MAFB modulates the maturation of lymphatic vascular networks in mice.

BACKGROUND: Lymphatic vessels play key roles in tissue fluid homeostasis, immune cell trafficking and in diverse disease settings. Lymphangiogenesis requires lymphatic endothelial cell (LEC) differentiation, proliferation, migration, and co-ordinated network formation, yet the transcriptional regulators underpinning these processes remain to be fully understood. The transcription factor MAFB was recently identified as essential for lymphangiogenesis in zebrafish and in cultured human LECs. MAFB is activated in response to VEGFC-VEGFR3 signaling and acts as a downstream effector. However, it remains unclear if the role of MAFB in lymphatic development is conserved in the mammalian embryo. RESULTS: We generated a Mafb loss-of-function mouse using CRISPR/Cas9 gene editing. Mafb mutant mice presented with perinatal lethality associated with cyanosis. We identify a role for MAFB in modifying lymphatic network morphogenesis in the developing dermis, as well as developing and postnatal diaphragm. Furthermore, mutant vessels displayed excessive smooth muscle cell coverage, suggestive of a defect in the maturation of lymphatic networks. CONCLUSIONS: This work confirms a conserved role for MAFB in murine lymphatics that is subtle and modulatory and may suggest redundancy in MAF family transcription factors during lymphangiogenesis.

Maf and Mafb control mouse pallial interneuron fate and maturation through neuropsychiatric disease gene regulation.

​Maf (c-Maf) and Mafb transcription factors (TFs) have compensatory roles in repressing somatostatin (SST+) interneuron (IN) production in medial ganglionic eminence (MGE) secondary progenitors in mice. Maf and Mafb conditional deletion (cDKO) decreases the survival of MGE-derived cortical interneurons (CINs) and changes their physiological properties. Herein, we show that (1) Mef2c and Snap25 are positively regulated by Maf and Mafb to drive IN morphological maturation; (2) Maf and Mafb promote Mef2c expression which specifies parvalbumin (PV+) INs; (3) Elmo1, Igfbp4 and Mef2c are candidate markers of immature PV+ hippocampal INs (HIN). Furthermore, Maf/Mafb neonatal cDKOs have decreased CINs and increased HINs, that express Pnoc, an HIN specific marker. Our findings not only elucidate key gene targets of Maf and Mafb that control IN development, but also identify for the first time TFs that differentially regulate CIN vs. HIN production.

Transcriptome profiling of the cardiac neural crest reveals a critical role for MafB.

The cardiac neural crest originates in the caudal hindbrain, migrates to the heart, and contributes to septation of the cardiac outflow tract and ventricles, an ability unique to this neural crest subpopulation. Here we have used a FoxD3 neural crest enhancer to isolate a pure population of cardiac neural crest cells for transcriptome analysis. This has led to the identification of transcription factors, signaling receptors/ligands, and cell adhesion molecules upregulated in the early migrating cardiac neural crest. We then functionally tested the role of one of the upregulated transcription factors, MafB, and found that it acts as a regulator of Sox10 expression specifically in the cardiac neural crest. Our results not only reveal the genome-wide profile of early migrating cardiac neural crest cells, but also provide molecular insight into what makes the cardiac neural crest unique.

MAFb protein confers intrinsic resistance to proteasome inhibitors in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) patients with t(14;20) have a poor prognosis and their outcome has not improved following the introduction of bortezomib (Bzb). The mechanism underlying the resistance to proteasome inhibitors (PIs) for this subset of patients is unknown. METHODS: IC50 of Bzb and carfilzomib (CFZ) in human myeloma cell lines (HMCLs) were established by MTT assay. Gene Expression profile (GEP) analysis was used to determine gene expression in primary myeloma cells. Immunoblotting analysis was performed for MAFb and caspase family proteins. Immunofluorescence staining was used to detect the location of MAFb protein in MM cells. Lentiviral infections were used to knock-down MAFb expression in two lines. Apoptosis detection by flow cytometry and western blot analysis was performed to determine the molecular mechanism MAFb confers resistance to proteasome inhibitors. RESULTS: We found high levels of MAFb protein in cell lines with t(14;20), in one line with t(6;20), in one with Igλ insertion into MAFb locus, and in primary plasma cells from MM patients with t(14;20). High MAFb protein levels correlated with higher IC50s of PIs in MM cells. Inhibition of GSK3ß activity or treatment with Bzb or CFZ prevented MAFb protein degradation without affecting the corresponding mRNA level indicating a role for GSK3 and proteasome inhibitors in regulation of MAFb stability. Silencing MAFb restored sensitivity to Bzb and CFZ, and enhanced PIs-induced apoptosis and activation of caspase-3, - 8, - 9, PARP and lamin A/C suggesting that high expression of MAFb protein leads to insensitivity to proteasome inhibitors. CONCLUSION: These results highlight the role of post-translational modification of MAFb in maintaining its protein level, and identify a mechanism by which proteasome inhibitors induced stabilization of MAFb confers resistance to proteasome inhibitors, and provide a rationale for the development of targeted therapeutic strategies for this subset of patients.

Beyond regulation of pol III: Role of MAF1 in growth, metabolism, aging and cancer.

MAF1 was discovered as a master repressor of Pol III-dependent transcription in response to diverse extracellular signals, including growth factor, nutrient and stress. It is regulated through posttranslational mechanisms such as phosphorylation. A prominent upstream regulator of MAF1 is the mechanistic target of rapamycin (mTOR) pathway. mTOR kinase directly phosphorylates MAF1, controlling its localization and transcriptional activity. In mammals, MAF1 has also been shown to regulate Pol I- and Pol II-dependent transcription. Interestingly, MAF1 modulates Pol II activity both as a repressor and activator, depending on specific target genes, to impact on cellular growth and metabolism. While MAF1 represses genes such as TATA-binding protein (TBP) and fatty acid synthase (FASN), it activates the expression of PTEN, a major tumor suppressor and an inhibitor of the mTOR signaling. Increasing evidence indicates that MAF1 plays an important role in different aspects of normal physiology, lifespan and oncogenesis. Here we will review the current knowledge on MAF1 in growth, metabolism, aging and cancer. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

Maf1 phenotypes and cell physiology.

As a master regulator of transcription by RNA polymerase (Pol) III, Maf1 represses the synthesis of highly abundant non-coding RNAs as anabolic signals dissipate, as the quality or quantity of nutrients decreases, and under a wide range of cellular and environmental stress conditions. Thus, Maf1 responds to changes in cell physiology to conserve metabolic energy and to help maintain appropriate levels of tRNAs and other essential non-coding RNAs. Studies in different model organisms and cell-based systems show that perturbations of Maf1 can also impact cell physiology and metabolism. These effects are mediated by changes in Pol III transcription and/or by effects of Maf1 on the expression of select Pol II-transcribed genes. Maf1 phenotypes can vary between different systems and are sometimes conflicting as in comparisons between Maf1 KO mice and cultured mammalian cells. These studies are reviewed in an effort to better appreciate the relationship between Maf1 function and cell physiology. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

MafB is required for development of the hindbrain choroid plexus.

The choroid plexus (ChP) is a non-neural epithelial tissue that produces cerebrospinal fluid (CSF). The ChP differentiates from the roof plate, a dorsal midline structure of the neural tube. However, molecular mechanisms underlying ChP development are poorly understood compared to neural development. MafB is a bZip transcription factor that is known to be expressed in the roof plate. Here we investigated the role of MafB in embryonic development of the hindbrain ChP (hChP) using Mafb-deficient mice. Immunohistochemical analyses revealed that MafB is expressed in the roof plate and early hChP epithelial cells but its expression disappears at a later embryonic stage. We also found that the Mafb-deficient hChP exhibits delayed differentiation and results in hypoplasia compared to the wild-type hChP. Furthermore, the Mafb-deficient hChP exhibits increased apoptotic cell death and decreased proliferating cells at E12.5, an early stage of hChP development. Collectively, our findings reveal that MafB play an important role in promoting hChP development during embryogenesis.

The role of macrophage transcription factor MafB in atherosclerotic plaque stability.

BACKGROUND AND AIMS: Macrophage differentiation is associated with the development of atherosclerosis and plaque vulnerability and is regulated by transcription factor MafB. We previously reported that MafB attenuates macrophage apoptosis, which is associated with atherosclerotic plaque instability. The aim of this study was to elucidate the role of MafB in the progression of atherosclerotic plaque. METHODS: We generated macrophage-specific dominant-negative (DN) MafB transgenic mice and intercrossed DN-MafB mice with apolipoprotein E (ApoE) knockout (KO) mice. RESULTS: There was no significant difference in advanced atherosclerotic lesion area between DN-MafB/ApoE KO mice and littermate control ApoE KO mice 9 weeks after high-cholesterol diet. However, DN-MafB/ApoE KO mice showed significantly larger necrotic cores and lower collagen content in atherosclerotic plaques than ApoE KO mice. Although there was no difference in intraplaque macrophage infiltration and efferocytosis, DN-MafB/ApoE KO mice showed significantly more apoptotic macrophages at the plaque edges than did ApoE KO mice. Real-time PCR analysis revealed that peritoneal macrophages of DN-MafB/ApoE KO mice had a greater increase in matrix metalloproteinase-9 and mRNA expression of inflammatory/M1 macrophage markers (tissue necrosis factor-α, interleukin-6, CD11c, and p47phox) after lipopolysaccharide stimulation than those of ApoE KO mice. CONCLUSION: Macrophage-specific inhibition of MafB may destabilize atherosclerotic plaques in advanced lesions.

Loss of MAFB Function in Humans and Mice Causes Duane Syndrome, Aberrant Extraocular Muscle Innervation, and Inner-Ear Defects.

Duane retraction syndrome (DRS) is a congenital eye-movement disorder defined by limited outward gaze and retraction of the eye on attempted inward gaze. Here, we report on three heterozygous loss-of-function MAFB mutations causing DRS and a dominant-negative MAFB mutation causing DRS and deafness. Using genotype-phenotype correlations in humans and Mafb-knockout mice, we propose a threshold model for variable loss of MAFB function. Postmortem studies of DRS have reported abducens nerve hypoplasia and aberrant innervation of the lateral rectus muscle by the oculomotor nerve. Our studies in mice now confirm this human DRS pathology. Moreover, we demonstrate that selectively disrupting abducens nerve development is sufficient to cause secondary innervation of the lateral rectus muscle by aberrant oculomotor nerve branches, which form at developmental decision regions close to target extraocular muscles. Thus, we present evidence that the primary cause of DRS is failure of the abducens nerve to fully innervate the lateral rectus muscle in early development.

Perturbed MafB/GATA1 axis after burn trauma bares the potential mechanism for immune suppression and anemia of critical illness.

Patients who survive initial burn injury are susceptible to nosocomial infections. Anemia of critical illness is a compounding factor in burn patients that necessitates repeated transfusions, which further increase their susceptibility to infections and sepsis. Robust host response is dependent on an adequate number and function of monocytes/macrophages and dendritic cells. In addition to impaired RBC production, burn patients are prone to depletion of dendritic cells and an increase in deactivated monocytes. In steady-state hematopoiesis, RBCs, macrophages, and dendritic cells are all generated from a common myeloid progenitor within the bone marrow. We hypothesized in a mouse model of burn injury that an increase in myeloid-specific transcription factor V-maf musculoaponeurotic fibrosarcoma oncogene homolog B at the common myeloid progenitor stage steers their lineage potential away from the megakaryocyte erythrocyte progenitor production and drives the terminal fate of common myeloid progenitors to form macrophages vs. dendritic cells, with the consequences being anemia, monocytosis, and dendritic cell deficits. Results indicate that, even though burn injury stimulated bone marrow hematopoiesis by increasing multipotential stem cell production (LinnegSca1poscKitpos), the bone marrow commitment is shifted away from the megakaryocyte erythrocyte progenitor and toward granulocyte monocyte progenitors with corresponding alterations in peripheral blood components, such as hemoglobin, hematocrit, RBCs, monocytes, and granulocytes. Furthermore, burn-induced V-maf musculoaponeurotic fibrosarcoma oncogene homolog B in common myeloid progenitors acts as a transcriptional activator of M-CSFR and a repressor of transferrin receptors, promoting macrophages and inhibiting erythroid differentiations while dictating a plasmacytoid dendritic cell phenotype. Results from small interfering RNA and gain-of-function (gfp-globin transcription factor 1 retrovirus) studies indicate that targeted interventions to restore V-maf musculoaponeurotic fibrosarcoma oncogene homolog B/globin transcription factor 1 balance can mitigate both immune imbalance and anemia of critical illness.

The MAFB transcription factor impacts islet α-cell function in rodents and represents a unique signature of primate islet ß-cells.

Analysis of MafB(-/-) mice has suggested that the MAFB transcription factor was essential to islet α- and ß-cell formation during development, although the postnatal physiological impact could not be studied here because these mutants died due to problems in neural development. Pancreas-wide mutant mice were generated to compare the postnatal significance of MafB (MafB(Δpanc)) and MafA/B (MafAB(Δpanc)) with deficiencies associated with the related ß-cell-enriched MafA mutant (MafA(Δpanc)). Insulin(+) cell production and ß-cell activity were merely delayed in MafB(Δpanc) islets until MafA was comprehensively expressed in this cell population. We propose that MafA compensates for the absence of MafB in MafB(Δpanc) mice, which is supported by the death of MafAB(Δpanc) mice soon after birth from hyperglycemia. However, glucose-induced glucagon secretion was compromised in adult MafB(Δpanc) islet α-cells. Based upon these results, we conclude that MafB is only essential to islet α-cell activity and not ß-cell. Interestingly, a notable difference between mice and humans is that MAFB is coexpressed with MAFA in adult human islet ß-cells. Here, we show that nonhuman primate (NHP) islet α- and ß-cells also produce MAFB, implying that MAFB represents a unique signature and likely important regulator of the primate islet ß-cell.

DeepCAGE Transcriptomics Reveal an Important Role of the Transcription Factor MAFB in the Lymphatic Endothelium.

VEGF-C/VEGFR-3 signaling plays a central role in lymphatic development, regulating the budding of lymphatic progenitor cells from embryonic veins and maintaining the expression of PROX1 during later developmental stages. However, how VEGFR-3 activation translates into target gene expression is still not completely understood. We used cap analysis of gene expression (CAGE) RNA sequencing to characterize the transcriptional changes invoked by VEGF-C in LECs and to identify the transcription factors (TFs) involved. We found that MAFB, a TF involved in differentiation of various cell types, is rapidly induced and activated by VEGF-C. MAFB induced expression of PROX1 as well as other TFs and markers of differentiated LECs, indicating a role in the maintenance of the mature LEC phenotype. Correspondingly, E14.5 Mafb(-/-) embryos showed impaired lymphatic patterning in the skin. This suggests that MAFB is an important TF involved in lymphangiogenesis.

MafB antagonizes phenotypic alteration induced by GM-CSF in microglia.

Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB by analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia.

Role of large MAF transcription factors in the mouse endocrine pancreas.

The members of the MAF family of transcription factors are homologs of v-Maf -the oncogenic component of the avian retrovirus AS42. The MAF family is subdivided into 2 groups, small and large MAFs. To elucidate the role of the large MAF transcription factors in the endocrine pancreas, we analyzed large MAF gene knockout mice. It has been shown that Mafa(-/-) mice develop phenotypes including abnormal islet structure soon after birth. This study revealed that Ins1 and Ins2 transcripts and the protein contents were significantly reduced in Mafa(-/-) mice at embryonic day 18.5. In addition, Mafa(-/-);Mafb(-/-) mice contained less than 10% of the insulin transcript and protein of those of wild-type mice, suggesting that Mafa and Mafb cooperate to maintain insulin levels at the embryonic stage. On the other hand, the number of insulin-positive cells in Mafa(-/-) mice was comparable to that of wild-type mice, and even under a Mafb-deficient background the number of insulin-positive cells was not decreased, suggesting that Mafb plays a dominant role in embryonic ß-cell development. We also found that at 20 weeks of age Mafa(-/-);Mafb(+/-) mice showed a higher fasting blood glucose level than single Mafa(-/-) mice. In summary, our results indicate that Mafa is necessary for the maintenance of normal insulin levels even in embryos and that Mafb is important for the maintenance of fasting blood glucose levels in the Mafa-deficient background in adults.

Sexually dimorphic expression of Mafb regulates masculinization of the embryonic urethral formation.

Masculinization of external genitalia is an essential process in the formation of the male reproductive system. Prominent characteristics of this masculinization are the organ size and the sexual differentiation of the urethra. Although androgen is a pivotal inducer of the masculinization, the regulatory mechanism under the control of androgen is still unknown. Here, we address this longstanding question about how androgen induces masculinization of the embryonic external genitalia through the identification of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) gene. Mafb is expressed prominently in the mesenchyme of male genital tubercle (GT), the anlage of external genitalia. MAFB expression is rarely detected in the mesenchyme of female GTs. However, exposure to exogenous androgen induces its mesenchymal expression in female GTs. Furthermore, MAFB expression is prominently down-regulated in male GTs of androgen receptor (Ar) KO mice, indicating that AR signaling is necessary for its expression. It is revealed that Mafb KO male GTs exhibit defective embryonic urethral formation, giving insight into the common human congenital anomaly hypospadias. However, the size of Mafb KO male GTs is similar with that of wild-type males. Moreover, androgen treatment fails to induce urethral masculinization of the GTs in Mafb KO mice. The current results provide evidence that Mafb is an androgen-inducible, sexually dimorphic regulator of embryonic urethral masculinization.

MafB promotes atherosclerosis by inhibiting foam-cell apoptosis.

MafB is a transcription factor that induces myelomonocytic differentiation. However, the precise role of MafB in the pathogenic function of macrophages has never been clarified. Here we demonstrate that MafB promotes hyperlipidemic atherosclerosis by suppressing foam-cell apoptosis. Our data show that MafB is predominantly expressed in foam cells found within atherosclerotic lesions, where MafB mediates the oxidized LDL-activated LXR/RXR-induced expression of apoptosis inhibitor of macrophages (AIM). In the absence of MafB, activated LXR/RXR fails to induce the expression of AIM, a protein that is normally responsible for protecting macrophages from apoptosis; thus, Mafb-deficient macrophages are prone to apoptosis. Haematopoietic reconstitution with Mafb-deficient fetal liver cells in recipient LDL receptor-deficient hyperlipidemic mice revealed accelerated foam-cell apoptosis, which subsequently led to the attenuation of the early atherogenic lesion. These findings represent the first evidence that the macrophage-affiliated MafB transcription factor participates in the acceleration of atherogenesis.

A Gata3-Mafb transcriptional network directs post-synaptic differentiation in synapses specialized for hearing.

Information flow through neural circuits is determined by the nature of the synapses linking the subtypes of neurons. How neurons acquire features distinct to each synapse remains unknown. We show that the transcription factor Mafb drives the formation of auditory ribbon synapses, which are specialized for rapid transmission from hair cells to spiral ganglion neurons (SGNs). Mafb acts in SGNs to drive differentiation of the large postsynaptic density (PSD) characteristic of the ribbon synapse. In Mafb mutant mice, SGNs fail to develop normal PSDs, leading to reduced synapse number and impaired auditory responses. Conversely, increased Mafb accelerates synaptogenesis. Moreover, Mafb is responsible for executing one branch of the SGN differentiation program orchestrated by the Gata3 transcriptional network. Remarkably, restoration of Mafb rescues the synapse defect in Gata3 mutants. Hence, Mafb is a powerful regulator of cell-type specific features of auditory synaptogenesis that offers a new entry point for treating hearing loss. DOI: http://dx.doi.org/10.7554/eLife.01341.001.

High MafB expression following burn augments monocyte commitment and inhibits DC differentiation in hemopoietic progenitors.

We have previously shown that perturbed bone marrow progenitor development promotes hyporesponsive monocytes following experimental burn sepsis. Clinical and experimental sepsis is associated with monocyte deactivation and depletion of mDCs. Decrease in circulating DCs is reported in burn patients who develop sepsis. In our 15% TBSA scald burn model, we demonstrate a significant reduction in the circulating MHC-II(+) population and mDCs (Gr1(neg)CD11b(+)CD11c(+)) with a corresponding decrease in bone marrow MHC-II(+) cells and mDCs for up to 14 days following burn. We explored the underlying mechanism(s) that regulate bone marrow development of monocytes and DCs following burn injury. We found a robust bone marrow response with a significant increase in multipotential HSCs (LSK) and bipotential GMPs following burn injury. GMPs from burn mice exhibit a significant reduction in GATA-1, which is essential for DC development, but express high levels of MafB and M-CSFRs, both associated with monocyte production. GMPs obtained from burn mice differentiated 1.7 times more into Mϕ and 1.6-fold less into DCs compared with sham. Monocytes and DCs expressed 50% less MHC-II in burn versus sham. Increased monocyte commitment in burn GMPs was a result of high MafB and M-CSFR expressions. Transient silencing of MafB (siRNA) in GMP-derived monocytes from burn mice partially restored DC differentiation deficits and increased GATA-1 expression. We provide evidence that high MafB following burn plays an inhibitory role in monocyte-derived DC differentiation by regulating M-CSFR and GATA-1 expressions.

The kreisler mutation leads to the loss of intrinsically hypoxia-activated spots in the region of the retrotrapezoid nucleus/parafacial respiratory group.

Acute hypoxia elicits a biphasic respiratory response characterized in the newborn by a transient hyperventilation followed by a severe decrease in respiratory drive known as hypoxic respiratory depression. Medullary O(2) chemosensitivity is known to contribute to respiratory depression induced by hypoxia, although precise involvement of cell populations remains to be determined. Having a thorough knowledge of these populations is of relevance because perturbations in the respiratory response to hypoxia may participate in respiratory diseases in newborns. We aimed to analyze the hypoxic response of ponto-medullary cell populations of kreisler mutant mice. These mice have defects in a gene expressed in two rhombomeres encompassing a part of the medulla oblongata implicated in hypoxic respiratory depression. Central responses to hypoxia were analyzed in newborn mice by measuring respiratory rhythm in ex vivo caudal pons-medullary-spinal cord preparations and c-fos expression in wild-type and kreisler mutants. The homozygous kreisler mutation, which eliminates most of rhombomere 5 and mis-specifies rhombomere 6, abolished (1) an early decrease in respiratory frequency within 10 min of hypoxia and (2) an intrinsic hypoxic activation, which is characterized by an increase in c-fos expression in the region of the ventral medullary surface encompassing the retrotrapezoid nucleus/parafacial respiratory group expressing Phox2b. This increase in c-fos expression persisted in wild-type Phox2b-negative and Phox2b-positive cells after blockade of synaptic transmission and rhythmogenesis by a low [Ca(2+)](0). Another central response was retained in homozygous kreisler mutant mice; it was distinguished by (1) a delayed (10-30 min) depression of respiratory frequency and (2) a downregulation of c-fos expression in the ventrolateral reticular nucleus of the medulla, the nucleus of the solitary tract, and the area of the A5 region. Thus, two types of ponto-medullary cell groups, with distinct anatomical locations, participate in central hypoxic respiratory depression in newborns.