MAFB in Macrophages Regulates Prostaglandin E2-Mediated Lipid Mediator Class Switch through ALOX15 in Ischemic Acute Kidney Injury.

Monocytes and macrophages express the transcription factor MAFB (V-maf musculoaponeurotic fibrosarcoma oncogene homolog B) and protect against ischemic acute kidney injury (AKI). However, the mechanism through which MAFB alleviates AKI in macrophages remains unclear. In this study, we induced AKI in macrophage lineage-specific Mafb-deficient mice (C57BL/6J) using the ischemia-reperfusion injury model to analyze these mechanisms. Our results showed that MAFB regulates the expression of Alox15 (arachidonate 15-lipoxygenase) in macrophages during ischemic AKI. The expression of ALOX15 was significantly decreased at the mRNA and protein levels in macrophages that infiltrated the kidneys of macrophage-specific Mafb-deficient mice at 24 h after ischemia-reperfusion injury. ALOX15 promotes the resolution of inflammation under acute conditions by producing specialized proresolving mediators by oxidizing essential fatty acids. Therefore, MAFB in macrophages promotes the resolution of inflammation in ischemic AKI by regulating the expression of Alox15. Moreover, MAFB expression in macrophages is upregulated via the COX-2/PGE2/EP4 pathway in ischemic AKI. Our in vitro assay showed that MAFB regulates the expression of Alox15 under the COX-2/PGE2/EP4 pathway in macrophages. PGE2 mediates the lipid mediator (LM) class switch from inflammatory LMs to specialized proresolving mediators. Therefore, MAFB plays a key role in the PGE2-mediated LM class switch by regulating the expression of Alox15. Our study identified a previously unknown mechanism by which MAFB in macrophages alleviates ischemic AKI and provides new insights into regulating the LM class switch in acute inflammatory conditions.

Natto consumption suppresses atherosclerotic plaque progression in LDL receptor-deficient mice transplanted with iRFP-expressing hematopoietic cells.

Natto, known for its high vitamin K content, has been demonstrated to suppress atherosclerosis in large-scale clinical trials through a yet-unknown mechanism. In this study, we used a previously reported mouse model, transplanting the bone marrow of mice expressing infra-red fluorescent protein (iRFP) into LDLR-deficient mice, allowing unique and non-invasive observation of foam cells expressing iRFP in atherosclerotic lesions. Using 3 natto strains, we meticulously examined the effects of varying vitamin K levels on atherosclerosis in these mice. Notably, high vitamin K natto significantly reduced aortic staining and iRFP fluorescence, indicative of decreased atherosclerosis. Furthermore, mice administered natto showed changes in gut microbiota, including an increase in natto bacteria within the cecum, and a significant reduction in serum CCL2 expression. In experiments with LPS-stimulated macrophages, adding natto decreased CCL2 expression and increased anti-inflammatory cytokine IL-10 expression. This suggests that natto inhibits atherosclerosis through suppression of intestinal inflammation and reduced CCL2 expression in macrophages.

Analysis of Notch1 signaling in mammalian sperm development.

OBJECTIVE: A mammalian Delta-Notch signaling component, Notch1, has been suggested for its expression during the normal sperm development although its conditional deletion caused no apparent abnormalities. Since we established our original transgenic mouse system that enabled labeling of past and ongoing Notch1 signaling at a cellular level, we tried to validate that observation in vivo. Our transgenic mouse system used Cre/loxP system to induce tandem dsRed expression upon Notch1 signaling. RESULTS: To our surprise, we were unable to observe tandem dsRed expression in the seminiferous tubules where the sperms developed. In addition, tandem dsRed expression was lacking in the somatic cells of the next generation in our transgenic mouse system, suggesting that sperms received no Notch1 signaling during their development. To validate this result, we conducted re-analysis of four single-cell RNA-seq datasets from mouse and human testes and showed that Notch1 expression was little in the sperm cell lineage. Collectively, our results posed a question into the involvement of Notch1 in the normal sperm development although this observation may help the interpretation of the previous result that Notch1 conditional deletion caused no apparent abnormalities in murine spermatogenesis.

Notch1 signaling is limited in healthy mature kidneys in vivo.

OBJECTIVE: A Delta-Notch signaling component, Notch1, is involved in the normal development and multiple disorders of the kidney. Although the increase in Notch1 signaling is crucial to these pathogeneses, the basal signaling level in 'healthy' mature kidneys is still unclear. To address this question, we used an artificial Notch1 receptor fused with Gal4/UAS components in addition to the Cre/loxP system and fluorescent proteins in mice. This transgenic reporter mouse system enabled labeling of past and ongoing Notch1 signaling with tdsRed or Cre recombinase, respectively. RESULTS: We confirmed that our transgenic reporter mouse system mimicked the previously reported Notch1 signaling pattern. Using this successful system, we infrequently observed cells with ongoing Notch1 signaling only in Bowman's capsule and tubules. We consider that Notch1 activation in several lines of disease model mice was pathologically significant itself.

Transcription Factor MAFB as a Prognostic Biomarker for the Lung Adenocarcinoma.

MAFB is a basic leucine zipper (bZIP) transcription factor specifically expressed in macrophages. We have previously identified MAFB as a candidate marker for tumor-associated macrophages (TAMs) in human and mouse models. Here, we analyzed single-cell sequencing data of patients with lung adenocarcinoma obtained from the GEO database (GSE131907). Analyzed data showed that general macrophage marker CD68 and macrophage scavenger receptor 1 (CD204) were expressed in TAM and lung tissue macrophage clusters, while transcription factor MAFB was expressed specifically in TAM clusters. Clinical records of 120 patients with lung adenocarcinoma stage I (n = 57), II (n = 21), and III (n = 42) were retrieved from Tsukuba Human Tissue Biobank Center (THB) in the University of Tsukuba Hospital, Japan. Tumor tissues from these patients were extracted and stained with anti-human MAFB antibody, and then MAFB-positive cells relative to the tissue area (MAFB+ cells/tissue area) were morphometrically quantified. Our results indicated that higher numbers of MAFB+ cells significantly correlated to increased local lymph node metastasis (nodal involvement), high recurrence rate, poor pathological stage, increased lymphatic permeation, higher vascular invasion, and pleural infiltration. Moreover, increased amounts of MAFB+ cells were related to poor overall survival and disease-free survival, especially in smokers. These data indicate that MAFB may be a suitable prognostic biomarker for smoker lung cancer patients.

Identifying potential regulators of JAGGED1 expression in portal mesenchymal cells.

OBJECTIVE: Portal mesenchymal cells induce the epithelial differentiation of the bile ducts in the developing liver via one of the Delta-Notch signaling components, JAGGED1. Although this differential induction is crucial for normal liver physiology as its genetic disorder (Alagille syndrome) causes jaundice, the molecular mechanism behind JAGGED1 expression remains unknown. Here, we searched for upstream regulatory transcription factors of JAGGED1 using an integrated bioinformatics method. RESULTS: According to the DoRothEA database, which integrates multiple lines of evidence on the relationship between transcription factors and their downstream target genes, three transcription factors were predicted to be upstream of JAGGED1: SLUG, SOX2, and EGR1. Among these, SLUG and EGR1 were enriched in ACTA2-expressing portal mesenchymal cells in two previously reported human fetal liver single-cell RNA-seq datasets. JAGGED1-expressing portal mesenchymal cells tended to express SLUG rather than EGR1, supporting that SLUG induced JAGGED1 expression. Together with the higher confidentiality of SLUG (DoRothEA level A) over EGR1 (DoRothEA level D), we concluded that SLUG was one of the most important candidate transcription factors upstream of JAGGED1. These results add mechanistic insights into the developmental biology of how portal mesenchymal cells support biliary development in the liver.

Generation of a Gal4-dependent gene recombination and illuminating mouse.

Cell labeling technologies, including the Cre/loxP system, are powerful tools in developmental biology. Although the conventional Cre/loxP system has been extensively used to label the expression of specific genes, it is less frequently used for labeling protein-protein interactions owing to technical difficulties. In the present study, we generated a new Gal4-dependent transgenic reporter mouse line that expressed Cre recombinase and a near-infrared fluorescent protein, miRFP670. To examine whether this newly generated transgenic mouse line is applicable in labeling of protein-protein interaction, we used a previously reported transgenic mouse lines that express Notch1 receptor with its intracellular domain replaced with a yeast transcription factor, Gal4. Upon the binding of this artificial Notch1 receptor and endogenous Notch1 ligands, Gal4 would be cleaved from the cell membrane to induce expression of Cre recombinase and miRFP670. Indeed, we observed miRFP670 signal in the mouse embryos (embryonic day 14.5). In addition, we examined whether our Cre recombinase was functional by using another transgenic mouse line that express dsRed after Cre-mediated recombination. We observed dsRed signal in small intestine epithelial cells where Notch1 signal was suggested to be involved in the crypt stem cell maintenance, suggesting that our Cre recombinase was functional. As our newly generated mouse line required only the functioning of Gal4, it could be useful for labeling several types of molecular activities in vivo.

Mathematical analysis of the effect of portal vein cells on biliary epithelial cell differentiation through the Delta-Notch signaling pathway.

OBJECTIVE: The Delta-Notch signaling pathway induces fine-grained patterns of differentiation from initially homogeneous progenitor cells in many biological contexts, including Drosophila bristle formation, where mathematical modeling reportedly suggests the importance of production rate of the components of this signaling pathway. In contrast, the epithelial differentiation of bile ducts in the developing liver is unique in that it occurs around the portal vein cells, which express extremely high amounts of Delta ligands and act as a disturbance for the amount of Delta ligands in the field by affecting the expression levels of downstream target genes in the cells nearby. In the present study, we mathematically examined the dynamics of the Delta-Notch signaling pathway components in disturbance-driven biliary differentiation, using the model for fine-grained patterns of differentiation. RESULTS: A portal vein cell induced a high Notch signal in its neighboring cells, which corresponded to epithelial differentiation, depending on the production rates of Delta ligands and Notch receptors. In addition, this epithelial differentiation tended to occur in conditions where fine-grained patterning was reported to be lacking. These results highlighted the potential importance of the stability towards homogeneity determined by the production rates in Delta ligands and Notch receptors, in a disturbance-dependent epithelial differentiation.

Phenotypic analysis of mice carrying human-type MAFB p.Leu239Pro mutation.

The transcription factor, MafB, plays important role in the differentiation and functional maintenance of various cells and tissues, such as the inner ear, kidney podocyte, parathyroid gland, pancreatic islet, and macrophages. The rare heterozygous substitution (p.Leu239Pro) of the DNA binding domain in MAFB is the cause of Focal Segmental Glomerulosclerosis associated with Duane Retraction Syndrome, which is characterized by impaired horizontal eye movement due to cranial nerve maldevelopment in humans. In this research, we generated mice carrying MafB p.Leu239Pro (Mafbmt/mt) and retrieved their tissues for analysis. As a result, we found that the phenotype of Mafbmt/mt mouse was similar to that of the conventional Mafb deficient mouse. This finding suggests that the Leucine residue at 239 in the DNA binding domain plays a key role in MafB function and could contribute to the diagnosis or development of treatment for patients carrying the MafB p.Leu239Pro missense variant.