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Objective: This study aimed to clarify the association of MAFK polymorphisms (rs4268033, rs3735656, and rs10226620) with the degree of gastric mucosal atrophy and CDKN2A CpG methylation status. Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Results: Either rs3735656 or rs10226620, located in the 3'-UTR of MAFK , was significantly associated with the severity of gastric mucosal atrophy using a dominant genetic model (odds ratio [OR], 2.10; p  = 0.0012, and OR, 1.98; p  = 0.0027, respectively). However, using a recessive genetic model, no significant association was found between three polymorphisms and gastric mucosal atrophy. The serum pepsinogen I/II ratio was significantly lower in subjects with minor alleles of rs3735656 and rs10226620 than in subjects with the wild homozygous allele ( p  = 0.018 and 0.013, respectively). In a subgroup including 400 of the 491 subjects, the CpG of p14 ARF and p16 INK4a were methylated in 132 and 112 subjects, respectively. Fifty subjects had both CpG methylations and 206 subjects had neither methylation. When comparing the groups with both and neither methylations, there were no significant associations between three polymorphisms and CDKN2A methylation using a dominant genetic model. However, all polymorphisms investigated in this study (rs4268033, rs3735656, and rs10226620) were significantly associated with CDKN2A methylation in a recessive genetic model (OR, 3.58; p  = 0.0071, OR, 4.49; p  = 0.0004, and OR, 3.45; p  = 0.0027, respectively). Conclusions: Our results indicate that carrying the minor allele of the MAFK polymorphisms, particularly when they are located in the 3'-UTR, has a high risk for the severity of gastric mucosal atrophy; furthermore, CDKN2A CpG methylation may develop in subjects with homozygous minor allele of these polymorphisms.

The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors, and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific transcription factor and its expression is augmented by interferon-γ. Therefore, it serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. Ahigh-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-Seq analysis revealed three MafK binding regions (-25 kb, -20 kb, and IRF8 6th intron) within the IRF8 locus. These MafK binding sites are sufficient to repress a reporter gene when cloned in genome-integrated lentiviral reporter constructs in only expression-restrictive cells. Conversely, plasmid-based constructs do not demonstrate such repressive effect. These results highlight the role of these MafK binding sites in mediating repressed chromatin assembly. Finally, a more thorough genomic analysis was performed, using CRISPR-Cas9 to delete MafK-int6 binding region in IRF8 expression-restrictive cells. Deleted clones exhibited an accessible chromatin conformation within the IRF8 locus that was accompanied by a significant increase in basal expression of IRF8 that was further induced by interferon-γ. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression in a celltype-specific manner., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Reactive metabolites of anti-tuberculosis (anti-TB) drugs can result in excessive reactive oxygen species (ROS), which are responsible for drug-induced liver injury. The nuclear factor erythroid 2-related factor 2 (Nrf2) - antioxidant response elements (ARE) (Nrf2-ARE) signaling pathway plays a crucial role in protecting liver cells from ROS, inducing enzymes such as phase II metabolizing enzymes and antioxidant enzymes. Based on a Chinese anti-TB treatment cohort, a nested case-control study was performed to explore the association between 13 tag single-nucleotide polymorphisms (tagSNPs) in the NRF2, KEAP1, MAFF, MAFK genes in Nrf2-ARE signaling pathway and the risk of anti-TB drug-induced liver injury (ATLI) in 314 cases and 628 controls. Conditional logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) after adjusting weight and usage of hepatoprotectant. Patients carrying the TC genotype at rs4243387 or haplotype C-C (rs2001350-rs6726395) in NRF2 were at an increased risk of ATLI (adjusted OR = 1.362, 95% CI: 1.017-1.824, P = 0.038; adjusted OR = 2.503, 95% CI: 1.273-4.921, P = 0.008, respectively), whereas patients carrying TC genotype at rs2267373 or haplotype C-G-C (rs2267373-rs4444637-rs4821767) in MAFF were at a reduced risk of ATLI (adjusted OR = 0.712, 95% CI: 0.532-0.953, P = 0.022; adjusted OR = 0.753, 95% CI: 0.587-0.965, P = 0.025, respectively). Subgroup analysis also detected a significant association between multiple tagSNPs (rs4821767 and rs4444637 in MAFF, rs4720833 in MAFK) and specific clinical patterns of liver injury under different genetic models. This study shows that genetic polymorphisms of NRF2, MAFF and MAFK may contribute to the susceptibility to ATLI in the Chinese anti-TB treatment population.

Aim: To investigate whether single nucleotide polymorphisms in maf protein K ( MAFK ), which encodes the MAFK , lead to increased susceptibility to ulcerative colitis in the Japanese population., Methods: This case control study examined the associations between MAFK single nucleotide polymorphisms (rs4268033 G>A, rs3735656 T>C and rs10226620 C>T) and ulcerative colitis susceptibility in 174 patients with ulcerative colitis (UC) cases, and 748 subjects without no lower abdominal symptoms, diarrhea or hematochezia (controls). In addition, as the second controls, we set 360 subjects, who have an irregular bowel movement without abnormal lower endoscopic findings (IBM controls)., Results: The genotype frequency of rs4268033 AA and allelic frequency of the rs4268033A allele were significantly higher in the UC cases than in both controls ( P = 0.0005 and < 0.0001, P = 0.015 and 0.0027 vs controls and IBM controls, respectively). Logistic regression analysis after adjustment for age and gender showed that the rs4268033 AA and rs3735656 CC genotypes were significantly associated with susceptibility to UC development (OR = 2.63, 95%CI: 1.61-4.30, P = 0.0001 and OR = 1.81; 95%CI: 1.12-2.94, P = 0.015, respectively). Similar findings were observed by the comparison with IBM controls. In addition, the rs4268033 AA genotype was significantly associated with all phenotypes of UC except early onset. There was no significant association between rs10226620 and ulcerative colitis., Conclusion: Our results provide the first evidence that MAFK genetic polymorphisms are significantly associated with susceptibility to UC development. In particular, rs4268033 is closely associated with an increased risk for the development of UC., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons and lacks an effective treatment. The disease pathogenesis has not been clarified at present. Pathological transactive response DNA-binding protein 43 (TDP-43) plays an important role in the pathogenesis of ALS. Nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is found in a mutant TDP-43 transgenic cell model, but its downstream antioxidant enzyme expression is decreased. To elucidate the specific mechanism of Nrf2/ARE (antioxidant responsive element) signaling dysfunction, we constructed an ALS cell model with human mutant TDP-43 using the NSC-34 cell line to evaluate the impact of the TDP-43 mutation on the Nrf2/ARE pathway. We found the nuclear translocation of Nrf2, but the expression of total Nrf2, cytoplasmic Nrf2, and downstream phase II detoxifying enzyme (NQO1) was decreased in NSC-34 cells transfected with the TDP-43-M337V plasmid. Besides, TDP-43-M337V plasmid-transfected NSC-34 cells were rounded with reduced neurites, shortened axons, increased levels of intracellular lipid peroxidation products, and decreased viability, which suggests that the TDP-43-M337V plasmid weakened the antioxidant capacity of NSC-34 cells and increased their susceptibility to oxidative damage. We further showed that expression of the MafK protein and the Jun dimerization protein 2 (JDP2) was reduced in TDP-43-M337V plasmid-transfected NSC-34 cells, which might cause accumulation of Nrf2 in nuclei but a decrease in NQO1 expression. Taken together, our results confirmed that TDP-43-M337V impaired the Nrf2/ARE pathway by reducing the expression of MafK and JDP2 proteins, and provided information for further research on the molecular mechanisms of TDP-43-M337V in ALS.

Triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat. For example, the transforming growth factor-β (TGF-β) pathway is implicated in TNBC progression and metastasis, but its opposing role in tumor suppression in healthy tissues and early-stage lesions makes it a challenging target. Therefore, additional molecular characterization of TNBC may lead to improved patient prognosis by informing the development and optimum use of targeted therapies. We found that musculoaponeurotic fibrosarcoma (MAF) oncogene family protein K (MAFK), a member of the small MAF family of transcription factors that are induced by the TGF-β pathway, was abundant in human TNBC and aggressive mouse mammary tumor cell lines. MAFK promoted tumorigenic growth and metastasis by 4T1 cells when implanted subcutaneously in mice. Overexpression of MAFK in mouse breast epithelial NMuMG cells induced epithelial-mesenchymal transition (EMT) phenotypes and promoted tumor formation and invasion in mice. MAFK induced the expression of the gene encoding the transmembrane glycoprotein nmb (GPNMB). Similar to MAFK, GPNMB overexpression in NMuMG cells induced EMT, tumor formation, and invasion, in mice, whereas knockdown of MAFK in tumor cells before implantation suppressed tumor growth and progression. MAFK and GPNMB expression correlated with poor prognosis in TNBC patients. These findings suggest that MAFK and its target gene GPNMB play important roles in the malignant progression of TNBC cells, offering potentially new therapeutic targets for TNBC patients., (Copyright © 2017, American Association for the Advancement of Science.)

BACKGROUND The aim of this study was to compare the transcriptome between impaired fasting glucose (IFG) and type 2 diabetes mellitus (T2DM), and further research their molecular mechanisms. MATERIAL AND METHODS The original microarray GSE21321, including miRNA and mRNA expression profiles, was downloaded from the GEO database. Data preprocessing was processed by limma package, and differentially expressed genes (DGs) and miRNA (DMs) were screened. Then, the regulatory relationships among miRNA, TF, and genes were screened and the regulatory network was constructed. Finally, DAVID was used for KEGG enrichment analysis. RESULTS There were 11 upregulated IFG-related DMs and five upregulated T2DM-related DMs. Three of the DMs overlapped. In addition, there were eight downregulated IFG-related DMs and two downregulated T2DM-related DMs. Only one downregulated DM overlapped. Similarly, there were 264 upregulated IFG-related DGs and 331 upregulated T2DM-related DGs; and 196 overlapping genes were obtained. In addition, there were 400 downregulated IFG-related DMs and 568 downregulated T2DM-related DMs. A total of 326 downregulated DMs were overlapped. The overlapped DGs were enriched in various pathways, including hematopoietic cell lineage, Fc gamma R-mediated phagocytosis, and MAPK signaling pathway. TAF1 (upregulated gene) and MAFK (downregulated gene) were hub nodes both in IFG- and T2DM-related miRNA-TF-gene regulatory network. In addition, miRNAs, including hsa-miR-29a, hsa-miR-192, and hsa-miR-144, were upregulated hub nodes in the two regulatory networks. CONCLUSIONS Genes including TAF1 and MAFK, and miRNAs including hsa-miR-29a, hsa-miR-192, and hsa-miR-144 might be potential target genes and important miRNAs for IFG and T2DM., Competing Interests: Conflict of interests All authors declare that they have no conflict of interests to state.

RNA-binding proteins provide a new layer of posttranscriptional regulation of RNA during cancer progression. We identified RNA-binding motif protein 47 (RBM47) as a target gene of transforming growth factor (TGF)-β in mammary gland epithelial cells (NMuMG cells) that have undergone the epithelial-to-mesenchymal transition. TGF-β repressed RBM47 expression in NMuMG cells and lung cancer cell lines. Expression of RBM47 correlated with good prognosis in patients with lung, breast and gastric cancer. RBM47 suppressed the expression of cell metabolism-related genes, which were the direct targets of nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2). RBM47 bound to KEAP1 and Cullin 3 mRNAs, and knockdown of RBM47 inhibited their protein expression, which led to enhanced binding of Nrf2 to target genomic regions. Knockdown of RBM47 also enhanced the expression of some Nrf2 activators, p21/CDKN1A and MafK induced by TGF-β. Both mitochondrial respiration rates and the side population cells in lung cancer cells increased in the absence of RBM47. Our findings, together with the enhanced tumor formation and metastasis of xenografted mice by knockdown of the RBM47 expression, suggested tumor-suppressive roles for RBM47 through the inhibition of Nrf2 activity.

The small Maf proteins (sMafs) are basic region leucine zipper (bZIP)-type transcription factors. The basic region of the Maf family is unique among the bZIP factors, and it contributes to the distinct DNA-binding mode of this class of proteins. MafF, MafG and MafK are the three vertebrate sMafs, and no functional differences have been observed among them in terms of their bZIP structures. sMafs form homodimers by themselves, and they form heterodimers with cap 'n' collar (CNC) proteins (p45 NF-E2, Nrf1, Nrf2, and Nrf3) and also with Bach proteins (Bach1 and Bach2). Because CNC and Bach proteins cannot bind to DNA as monomers, sMafs are indispensable partners that are required by CNC and Bach proteins to exert their functions. sMafs lack the transcriptional activation domain; hence, their homodimers act as transcriptional repressors. In contrast, sMafs participate in transcriptional activation or repression depending on their heterodimeric partner molecules and context. Mouse genetic analyses have revealed that various biological pathways are under the regulation of CNC-sMaf heterodimers. In this review, we summarize the history and current progress of sMaf studies in relation to their partners., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Osteosarcoma is one of the most common primary bone tumors in children and young adults. In this study, we investigated the role of musculoaponeurotic fibrosarcoma oncogene homolog K (MAFK) in osteosarcoma cell proliferation in vitro and the possible pathways that contributed to MAFK-related osteosarcoma development. We first reported that MAFK was expressed at low levels in an osteosarcoma cell line. Furthermore, a significant correlation between MAFK and the Wnt signaling pathway was observed in osteosarcoma by using a gene microarray assay. We found that expression of MAFK could be induced by Wnt1 in a dose-dependent manner. Furthermore, Wnt1-induced MAFK expression caused a significant increase of cell viability, whereas a Wnt pathway inhibitor, IWR-1-endo, abolished Wnt1-induced effects on MAFK. Finally, cell cycle analysis showed that enhanced cell proliferation might be attributed to re-distribution of the cell cycle. Together, our results suggested that Wnt1-induced MAFK expression promoted cell proliferation in MG63 cells, and that the role of MAFK in osteosarcoma might be closely linked to the Wnt signaling pathway.

Although majority of the genes linked to early-onset cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. We report on small Maf transcription factors Mafg and Mafk as regulators of several non-crystallin human cataract-associated genes in fiber cells and establish their significance to this disease. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify Mafg and its co-regulators in the lens, and generated various null-allelic combinations of Mafg:Mafk mouse mutants for phenotypic and molecular analysis. By age 4 months, Mafg-/-:Mafk+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of Mafg-/-:Mafk+/- mouse lens reveals severely disorganized fiber cells, while microarray-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of Mafg-/-:Mafk+/- lens-DRGs with (1) binding motifs and genomic targets of small Mafs and their regulatory partners, (2) iSyTE lens expression data, and (3) interactions between DRGs in the String database, unravel a detailed small Maf regulatory network in the lens, several nodes of which are linked to cataract. This approach identifies 36 high-priority candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes oxidative stress and misregulation of sterol synthesis. These data identify Mafg and Mafk as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to human cataract.

Cystic fibrosis transmembrane conductance regulator gene (CFTR) expression in human airway epithelial cells involves the recruitment of distal cis-regulatory elements, which are associated with airway-selective DNase hypersensitive sites at -44 kb and -35 kb from the gene. The -35-kb site encompasses an enhancer that is regulated by the immune mediators interferon regulatory factor 1 and 2 and by nuclear factor Y. Here we investigate the -44-kb element, which also has enhancer activity in vitro in airway epithelial cells but is inactive in intestinal epithelial cells. This site contains an antioxidant response element (ARE) that plays a critical role in its function in airway cell lines and primary human bronchial epithelial cells. The natural antioxidant sulforaphane (SFN) induces nuclear translocation of nuclear factor, erythroid 2-like 2 (Nrf2), a transcription factor that regulates genes with AREs in their promoters, many of which are involved in response to injury. Under normal conditions, the -44-kb ARE is occupied by the repressor BTB and CNC homology 1, basic leucine zipper transcription factor (Bach1), and v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) heterodimers. After 2 hours of SFN treatment, Nrf2 displaces these repressive factors and activates CFTR expression. Site-directed mutagenesis shows that both the ARE and an adjacent NF-κB binding site are required for activation of the -44-kb element in airway epithelial cells. Moreover, this element is functionally linked to the -35-kb enhancer in modulating CFTR expression in response to environmental stresses in the airway.

Oxidative stress and inflammation are common to many pathological conditions. Defense mechanisms protect cells from oxidative stress, but can become over-activated following injury and inflammation. NF-κB and Nrf2 transcription factors regulate proinflammatory and antioxidant gene expression, respectively. Studies have shown that many natural dietary compounds regulate NF-κB and Nrf2, preventing inflammation and oxidative stress. Here, we report major compounds of Prunella vulgaris var. lilacina such as rosmarinic acid, oleanolic acid, ursolic acid and caffeic acid as a potential therapeutic for oxidative stress and inflammation. The major compounds exhibited high anti-inflammatory activity, inhibiting NO, PGE2 production, NF-κB expression and activating Nrf2 expression. In addition, we examined the effect of major compounds on MafK expression. Among the compounds, oleanolic acid significantly decreased MafK expression and MafK-mediated p65 acetylation. These findings suggest that oleanolic acid as NF-κB inhibitors can potentially be used in therapeutic applications for the treatment of oxidative stressnduced diseases.

Chronic myeloid leukemia (CML) is one of the most frequent hematological neoplasia worldwide. The abnormal accumulation of reactive oxygen species may be an important factor in CML development. The transcription factor NRF2 can regulate the transcription of a battery of antioxidant and detoxificant genes after heterodimerizing with small-Maf proteins. Although the participation of NRF2 in the development of chronic degenerative diseases has been thoroughly studied, the role of small-Maf genes has not been documented. We have identified polymorphisms in the three MAF genes (F, G and K) and assessed their association with CML. Over 266 subjects with CML and 399 unrelated healthy donors have been studied. After sequencing each MAF gene by Sanger technology, we found 17 variants in MAFF gene, eight in MAFG and seven in MAFK. In the case-control study, the homozygote genotype CC for the rs9610915 SNP of MAFF was significantly associated with CML. The frequency of the ACC haplotype from MAFK was significantly lower than controls. After stratification by gender, the ACC and GTG haplotypes were associated only with males with CML. These novel data suggest an association between MAFF and MAFG and the development of CML., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Reactive oxygen species, produced by oxidative stress, initiate and promote many metabolic diseases through activation/suppression of redox-sensitive transcription factors. NF-κB and Nrf2 are important regulators of oxidation resistance and contribute to the pathogenesis of many diseases. We identified MafK, a novel transcriptional regulator that modulates NF-κB activity. MafK knockdown reduced NF-κB activation, whereas MafK overexpression enhanced NF-κB function. MafK mediated p65 acetylation by CBP upon LPS stimulation, thereby facilitating recruitment of p65 to NF-κB promoters such as IL-8 and TNFα. Consistent with these results, MafK-depleted mice showed prolonged survival with a reduced hepatic inflammatory response after LPS and D-GalN injection. Thus, our findings reveal a novel mechanism by which MafK controls NF-κB activity via CBP-mediated p65 acetylation.

Oxidative stress and reactive oxygen species (ROS) are associated with diseases such as cancer, cardiovascular complications, inflammation and neurodegeneration. Cellular defense systems must work constantly to control ROS levels and to prevent their accumulation. We report here that the Jun dimerization protein 2 (JDP2) has a critical role as a cofactor for transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and small Maf protein family K (MafK) in the regulation of the antioxidant-responsive element (ARE) and production of ROS. Chromatin immunoprecipitation-quantitative PCR (qPCR), electrophoresis mobility shift and ARE-driven reporter assays were carried out to examine the role of JDP2 in ROS production. JDP2 bound directly to the ARE core sequence, associated with Nrf2 and MafK (Nrf2-MafK) via basic leucine zipper domains, and increased DNA-binding activity of the Nrf2-MafK complex to the ARE and the transcription of ARE-dependent genes. In mouse embryonic fibroblasts from Jdp2-knockout (Jdp2 KO) mice, the coordinate transcriptional activation of several ARE-containing genes and the ability of Nrf2 to activate expression of target genes were impaired. Moreover, intracellular accumulation of ROS and increased thickness of the epidermis were detected in Jdp2 KO mice in response to oxidative stress-inducing reagents. These data suggest that JDP2 is required to protect against intracellular oxidation, ROS activation and DNA oxidation. qPCR demonstrated that several Nrf2 target genes such as heme oxygenase-1, glutamate-cysteine ligase catalytic and modifier subunits, the notch receptor ligand jagged 1 and NAD(P)H dehydrogenase quinone 1 are also dependent on JDP2 for full expression. Taken together, these results suggest that JDP2 is an integral component of the Nrf2-MafK complex and that it modulates antioxidant and detoxification programs by acting via the ARE.

Transforming growth factor-β (TGF-β) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-β suppresses the transcriptional activation of the heme oxygenase-1 (HO-1) gene, which is implicated in protection against oxidative injury and lung carcinogenesis. HO-1 is a target of the oxidative stress-responsive transcription factor Nrf2. TGF-β did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-β induced expression of transcription factors MafK and Bach1. Enhanced expression of either MafK or Bach1 was enough to suppress the electrophile-inducible expression of HO-1 even in the presence of accumulated Nrf2 in the nucleus. Knockdown of MafK and Bach1 by siRNA abolished TGF-β-dependent suppression of HO-1. Furthermore, chromatin immunoprecipitation assays revealed that Nrf2 substitutes for Bach1 at the antioxidant response elements (E1 and E2), which are responsible for the induction of HO-1 in response to oxidative stress. On the other hand, pretreatment with TGF-β suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-β is activated after tissue injury and in the process of cancer development, these findings suggest a novel mechanism by which damaged tissue becomes vulnerable to oxidative stress and xenobiotics.

Tuberculosis (TB) treatment can cause serious sequelae including adverse effects such as anti-TB drug-induced hepatotoxicity (ATDH). We performed a candidate gene-based association study between single nucleotide polymorphisms (SNPs) in 10 genes in the antioxidant pathway and ATDH susceptibility. The subjects comprised 100 Japanese patients with pulmonary TB who received a treatment regimen including isoniazid and rifampicin. Out of them, 18 patients had ATDH. Thirty-four tag SNPs in 10 genes were analyzed by PCR-restriction fragment length polymorphism or PCR-direct DNA sequencing. The frequencies of alleles and genotypes between patients with and without ATDH were compared in three different genetic models. Statistical analyses revealed that a C/C genotype at rs11080344 in NOS2A, a C/C genotype at rs2070401 in BACH1, and a G/A or A/A genotype at rs4720833 in MAFK independently conferred ATDH susceptibility. Remarkably, the association of the latter two tag SNPs with ATDH susceptibility was highly statistically significant (P = 0.0006) with an odds ratio of 9.730. This study is the first report to demonstrate that NOS2A, BACH1, and MAFK appear to be genetic determinants of ATDH in Japanese patients with TB. Furthermore, a combination of BACH1 and MAFK polymorphisms may be useful as new biomarkers to identify high-risk Japanese TB patients for ATDH., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.

Many models of diabetic nephropathy have been reported. However, it is rare that the characteristic findings of severe human diabetic nephropathy, such as diffuse, nodular, and exudative lesions, are all detected in one model mouse. Previously, we reported that MAFA-deficient and beta cell-specific MAFK-overexpressing hybrid transgenic (Mafa(-/-)Mafk (+)) mice develop diabetes mellitus and, after uninephrectomy, demonstrate these characteristic lesions. In this study, we administered TCV-116 (candesartan cilexetil) to Mafa(-/-)Mafk (+) mice after uninephrectomy and examined whether TCV-116 ameliorated the diabetic nephropathy. We also evaluated the utility of these mice as a model for developing treatments for diabetic nephropathy. We performed uninephrectomy of the Mafa(-/-)Mafk (+) mice at 8 weeks old. We then divided these mice into two groups as follows: 1) an untreated group and 2) a group treated with TCV-116 at 5 µg/g/day from 10 to 20 weeks. TCV-116 treatment did not affect serum glucose levels. However, in the treated group, urinary protein excretion, mesangial matrix expansion, enlargement of the kidney, and glomerular surface area were all improved relative to untreated mice. Oxidative stress is known to be increased in diabetic nephropathy and to be suppressed by TCV-116. The urinary level of 8-OHdG, an oxidative stress marker, at 20 weeks was lower in the TCV-116-treated group than in the untreated group. From these results, we concluded that the Mafa(-/-)Mafk (+) mouse is a useful model to analyze diabetic nephropathy and a useful tool for the development of new drugs to treat diabetic nephropathy.

Background: Akt regulates various cellular processes, including cell growth, survival, and metabolism. Recently, Akt's role in neurite outgrowth has also emerged. We thus aimed to identify neuronal function-related genes that are regulated by Akt., Methods: We performed suppression subtractive hybridization on two previously established PC12 sublines, one of which overexpresses the wild-type (WT) form and the other, the dominant-negative (DN) form of Akt. These sublines respond differently to NGF's neuronal differentiation effect., Results: A variety of genes was identified and could be classified into several functional groups, one of which was developmental processes. Two genes involved in neuronal differentiation and function were found in this group. v-Maf musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) induces the neuronal differentiation of PC12 cells and immature telencephalon neurons, and synaptotagmin I (SytI) is essential for neurotransmitter release. Another gene, syntenin-1 (Syn-1) was also recognized in the same functional group into which MafK and SytI were classified. Syn-1 has been reported to promote the formation of membrane varicosities in neurons. Quantitative reverse transcription polymerase chain reaction analyses show that the transcript levels of these three genes were lower in PC12 (WT-Akt) cells than in parental PC12 and PC12 (DN-Akt) cells. Furthermore, treatment of PC12 (WT-Akt) cells with an Akt inhibitor resulted in the increase of the expression of these genes and the improvement of neurite outgrowth. These results indicate that dominant-negative or pharmacological inhibition of Akt increases the expression of MafK, SytI, and Syn-1 genes. Using lentiviral shRNA to knock down endogenous Syn-1 expression, we demonstrated that Syn-1 promotes an increase in the numbers of neurites and branches., Conclusions: Taken together, these results indicate that Akt negatively regulates the expression of MafK, SytI, and Syn-1 genes that all participate in regulating neuronal integrity in some way or another.

Transcription factor MafA is a key molecule in insulin secretion and the development of pancreatic islets. Previously, we demonstrated that some of the MafA-deficient mice develop overt diabetes mellitus, and the phenotype of these mice seems to be mild probably because of redundant functions of other Maf proteins. In this study, we generated hybrid transgenic mice that were MafA-deficient and also over-expressed MafK specifically in beta cells (MafA(-/-)MafK(+)). MafA(-/-)MafK(+) mice developed severe overt diabetes mellitus within 5weeks old, and showed higher levels of proteinuria and serum creatinine. Histological analysis revealed that embryonic development of beta cells in the MafA(-/-)MafK(+) mice was significantly suppressed and the reduced number of beta cells was responsible for the early onset of diabetes. Furthermore, after uninephrectomy, these mice demonstrated three characteristics of human diabetic nephropathy: diffuse, nodular, and exudative lesions. MafA(-/-)MafK(+) mice might be a useful model for the analysis of human diabetic nephropathy.

HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.

MafA is a member of large Maf transcription factors and activates the insulin gene in pancreatic beta-cells. Other large Maf transcription factors, MafB and c-Maf, also express and activate insulin transcription in beta-cells. However, the functional relationship between MafA and other Maf proteins in beta-cells has not been established. In order to suppress the function of large Maf proteins, we generated transgenic mice overexpressing MafK, which act as dominant negative protein in pancreatic beta-cells. These mice showed hyperglycemia at a young age due to impairment of glucose-stimulated insulin secretion. Although the transgenic mice showed an abnormal response in the glucose tolerance test, hyperglycemia was restored in adulthood. Histological analysis revealed islet hypertrophy in adult transgenic mice. Finally, an electrophoretic gel mobility shift assay showed that the DNA-binding activity of endogenous MafA was significantly increased in the MafK transgenic mice. These results indicated that MafA may have relevance to compensatory response.

A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity.

Placental glutathione S-transferase (GST-P), a member of glutathione S-transferase, is known for its specific expression during rat hepatocarcinogenesis and has been used as a reliable tumor marker for experimental rat hepatocarcinogenesis. To explain the molecular mechanism underlying its specific expression concomitant with the malignant transformation, we have analyzed the regulatory element of the GST-P gene and the transcription factor that binds to this element. From the extensive analyses by the establishment of the transgenic rat lines having various regions of GST-P gene, we could identify the GPE1 as an essential enhancer element for specific GST-P expression. Next, we examined the transcription factor that binds and activates the GPE1, specifically in the early stage of hepatocarcinogenesis and in the hepatoma. Electrophoresis gel mobility shift assay, reporter transfection analysis, and the chromatin immunoprecipitation analysis indicate that the Nrf2/MafK heterodimer binds and activates GPE1 element in preneoplastic lesions and hepatomas but not in the normal liver cells. In this chapter, we describe details of the transgenic rat analyses and the identification of a factor responsible for the specific expression of the GST-P gene and discuss a possible molecular scenario for malignant transformation and tumor marker gene expression.