Transcription factor MafB may play an important role in secondary hyperparathyroidism.

The transcription factor MafB is essential for development of the parathyroid glands, the expression of which persists after morphogenesis and in adult parathyroid glands. However, the function of MafB in adult parathyroid tissue is unclear. To investigate this, we induced chronic kidney disease (CKD) in wild-type and MafB heterozygote (MafB+/-) mice by feeding them an adenine-supplemented diet, leading to secondary hyperparathyroidism. The elevated serum creatinine and blood urea nitrogen levels in heterozygous and wild-type mice fed the adenine-supplemented diet were similar. Interestingly, secondary hyperparathyroidism, characterized by serum parathyroid hormone elevation and enlargement of parathyroid glands, was suppressed in MafB+/- mice fed the adenine-supplemented diet compared to similarly fed wild-type littermates. Quantitative RT-PCR and immunohistochemical analyses showed that the increased expression of parathyroid hormone and cyclin D2 in mice with CKD was suppressed in the parathyroid glands of heterozygous CKD mice. A reporter assay indicated that MafB directly regulated parathyroid hormone and cyclin D2 expression. To exclude an effect of a developmental anomaly in MafB+/- mice, we analyzed MafB tamoxifen-induced global knockout mice. Hypocalcemia-stimulated parathyroid hormone secretion was significantly impaired in MafB knockout mice. RNA-sequencing analysis indicated PTH, Gata3 and Gcm2 depletion in the parathyroid glands of MafB knockout mice. Thus, MafB appears to play an important role in secondary hyperparathyroidism by regulation of parathyroid hormone and cyclin D2 expression. Hence, MafB may represent a new therapeutic target in secondary hyperparathyroidism.

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 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.

AIM/CD5L attenuates DAMPs in the injured brain and thereby ameliorates ischemic stroke.

The sterile inflammation caused by damage-associated molecular patterns (DAMPs) worsens the prognosis following primary injury such as ischemic stroke. However, there are no effective treatments to regulate DAMPs. Here, we report that AIM (or CD5L) protein reduces sterile inflammation by attenuating DAMPs and that AIM administration ameliorates the deleterious effects of ischemic stroke. AIM binds to DAMPs via charge-based interactions and disulfide bond formation. This AIM association promotes the phagocytic removal of DAMPs and neutralizes DAMPs by impeding their binding to inflammatory receptors. In experimental stroke, AIM-deficient mice exhibit severe neurological damage and higher mortality with greater levels of DAMPs and associated inflammation in the brain than wild-type mice, in which brain AIM levels increase following stroke onset. Recombinant AIM administration reduces sterile inflammation in the infarcted region, leading to a profound reduction of animal mortality. Our findings provide a basis for the therapies targeting DAMPs to improve ischemic stroke.

Preferential reduction of beta cells derived from Pax6-MafB pathway in MafB deficient mice.

During pancreatic development insulin(+) cells co-express the transcription factors MafB and Pax6, and transition from a MafA(-) to MafA(+) state. To examine the role of Pax6 and MafB in the development of beta-cells, we analyzed embryonic pancreata from Pax6- and MafB-deficient mice. Pax6 deficiency, as manifest in the Pax6(Sey-Neu) allele, reduced not only the number of cells expressing insulin or glucagon, but also the number of MafB, PDX-1 and MafA expressing cells. We show that MafB can directly activate expression of insulin and glucagon, and a MafB protein engineered to contain N248S mutation in the MafB (kr(ENU)) results in significantly reduced activation. Furthermore, pancreata from MafB deficient (kr(ENU)/kr(ENU)) mice exhibited reduced number of cells expressing insulin, glucagon, PDX-1 and MafA, with only a minor reduction in MafB expressing cells. MafB deficiency does not affect endocrine specification but does affect the lineage commitment of the endocrine cells and their maturation. Similar to Pax6 deficient mice, MafB deficient mice showed reductions both in insulin and glucagon expressing cells and in the ability of MafB and PDX-1 expressing cells to activate expression of these hormones. However, MafB deficient mice exhibited no effect on Pax6 expression. These results suggest that MafB may function as a downstream mediator of Pax6 in regulating the specification of insulin and glucagon expressing cells. Interestingly, the remaining insulin(+) cells in these knockouts preferentially express Hb9, suggesting the existence of an alternate pathway for the generation of insulin expressing cells, even in the absence of Pax6 and MafB function. Thus, Pax6 acts upstream of MafB, which in turn may trigger the expression of insulin and regulate the PDX-1 and MafA expression required for beta-cell maturation.

Development of macrophages with altered actin organization in the absence of MafB.

In the hematopoietic system the bZip transcription factor MafB is selectively expressed at high levels in monocytes and macrophages and promotes macrophage differentiation in myeloid progenitors, whereas a dominant-negative allele can inhibit this process. To analyze the requirement of MafB for macrophage development, we generated MafB-deficient mice and, due to their neonatal lethal phenotype, analyzed macrophage differentiation in vitro, in the embryo, and in reconstituted mice. Surprisingly we observed in vitro differentiation of macrophages from E14.5 fetal liver (FL) cells and E18.5 splenocytes. Furthermore we found normal numbers of F4/80(+)/Mac-1(+) macrophages and monocytes in fetal liver, spleen, and blood as well as in bone marrow, spleen, and peritoneum of adult MafB(-/-) FL reconstituted mice. MafB(-/-) macrophages showed intact basic macrophage functions such as phagocytosis of latex beads or Listeria monocytogenes and nitric oxide production in response to lipopolysaccharide. By contrast, MafB(-/-) macrophages expressed increased levels of multiple genes involved in actin organization. Consistent with this, phalloidin staining revealed an altered morphology involving increased numbers of branched protrusions of MafB(-/-) macrophages in response to macrophage colony-stimulating factor. Together these data point to an unexpected redundancy of MafB function in macrophage differentiation and a previously unknown role in actin-dependent macrophage morphology.

Loss of MafA and MafB expression promotes islet inflammation.

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA-/-MafB+/-, but not MafA-/- islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

MafB is a critical regulator of complement component C1q.

The transcription factor MafB is expressed by monocytes and macrophages. Efferocytosis (apoptotic cell uptake) by macrophages is important for inhibiting the development of autoimmune diseases, and is greatly reduced in Mafb-deficient macrophages. Here, we show the expression of the first protein in the classical complement pathway C1q is important for mediating efferocytosis and is reduced in Mafb-deficient macrophages. The efferocytosis defect in Mafb-deficient macrophages can be rescued by adding serum from wild-type mice, but not by adding serum from C1q-deficient mice. By hemolysis assay we also show that activation of the classical complement pathway is decreased in Mafb-deficient mice. In addition, MafB overexpression induces C1q-dependent gene expression and signals that induce C1q genes are less effective in the absence of MafB. We also show that Mafb-deficiency can increase glomerular autoimmunity, including anti-nuclear antibody deposition. These results show that MafB is an important regulator of C1q.

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.

MafB/c-Maf deficiency enables self-renewal of differentiated functional macrophages.

In metazoan organisms, terminal differentiation is generally tightly linked to cell cycle exit, whereas the undifferentiated state of pluripotent stem cells is associated with unlimited self-renewal. Here, we report that combined deficiency for the transcription factors MafB and c-Maf enables extended expansion of mature monocytes and macrophages in culture without loss of differentiated phenotype and function. Upon transplantation, the expanded cells are nontumorigenic and contribute to functional macrophage populations in vivo. Small hairpin RNA inactivation shows that continuous proliferation of MafB/c-Maf deficient macrophages requires concomitant up-regulation of two pluripotent stem cell-inducing factors, KLF4 and c-Myc. Our results indicate that MafB/c-MafB deficiency renders self-renewal compatible with terminal differentiation. It thus appears possible to amplify functional differentiated cells without malignant transformation or stem cell intermediates.

Collapsing glomerulopathy: an inflammatory podocytopathy?

PURPOSE OF REVIEW: Collapsing glomerulopathy is a relatively new and debated podocytopathy. Among several conjectures, inflammatory injury orchestrated by podocytes is emerging to explain the pathogenesis of collapsing glomerulopathy. Here, we briefly summarize recent studies in support of this novel and intriguing hypothesis. RECENT FINDINGS: Immunohistochemical analyses of markers conventionally used to demarcate podocytes apart from parietal epithelium identified the parietal podocyte. MafB-deficient mice exhibited abnormal podocyte and macrophage differentiation, suggesting ancestral and functional overlap. These apparent developmental anomalies were detected in studies showing an admixture of hyperplastic podocytes with macrophage epitopes and hyperplastic parietal epithelium in pseudocrescents and in true crescents. Experimental antibody-mediated injury of podocytes could trigger capillary collapse and pseudocrescent formation marked by recruitment of epithelial cells from Bowman's capsule. In contrast, experimental stabilization of hypoxia-inducible factors within podocytes--a known inflammatory response by macrophages--could trigger podocyte proliferation and the formation of true necrotizing crescents. SUMMARY: Preliminary evidence suggests that visceral and parietal podocytes may become macrophage-like inflammatory mediators of proliferative epithelial injury within the glomerulus. This may manifest as collapsing glomerulopathy or crescentic glomerulonephritis--lesions that appear to be anatomically and pathogenically linked.