Mtc6/Ehg2 is a novel endoplasmic reticulum-resident glycoprotein essential for high-pressure tolerance.

Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that Maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrate that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain, and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS are essential for Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates proper folding of nutrient permeases in the ER under the stress condition. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.

Perturbed collagen metabolism underlies lymphatic recanalization failure in Gata2 heterozygous deficient mice.

Lymphedema has become a global health issue following the growing number of cancer surgeries. Curative or supportive therapeutics have long been awaited for this refractory condition. Transcription factor GATA2 is crucial in lymphatic development and maintenance, as GATA2 haploinsufficient disease often manifests as lymphedema. We recently demonstrated that Gata2 heterozygous deficient mice displayed delayed lymphatic recanalization upon lymph node resection. However, whether GATA2 contributes to lymphatic regeneration by functioning in the damaged lymph vessels' microenvironment remains explored. In this study, our integrated analysis demonstrated that dermal collagen fibers were more densely accumulated in the Gata2 heterozygous deficient mice. The collagen metabolism-related transcriptome was perturbed, and collagen matrix contractile activity was aberrantly increased in Gata2 heterozygous embryonic fibroblasts. Notably, soluble collagen placement ameliorated delayed lymphatic recanalization, presumably by modulating the stiffness of the extracellular matrix around the resection site of Gata2 heterozygous deficient mice. Our results provide valuable insights into mechanisms underlying GATA2-haploinsufficiency-mediated lymphedema and shed light on potential therapeutic avenues for this intractable disease.

Exogenous l-fucose attenuates neuroinflammation induced by lipopolysaccharide.

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost GlcNAc residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous KO (Fut8-/-) or heterozygous KO (Fut8+/-) mice, compared with the WT (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this bacterial artificial chromosome transgenic monitoring system. Then we examined the effects of l-fucose on neuroinflammation in the Fut8+/- mice. The lectin blot and mass spectrometry analysis showed that l-fucose preadministration increased the core fucosylation levels in the Fut8+/- mice. Notably, exogenous l-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by l-fucose preadministration. l-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2 (Janus kinase 2), Akt (protein kinase B), and STAT3 (signal transducer and activator of transcription 3). l-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/- mice, which was further confirmed in BV-2 cells. These results indicate that l-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/- mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that l-fucose is a potential prophylactic compound against neuroinflammation.

The Il6 -39 kb enhancer containing clustered GATA2- and PU.1-binding sites is essential for Il6 expression in murine mast cells.

Mast cells serve as a first-line defense of innate immunity. Interleukin-6 (IL-6) induced by bacterial lipopolysaccharide (LPS) in mast cells plays a crucial role in antibacterial protection. The zinc finger transcription factor GATA2 cooperatively functions with the ETS family transcription factor PU.1 in multiple mast cell activities. However, the regulatory landscape directed by GATA2 and PU.1 under inflammation remains elusive. We herein showed that a large proportion of GATA2-binding peaks were closely located with PU.1-binding peaks in distal cis-regulatory regions of inflammatory cytokine genes in mast cells. Notably, GATA2 and PU.1 played crucial roles in promoting LPS-mediated inflammatory cytokine production. Genetic ablation of GATA2-PU.1-clustered binding sites at the Il6 -39 kb region revealed its central role in LPS-induced Il6 expression in mast cells. We demonstrate a novel collaborative activity of GATA2 and PU.1 in cytokine induction upon inflammatory stimuli via the GATA2-PU.1 overlapping sites in the distal cis-regulatory regions.

Pleiotropic roles of N-glycans for enzyme activities and stabilities of MIPC synthases, Csh1 and Sur1/Csg1, in Saccharomyces cerevisiae.

Mannosyl phosphorylceramide (MIPC) is a membrane lipid classified as a complex sphingolipid in Saccharomyces cerevisiae. MIPC is synthesized by 2 redundant enzymes, Sur1/Csg1 and Csh1, in the Golgi lumen. MIPC consists of 5 subtypes (A, B', B, C, and D-type) according to the position and number of hydroxyl groups on the ceramide moiety. Sur1 exerts higher impact on synthesis of MIPC-B and MIPC-C than Csh1. In this study, we elucidated the roles played by N-glycans attached to Sur1 and Csh1, and dissected the mechanisms underlying substrate recognition by these 2 enzymes. Sur1 carries an N-glycan on Asn-224, whereas Csh1 has N-glycans on Asn-51 and Asn-247. Although intracellular proteins usually harbor core-type N-glycans, the N-glycan on Asn-51 of Csh1 exhibited a unique mannan-like structure containing a long backbone of mannose. Sur1 N224Q and Csh1 N51Q mutants exhibited a decrease in the activity to synthesize specific MIPC subtypes for each enzyme, suggesting that these N-glycans play a role in substrate recognition through their catalytic domains. Moreover, ectopic insertion of an N-glycosylation consensus sequence (NST) at codon 51 of Sur1 (Sur1-NST51) resulted in an artificial modification with mannan, which markedly decreased protein stability. Our results suggest that the diminished stability of the Sur1-NST51 mutant protein could be attributable to potential structural alterations by the mannan. Collectively, the present study reveals essential luminal domains of Sur1 and Csh1 that dictate substrate specificity and/or the protein stabilities via mannan modification.

Genomic landscape of chemical-induced lung tumors under Nrf2 different expression levels.

The transcription factor Nrf2 plays a crucial role in the anti-oxidative stress response, protection of DNA from injury and DNA repair mechanisms. Nrf2 activity reduces cancer initiation, but how Nrf2 affects whole-genome alterations upon carcinogenic stimulus remains unexplored. Although recent genome-wide analysis using next-generation sequencing revealed landscapes of nucleotide mutations and copy number alterations in various human cancers, genomic changes in murine cancer models have not been thoroughly examined. We elucidated the relationship between Nrf2 expression levels and whole exon mutation patterns using an ethyl-carbamate (urethane)-induced lung carcinogenesis model employing Nrf2-deficient and Keap1-kd mice, the latter of which express high levels of Nrf2. Exome analysis demonstrated that single nucleotide and trinucleotide mutation patterns and the Kras mutational signature differed significantly and were dependent on the expression level of Nrf2. The Nrf2-deficient tumors exhibited fewer copy number alterations relative to the Nrf2-wt and Keap1-kd tumors. The observed trend in genomic alterations likely prevented the Nrf2-deficient tumors from progressing into malignancy. For the first time, we present whole-exome sequencing results for chemically-induced lung tumors in the Nrf2 gain or loss of function mouse models. Our results demonstrate that different Nrf2 expression levels lead to distinct gene mutation patterns that underly different oncogenic mechanisms in each tumor genotype.

Gata2 heterozygous mutant mice exhibit reduced inflammatory responses and impaired bacterial clearance.

Infectious diseases continually pose global medical challenges. The transcription factor GATA2 establishes gene networks and defines cellular identity in hematopoietic stem/progenitor cells and in progeny committed to specific lineages. GATA2-haploinsufficient patients exhibit a spectrum of immunodeficiencies associated with bacterial, viral, and fungal infections. Despite accumulating clinical knowledge of the consequences of GATA2 haploinsufficiency in humans, it is unclear how GATA2 haploinsufficiency compromises host anti-infectious defenses. To address this issue, we examined Gata2-heterozygous mutant (G2 Het) mice as a model for human GATA2 haploinsufficiency. In vivo inflammation imaging and cytokine multiplex analysis demonstrated that G2 Het mice had attenuated inflammatory responses with reduced levels of inflammatory cytokines, particularly IFN-γ, IL-12p40, and IL-17A, during lipopolysaccharide-induced acute inflammation. Consequently, bacterial clearance was significantly impaired in G2 Het mice after cecal ligation and puncture-induced polymicrobial peritonitis. These results provide direct molecular insights into GATA2-directed host defenses and the pathogenic mechanisms underlying observed immunodeficiencies in GATA2-haploinsufficient patients.

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation.

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of ß-globinopathies (sickle cell disease and ß-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor-like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

GATA2 participates in the recanalization of lymphatic vessels after surgical lymph node extirpation.

Lymphatic recanalization failure after lymphadenectomy constitutes a major risk of lymphedema in cancer surgery. It has been reported that GATA2, a zinc finger transcription factor, is expressed in lymphatic endothelial cells and is involved in the development of fetal lymphatic vessels. GATA3, another member of the GATA family of transcription factors, is required for the differentiation of lymphoid tissue inducer (LTi) cells and is essential for lymph node formation. However, how GATA2 and GATA3 function in recanalization after the surgical extirpation of lymphatic vessels has not been elucidated. Employing a new model of lymphatic recanalization, we examined the lymphatic reconnection process in Gata2 heterozygous deficient (Gata2+/- ) and Gata3 heterozygous deficient (Gata3+/- ) mice. We found that lymphatic recanalization was significantly impaired in Gata2+/- mice, while Gata3+/- mice rarely showed such abnormalities. Notably, the perturbed lymphatic recanalization in the Gata2+/- mice was partially restored by crossing with the Gata3+/- mice. Our results demonstrate for the first time that GATA2 participates in the regeneration of damaged lymphatic vessels and the unexpected suppressive activity of GATA3 against lymphatic recanalization processes.

Development and Carcinogenesis: Roles of GATA Factors in the Sympathoadrenal and Urogenital Systems.

The GATA family of transcription factors consists of six proteins (GATA1-6) that control a variety of physiological and pathological processes. In particular, GATA2 and GATA3 are coexpressed in a number of tissues, including in the urogenital and sympathoadrenal systems, in which both factors participate in the developmental process and tissue maintenance. Furthermore, accumulating studies have demonstrated that GATA2 and GATA3 are involved in distinct types of inherited diseases as well as carcinogenesis in diverse tissues. This review summarizes our current knowledge of how GATA2 and GATA3 participate in the transcriptional regulatory circuitry during the development of the sympathoadrenal and urogenital systems, and how their dysregulation results in the carcinogenesis of neuroblastoma, renal urothelial, and gynecologic cancers.

GATA2 functions in adrenal chromaffin cells.

Catecholamine synthesized in the sympathoadrenal system, including sympathetic neurons and adrenal chromaffin cells, is vital for cardiovascular homeostasis. It has been reported that GATA2, a zinc finger transcription factor, is expressed in murine sympathoadrenal progenitor cells. However, a physiological role for GATA2 in adrenal chromaffin cells has not been established. In this study, we demonstrate that GATA2 is specifically expressed in adrenal chromaffin cells. We examined the consequences of Gata2 loss-of-function mutations, exploiting a Gata2 conditional knockout allele crossed to neural crest-specific Wnt1-Cre transgenic mice (Gata2 NC-CKO). The vast majority of Gata2 NC-CKO embryos died by embryonic day 14.5 (e14.5) and exhibited a decrease in catecholamine-producing adrenal chromaffin cells, implying that a potential catecholamine defect might lead to the observed embryonic lethality. When intercrossed pregnant dams were fed with synthetic adrenaline analogs, the lethality of the Gata2 NC-CKO embryos was partially rescued, indicating that placental transfer of the adrenaline analogs complements the lethal catecholamine deficiency in the Gata2 NC-CKO embryos. These results demonstrate that GATA2 participates in the development of neuroendocrine adrenaline biosynthesis, which is essential for fetal survival.

Histamine and histidine decarboxylase: Immunomodulatory functions and regulatory mechanisms.

Histamine is a bioactive monoamine that is synthesized by the enzymatic activity of histidine decarboxylase (HDC) in basophils, mast cells, gastric enterochromaffin-like (ECL) cells and histaminergic neuronal cells. Upon a series of cellular stimuli, these cells release stored histamine, which elicits allergies, inflammation, and gastric acid secretion and regulates neuronal activity. Recent studies have shown that certain other types of myeloid lineage cells also produce histamine with HDC induction under various pathogenic stimuli. Histamine has been shown to play a series of pathophysiological roles by modulating immune and inflammatory responses in a number of disease conditions, whereas the mechanistic aspects underlying induced HDC expression remain elusive. In the present review, we summarize the current understanding of the regulatory mechanism of Hdc gene expression and the roles played by histamine in physiological contexts as well as pathogenic processes. We also introduce a newly developed histaminergic cell-monitoring transgenic mouse line (Hdc-BAC-GFP) that serves as a valuable experimental tool to identify the source of histamine and dissect upstream regulatory signals.

Lipopolysaccharide-induced expansion of histidine decarboxylase-expressing Ly6G+ myeloid cells identified by exploiting histidine decarboxylase BAC-GFP transgenic mice.

Histamine is a biogenic amine that is chiefly produced in mast cells and basophils and elicits an allergic response upon stimulation. Histidine decarboxylase (HDC) is a unique enzyme that catalyzes the synthesis of histamine. Therefore, the spatiotemporally specific Hdc gene expression profile could represent the localization of histamine-producing cells under various pathophysiological conditions. Although the bioactivity of histamine is well defined, the regulatory mechanism of Hdc gene expression and the distribution of histamine-producing cell populations in various disease contexts remains unexplored. To address these issues, we generated a histidine decarboxylase BAC (bacterial artificial chromosome) DNA-directed GFP reporter transgenic mouse employing a 293-kb BAC clone containing the entire Hdc gene locus and extended flanking sequences (Hdc-GFP). We found that the GFP expression pattern in the Hdc-GFP mice faithfully recapitulated that of conventional histamine-producing cells and that the GFP expression level mirrored the increased Hdc expression in lipopolysaccharide (LPS)-induced septic lungs. Notably, a CD11b+Ly6G+Ly6Clow myeloid cell population accumulated in the lung during sepsis, and most of these cells expressed high levels of GFP and indeed contain histamine. This study reveals the accumulation of a histamine-producing myeloid cell population during sepsis, which likely participates in the immune process of sepsis.

Spiral ganglion cell degeneration-induced deafness as a consequence of reduced GATA factor activity.

Zinc-finger transcription factors GATA2 and GATA3 are both expressed in the developing inner ear, although their overlapping versus distinct activities in adult definitive inner ear are not well understood. We show here that GATA2 and GATA3 are co-expressed in cochlear spiral ganglion cells and redundantly function in the maintenance of spiral ganglion cells and auditory neural circuitry. Notably, Gata2 and Gata3 compound heterozygous mutant mice had a diminished number of spiral ganglion cells due to enhanced apoptosis, which resulted in progressive hearing loss. The decrease in spiral ganglion cellularity was associated with lowered expression of neurotrophin receptor TrkC that is an essential factor for spiral ganglion cell survival. We further show that Gata2 null mutants that additionally bear a Gata2 YAC (yeast artificial chromosome) that counteracts the lethal hematopoietic deficiency due to complete Gata2 loss nonetheless failed to complement the deficiency in neonatal spiral ganglion neurons. Furthermore, cochlea-specific Gata2 deletion mice also had fewer spiral ganglion cells and resultant hearing impairment. These results show that GATA2 and GATA3 redundantly function to maintain spiral ganglion cells and hearing. We propose possible mechanisms underlying hearing loss in human GATA2- or GATA3-related genetic disorders.

A Gata3 3' Distal Otic Vesicle Enhancer Directs Inner Ear-Specific Gata3 Expression.

Transcription factor GATA3 plays vital roles in inner ear development, while regulatory mechanisms controlling its inner ear-specific expression are undefined. We demonstrate that a cis-regulatory element lying 571 kb 3' to the Gata3 gene directs inner ear-specific Gata3 expression, which we refer to as the Gata3 otic vesicle enhancer (OVE). In transgenic murine embryos, a 1.5-kb OVE-directed lacZ reporter (TgOVE-LacZ) exhibited robust lacZ expression specifically in the otic vesicle (OV), an inner ear primordial tissue, and its derivative semicircular canal. To further define the regulatory activity of this OVE, we generated Cre transgenic mice in which Cre expression was directed by a 246-bp core sequence within the OVE element (TgcoreOVE-Cre). TgcoreOVE-Cre successfully marked the OV-derived inner ear tissues, including cochlea, semicircular canal and spiral ganglion, when crossed with ROSA26 lacZ reporter mice. Furthermore, Gata3 conditionally mutant mice, when crossed with the TgcoreOVE-Cre, showed hypoplasia throughout the inner ear tissues. These results demonstrate that OVE has a sufficient regulatory activity to direct Gata3 expression specifically in the otic vesicle and semicircular canal and that Gata3 expression driven by the OVE is crucial for normal inner ear development.

Reducing Inflammatory Cytokine Production from Renal Collecting Duct Cells by Inhibiting GATA2 Ameliorates Acute Kidney Injury.

Acute kidney injury (AKI) is a leading cause of chronic kidney disease. Proximal tubules are considered to be the primary origin of pathogenic inflammatory cytokines in AKI. However, it remains unclear whether other cell types, including collecting duct (CD) cells, participate in inflammatory processes. The transcription factor GATA2 is specifically expressed in CD cells and maintains their cellular identity. To explore the pathophysiological function of GATA2 in AKI, we generated renal tubular cell-specific Gata2 deletion (G2CKO) mice and examined their susceptibility to ischemia reperfusion injury (IRI). Notably, G2CKO mice exhibited less severe kidney damage, with reduced granulomacrophagic infiltration upon IRI. Transcriptome analysis revealed that a series of inflammatory cytokine genes were downregulated in GATA2-deficient CD cells, suggesting that GATA2 induces inflammatory cytokine expression in diseased kidney CD cells. Through high-throughput chemical library screening, we identified a potent GATA inhibitor. The chemical reduces cytokine production in CD cells and protects the mouse kidney from IRI. These results revealed a novel pathological mechanism of renal IRI, namely, that CD cells produce inflammatory cytokines and promote IRI progression. In injured kidney CD cells, GATA2 exerts a proinflammatory function by upregulating inflammatory cytokine gene expression. GATA2 can therefore be considered a therapeutic target for AKI.

Derepression of the DNA Methylation Machinery of the Gata1 Gene Triggers the Differentiation Cue for Erythropoiesis.

GATA1 is a critical regulator of erythropoiesis. While the mechanisms underlying the high-level expression of GATA1 in maturing erythroid cells have been studied extensively, the initial activation of the Gata1 gene in early hematopoietic progenitors remains to be elucidated. We previously identified a hematopoietic stem and progenitor cell (HSPC)-specific silencer element (the Gata1 methylation-determining region [G1MDR]) that recruits DNA methyltransferase 1 (Dnmt1) and provokes methylation of the Gata1 gene enhancer. In the present study, we hypothesized that removal of the G1MDR-mediated silencing machinery is the molecular basis of the initial activation of the Gata1 gene and erythropoiesis. To address this hypothesis, we generated transgenic mouse lines harboring a Gata1 bacterial artificial chromosome in which the G1MDR was deleted. The mice exhibited abundant GATA1 expression in HSPCs, in a GATA2-dependent manner. The ectopic GATA1 expression repressed Gata2 transcription and induced erythropoiesis and apoptosis of HSPCs. Furthermore, genetic deletion of Dnmt1 in HSPCs activated Gata1 expression and depleted HSPCs, thus recapitulating the HSC phenotype associated with GATA1 gain of function. These results demonstrate that the G1MDR holds the key to HSPC maintenance and suggest that release from this suppressive mechanism is a fundamental requirement for subsequent initiation of erythroid differentiation.

Gata3 Hypomorphic Mutant Mice Rescued with a Yeast Artificial Chromosome Transgene Suffer a Glomerular Mesangial Cell Defect.

GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease.

Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.

Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress response and also represses inflammation. However, the mechanisms how Nrf2 alleviates inflammation are still unclear. Here, we demonstrate that Nrf2 interferes with lipopolysaccharide-induced transcriptional upregulation of proinflammatory cytokines, including IL-6 and IL-1ß. Chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR analyses revealed that Nrf2 binds to the proximity of these genes in macrophages and inhibits RNA Pol II recruitment. Further, we found that Nrf2-mediated inhibition is independent of the Nrf2-binding motif and reactive oxygen species level. Murine inflammatory models further demonstrated that Nrf2 interferes with IL6 induction and inflammatory phenotypes in vivo. Thus, contrary to the widely accepted view that Nrf2 suppresses inflammation through redox control, we demonstrate here that Nrf2 opposes transcriptional upregulation of proinflammatory cytokine genes. This study identifies Nrf2 as the upstream regulator of cytokine production and establishes a molecular basis for an Nrf2-mediated anti-inflammation approach.

Transcription Factor MafB Coordinates Epidermal Keratinocyte Differentiation.

Mammalian epidermis is a stratified epithelium composed of distinct layers of keratinocytes. The outermost cornified layer is a primary barrier that consists of a cornified envelope, an insoluble structure assembled by cross-linked scaffold proteins, and a surrounding mixture of lipids. Skin keratinocytes undergo a multistep differentiation process, but the mechanism underlying this process is not fully understood. We demonstrate that the transcription factor MafB is expressed in differentiating keratinocytes in mice and is transcriptionally upregulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation was partially impaired and the cornified layer was thinner than in wild-type mice. On the basis of transcriptional profiling, we detected reduced expression levels of a subset of cornified envelope genes, for example, filaggrin and repetin, in the MafB(-/-) epidermis. By contrast, the expression levels of lipid metabolism-related genes, such as Alox12e and Smpd3, increased. The upregulated genes in the MafB(-/-) epidermis were enriched for putative target genes of the transcription factors Gata3, Grhl3, and Klf4. Immunohistochemical analysis of skin biopsy samples revealed that the expression levels of filaggrin and MafB were significantly reduced in patients with human atopic dermatitis and psoriasis vulgaris. Our results indicate that MafB is a component of the gene expression program that regulates epidermal keratinocyte differentiation.