxyterminal fusions to the transactivation domain of MyoD (myogenic differentiation 1)-indicated by *-for their effects on reprogramming mouse embryonic fibroblasts and human adult dermal fibroblasts to the smooth muscle cell fate as determined by the expression of specific markers. The combination of 3 transcription factors, Myocd (or Myocd*) with Mef2C (or Mef2C*) and Gata6, was the most efficient in enhancing the expression of smooth muscle marker genes and decreasing fibroblast gene expression. Additionally, the derived induced smooth muscle-like cells showed a contractile phenotype in response to carbachol. Conclusions- Combination of Myocd and Gata6 with Mef2C* (MG2*) could sufficiently and efficiently direct differentiation of mouse embryonic and human dermal fibroblasts into induced smooth muscle-like cells, thus opening new opportunities for disease modeling, tissue engineering, and personalized medicine.

J chain and myocyte enhancer factor 2B are useful in differentiating classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma and primary mediastinal large B-cell lymphoma

Although most classical Hodgkin lymphomas (CHLs) are easily distinguished from nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and primary mediastinal large B-cell lymphoma (PMBL), cases with significant CD20 expression cause diagnostic confusion. Although the absence of OCT-2 and BOB.1 are useful in these circumstances, a variable proportion of CHLs are positive for these antigens. We investigated the utility of J chain and myocyte enhancer factor 2B (MEF2B) in the diagnosis of CHL; NLPHL; PMBL; T-cell/histiocyte-rich large B-cell lymphoma (TCRLBL); and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, compared with OCT-2 and BOB.1. J chain and MEF2B highlighted lymphocyte predominant (LP) cells in 20/20 (100%) NLPHLs and were negative in 43/43 (100%) CHLs. Fourteen of 15 (93%) PMBLs and 4/4 (100%) TCRLBLs were MEF2B positive, whereas 67% of PMBLs and 50% of TCRLBLs were J chain positive. Three of 3 B-cell lymphomas, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, were negative for J chain and MEF2B. J chain and MEF2B were 100% sensitive and specific for NLPHL versus CHL. MEF2B was 100% sensitive and 98% specific for PMBL versus CHL. Whereas loss of OCT-2 and/or BOB.1 expression had a sensitivity of only 86% and specificity of 100% for CHL versus NLPHL, PMBL, and TCRLBL, lack of both J chain and MEF2B expression was 100% sensitive and 97% specific. J chain and MEF2B are highly sensitive and specific markers of NLPHL versus CHL; are particularly useful in highlighting LP cells; and, with rare exception, are of greater utility than OCT-2 and BOB.1 in differentiating CHL from NLPHL and other large B-cell lymphomas.

Association of growth rate with hormone levels and myogenic gene expression profile in broilers

BackgroundThe growth rate often varies among individual broilers of the same breed under a common management condition. To investigate whether a variation in the growth rate is associated with a difference in hormone levels and myogenic gene expression profile in broilers, a feeding trial was conducted with 10,000 newly hatched Ross 308 chicks in a commercial production facility under standard management. At 38 d of age, 30 fast-, 30 medium-, and 30 slow-growing broilers were selected among 600 healthy male individuals. The levels of insulin-like growth factor-1 (IGF-1), triiodothyronine (T3), thyroxine (T4), and growth hormone in the serum or breast muscle were assayed by ELISA or RIA kits, and the expression levels of several representative pro- and anti-myogenic genes in the breast muscle were also measured by real-time PCR.ResultsResults showed that both absolute and relative weights of the breast muscle were in linear positive correlations with the body weight of broilers (P < 0.001). Fast-growing broilers had higher concentrations of IGF-1 than slow-growing broilers (P < 0.05) in both the serum and breast muscle. The serum concentration of T3 was significantly higher in fast-growing birds than in slow-growing birds (P < 0.05). However, no difference was observed in growth hormone or T4 concentration among three groups of birds. Additionally, a decreased expression of an anti-myogenic gene (myostatin) and increased expressions of pro-myogenic genes such as myogenic differentiation factor 1, myogenin, muscle regulatory factor 4, myogenic factor 5, IGF-1, and myocyte enhancer factor 2B, C, and D were observed in fast-growing broilers (P < 0.05), relative to slow-growing broilers.ConclusionsCollectively, these findings suggested that the growth rate is linked to the hormone and myogenic gene expression levels in broiler chickens. Some of these parameters such as serum concentrations of IGF-1 and T3 could be employed to breed for enhanced growth.

DNA damage induced apoptosis suppressor (DDIAS) is upregulated via ERK5/MEF2B signaling and promotes β-catenin-mediated invasion

DNA damage induced apoptosis suppressor (DDIAS) is an anti-apoptotic protein that promotes cancer cell survival. We previously reported that DDIAS is transcriptionally activated by nuclear factor of activated T cells 2 (NFATc1). However, the upstream regulation of DDIAS expression by growth factors has not been studied. Here, we demonstrate that DDIAS expression is induced by extracellular signal-regulated kinase 5 (ERK5) and myocyte enhancer factor 2B (MEF2B) in response to epidermal growth factor (EGF) and that it positively regulates β-catenin signaling in HeLa cells. The genetic or pharmacological inhibition of ERK5 suppressed DDIAS induction following EGF exposure and the overexpression of constitutively active MEK5 (CA-MEK5) enhanced DDIAS expression. In chromatin immunoprecipitation assays, MEF2B, a downstream target of ERK5, exhibited sequence-specific binding to a MEF2 binding site in the DDIAS promoter following treatment with EGF. The overexpression of MEF2B increased the EGF-mediated induction of DDIAS expression, whereas the knockdown of MEF2B impaired this effect. Furthermore, DDIAS promoted invasion by increasing β-catenin expression at the post-translational level in response to EGF, suggesting that DDIAS plays a crucial role in the metastasis of cancer cells by regulating β-catenin expression. It is unlikely that MEF2B and NFATc1 cooperatively regulate DDIAS transcription in response to EGF. Collectively, EGF activates the ERK5/MEF2 pathway, which in turn induces DDIAS expression to promote cancer cell invasion by activating β-catenin target genes.

Methionine improves breast muscle growth and alters myogenic gene expression in broilers

To investigate the mechanism underlying the regulatory effect of Met on broiler growth, the growth performance, organ development, serum profile, myogenic gene expression, and methylation of myostatin gene exon 1 region in response to dietary Met status were evaluated. A total of 192 one-day-old Arbor Acres broiler chicks were housed in 3-layer cages in a temperature-controlled room with continuous lighting. The temperature of the room was maintained at 32 to 34°C for the first 3 d and then reduced by 2 to 3°C per week to a final temperature of 20°C. Cages were randomly allocated to 2 dietary treatments with 6 replicate cages (8 males and 8 females/cage) per treatment. Control starter and finisher diets contained 0.50 and 0.43% Met, respectively. Corresponding values for a +Met treatment were 0.60 and 0.53% Met, respectively. The birds receiving the +Met diets had a greater (P < 0.05) G:F throughout the experiment. The +Met diets increased (P < 0.05) the relative weight of breast muscle and the concentrations of uric acid and triglyceride in serum at 42 d of age, whereas other serum measurements were not affected by treatments. Increased myogenic factor 5 (Myf5) and myocyte enhancer factor 2B (MEF2B) and decreased myostatin mRNA expression were observed in broilers fed the +Met diets (P < 0.05). However, methylation of myostatin gene exon 1 region was not different between groups. In conclusion, broilers fed the +Met diets increased breast muscle growth that was reflected in the expected expression of myostatin, Myf5, and MEF2B genes.

The AP-1 site is essential for the promoter activity of NOX1/NADPH oxidase, a vascular superoxide-producing enzyme: Possible involvement of the ERK1/2-JunB pathway

NADPH oxidase is a major source of the superoxide produced in cardiovascular tissues. The expression of NOX1, a catalytic subunit of NADPH oxidase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F 2α, and platelet-derived growth factor (PDGF). It was reported previously that the inducible expression of NOX1 is governed by the activating transcription factor-1 (ATF-1)-myocyte enhancer factor 2B (MEF2B) cascade downstream of phosphoinositide 3 (PI3) kinase. It was also reported that extracellular signal-regulated kinase (ERK) 1/2 is involved in the expression of NOX1. To further clarify the factors involved in NOX1 induction downstream of ERK1/2, the promoter region of the NOX1 gene was analyzed. A consensus activator protein-1 (AP-1) site was found at −98/−92 in the 5′-flanking region of the rat NOX1 gene. The introduction of mutations at this site abolished PGF 2α-induced transcriptional activation in a luciferase assay. Electrophoresis mobility shift assays demonstrated that PGF 2α and PDGF augmented the binding of JunB to this sequence. PD98059, an inhibitor of MAPK/ERK kinase, suppressed the expression of JunB induced by PGF 2α or PDGF. These results suggest that the ERK1/2-JunB pathway is a key regulator of the inducible expression of the NOX1 gene in vascular smooth muscle cells.

Myocyte enhancer factor 2B is involved in the inducible expression of NOX1/NADPH oxidase, a vascular superoxide‐producing enzyme

NADPH oxidase is a major source of the superoxide produced in cardiovascular tissues. Expression of NOX1, a catalytic subunit of NADPH oxidase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F(2alpha) and platelet-derived growth factor (PDGF). To clarify the molecular basis of this transcriptional activation, we delineated the promoter region of the NOX1 gene. RT-PCR and 5'-rapid amplification of cDNA ends-based analyses revealed a novel 5'-terminal exon of the rat NOX1 gene located approximately 28 kb upstream of the exon containing the start codon. Both PGF(2alpha) and PDGF enhanced the transcriptional activity of the - 3.6 kb 5'-flanking region of the NOX1 gene in A7r5 cells, a rat vascular smooth muscle cell line. A PGF(2alpha)-response element was located between -146 and -125 in the 5'-flanking region containing a consensus binding site for myocyte enhancer factor 2 (MEF2), to which binding of MEF2 was augmented by PGF(2alpha). Gene silencing of MEF2B by RNA interference significantly suppressed the expression of NOX1, while silencing of activating transcription factor (ATF)-1, previously implicated in up-regulation of NOX1, abolished the PGF(2alpha)- or PDGF-induced expression of MEF2B. These results indicate that superoxide production in vascular smooth muscle cells is regulated by the ATF-1-MEF2B cascade by induction of the expression of the NOX1 gene.

Myocyte Enhancer Factors-2B and -2C Are Required for Adhesion Related Kinase Repression of Neuronal Gonadotropin Releasing Hormone Gene Expression

Synthesis of the hypothalamic peptide, gonadotropin releasing hormone (GnRH), is paramount for reproductive function. GnRH neurons originate in the olfactory region and migrate into the forebrain during development. We recently implicated adhesion related kinase (Ark) in GnRH neuron development based on its differential expression in two GnRH producing cell lines, GT1-7 and Gn10. The Ark membrane receptor encodes an extracellular domain resembling cell adhesion molecules and an intracellular tyrosine kinase. Ark is expressed in Gn10 cells derived from migrating GnRH neurons but not GT1-7 cells of the post-migratory phenotype. Here, we show that Ark and GnRH transcripts are colocalized in the cribriform plate at midgestation, suggesting that Ark is expressed in migrating GnRH neurons in vivo. Furthermore, we have identified the GnRH gene as a downstream target of Ark signaling. Ark inhibits GnRH gene expression in GnRH neuronal cells via the coordinated binding of myocyte enhancer factor-2B and -2C (MEF-2B and -2C) and a putative homeoprotein within the proximal rat GnRH promoter. Given that MEF-2 proteins are widely expressed in the brain, these studies provide further evidence for MEF-2 action during neuronal development. Moreover, our studies elucidate a potential role for Ark in regulating GnRH gene expression during GnRH neuronal migration.