Macrophage specific restoration of the Nrf2 gene in whole-body knockout mice ameliorates steatohepatitis induced by lipopolysaccharide from Porphyromonas gingivalis through enhanced hepatic clearance.

Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (P.g.), which causes periodontal disease, contributes to the development of non-alcoholic steatohepatitis (NASH). We investigated the role of Nrf2, an antioxidative stress sensor, in macrophages in the development of NASH induced by LPS from P.g. We generated macrophage-specific Nrf2 gene rescue mice (Nrf2-mRes), which express Nrf2 only in macrophages, using the cre/loxp system. Wild-type (WT) mice, whole body Nrf2-knockout (Nrf2-KO) mice, and Nrf2-mRes mice were fed a high-fat diet for 18 weeks, and LPS from P.g. was administered intraperitoneally for the last 6 weeks. Nrf2-KO mice developed severe steatohepatitis with liver inflammation and fibrosis compared with WT mice, and steatohepatitis was ameliorated in Nrf2-mRes mice. The mRNA expressions of Toll-like receptor (Tlr)-2, which activates inflammatory signaling pathways after LPS binding, and α-smooth muscle actin (αSma), which promotes hepatic fibrosis, were reduced in Nrf2-mRes mice compared with Nrf2-KO mice. The protein levels of LPS-binding protein in livers were increased in Nrf2-KO mice compared with WT mice; however, the levels were reduced in Nrf2-mRes mice despite similar numbers of F4/80 positive cells, which reflect macrophage/Kupffer cell infiltration into the livers. Nrf2 in macrophages ameliorates NASH through the increased hepatic clearance of LPS.

p62 Is a Potential Biomarker for Risk of Malignant Transformation of Oral Potentially Malignant Disorders (OPMDs).

To determine the intracellular behavior of p62, a marker of selective autophagy, in oral potentially malignant disorders (OPMDs). This retrospective study includes 70 patients who underwent biopsy or surgical resection and were definitively diagnosed with OPMDs. Immunohistochemical staining for p62, XPO1, p53, and ki67 was performed on all samples and positive cell occupancy was calculated. We statistically investigated the correlation between protein expression in OPMDs and the association between malignant transformation, clinicopathological characteristics, and occupancy. ki67 expression was negatively correlated with p62 expression in the nucleus (p < 0.01) and positively correlated with p62 expression in the cytoplasm (p < 0.01). For malignant transformation, the expression of p62 in the nucleus (p = 0.03) was significantly lower in malignant transformation cases, whereas the expression of p62 in the cytoplasm (p = 0.03) and the aggregation expression (p < 0.01) were significantly higher. Our results suggest that the function of p62 is altered by its subcellular localization. In addition, defects in selective autophagy occur in cases of malignant transformation, suggesting that p62 is a potential biomarker of the risk of malignant transformation of OPMDs.

Muscle p62 stimulates the expression of antioxidant proteins alleviating cancer cachexia.

Oxidative stress plays an important role in skeletal muscle atrophy during cancer cachexia, and more glycolytic muscles are preferentially affected. Sequestosome1/SQSTM1 (i.e., p62), particularly when phosphorylated at Ser 349 (Ser 351 in mice), competitively binds to the Kelch-like ECH-associated protein 1 (Keap1) activating Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 then stimulates the transcription of antioxidant/electrophile-responsive elements in target genes. However, a potential role for p62 in the protection of muscle wasting in cachexia remains to be determined. Here, using the well-established cachexia-inducing model of Lewis Lung Carcinoma (LLC) in mice we demonstrate higher expression of antioxidant proteins (i.e., NQO1, HO-1, GSTM1, CuZnSOD, MnSOD, and EcSOD) in the more oxidative and cachexia resistant soleus muscle than in the more glycolytic and cachexia prone extensor digitorum longus muscle. This was accompanied by higher p62 (total and phosphorylated) and nuclear Nrf2 levels in the soleus, which were paralleled by higher expression of proteins known to either phosphorylate or promote p62 phosphorylation (i.e., NBR1, CK1, PKCδ, and TAK1). Muscle-specific p62 gain-of-function (i.e., in p62 mTg mice) activated Nrf2 nuclear translocation and increased the expression of multiple antioxidant proteins (i.e., CuZnSOD, MnSOD, EcSOD, NQO1, and GSTM1) in glycolytic muscles. Interestingly, skeletal muscle Nrf2 haplodeficiency blunted the increases of most of these proteins (i.e., CuZnSOD, EcSOD, and NQO1) suggesting that muscle p62 stimulates antioxidant protein expression also via additional, yet to be determined mechanisms. Of note, p62 gain-of-function mitigated glycolytic muscle wasting in LLC-affected mice. Collectively, our findings identify skeletal muscle p62 as a potential therapeutic target for cancer cachexia.

Large Maf transcription factor family is a major regulator of fast type IIb myofiber determination.

Myofibers are broadly characterized as fatigue-resistant slow-twitch (type I) fibers and rapidly fatiguing fast-twitch (type IIa/IIx/IIb) fibers. However, the molecular regulation of myofiber type is not entirely understood; particularly, information on regulators of fast-twitch muscle is scarce. Here, we demonstrate that the large Maf transcription factor family dictates fast type IIb myofiber specification in mice. Remarkably, the ablation of three large Mafs leads to the drastic loss of type IIb myofibers, resulting in enhanced endurance capacity and the reduction of muscle force. Conversely, the overexpression of each large Maf in the type I soleus muscle induces type IIb myofibers. Mechanistically, a large Maf directly binds to the Maf recognition element on the promoter of myosin heavy chain 4, which encodes the type IIb myosin heavy chain, driving its expression. This work identifies the large Maf transcription factor family as a major regulator for fast type IIb muscle determination.

Stress Activated MAP Kinases and Cyclin-Dependent Kinase 5 Mediate Nuclear Translocation of Nrf2 via Hsp90α-Pin1-Dynein Motor Transport Machinery.

Non-lethal low levels of oxidative stress leads to rapid activation of the transcription factor nuclear factor-E2-related factor 2 (Nrf2), which upregulates the expression of genes important for detoxification, glutathione synthesis, and defense against oxidative damage. Stress-activated MAP kinases p38, ERK, and JNK cooperate in the efficient nuclear accumulation of Nrf2 in a cell-type-dependent manner. Activation of p38 induces membrane trafficking of a glutathione sensor neutral sphingomyelinase 2, which generates ceramide upon depletion of cellular glutathione. We previously proposed that caveolin-1 in lipid rafts provides a signaling hub for the phosphorylation of Nrf2 by ceramide-activated PKCζ and casein kinase 2 to stabilize Nrf2 and mask a nuclear export signal. We further propose a mechanism of facilitated Nrf2 nuclear translocation by ERK and JNK. ERK and JNK phosphorylation of Nrf2 induces the association of prolyl cis/trans isomerase Pin1, which specifically recognizes phosphorylated serine or threonine immediately preceding a proline residue. Pin1-induced structural changes allow importin-α5 to associate with Nrf2. Pin1 is a co-chaperone of Hsp90α and mediates the association of the Nrf2-Pin1-Hsp90α complex with the dynein motor complex, which is involved in transporting the signaling complex to the nucleus along microtubules. In addition to ERK and JNK, cyclin-dependent kinase 5 could phosphorylate Nrf2 and mediate the transport of Nrf2 to the nucleus via the Pin1-Hsp90α system. Some other ERK target proteins, such as pyruvate kinase M2 and hypoxia-inducible transcription factor-1, are also transported to the nucleus via the Pin1-Hsp90α system to modulate gene expression and energy metabolism. Notably, as malignant tumors often express enhanced Pin1-Hsp90α signaling pathways, this provides a potential therapeutic target for tumors.

p62/Sqstm1 rescue in muscle retards the progression of steatohepatitis in p62/Sqstm1-null mice fed a high-fat diet.

Introduction: Obesity is a risk factor for many diseases because it leads to a reduction in skeletal muscle mass and promotes insulin resistance. p62/Sqstm1-knockout mice are a model of metabolic syndrome; show obesity, insulin resistance, and non-alcoholic fatty liver (NAFL); and develop non-alcoholic steatohepatitis (NASH) in response to the feeding of a high-fat diet (HFD). These phenotypes suggest that muscle p62 may prevent obesity-induced muscle dysfunction. In the present study, we aimed to determine the effects of muscle p62 on skeletal muscle mass, muscle strength, insulin resistance, and NASH pathology. Methods: We generated muscle-specific p62 gene rescue mice (p62-mRes), which express p62 only in muscle and were derived from p62-knock out mice (p62 KIKI ) using the cre/loxp system. p62 KIKI and p62-mRes mice were fed an HFD for 20 weeks and their phenotypes were compared. Results: HFD-feeding caused severe obesity in both p62 KIKI and p62-mRes mice, but there was no effect of muscle p62 on body mass. Limb skeletal muscle mass, grip strength, and the cross-sectional area of muscle fibers were higher in p62-mRes mice than in p62 KIKI . The glucose tolerance and insulin sensitivity of the p62-mRes mice were also superior. The protein expression of mechanistic target of rapamycin, which promotes muscle protein synthesis, and GLUT4, a glucose transporter in skeletal muscle, were higher in the p62-mRes mice. p62 KIKI mice developed severe NASH when fed an HFD, but the progression of NASH was retarded by p62 gene rescue in muscle, and the expression of Tgf-ß1, which encodes a factor that promotes hepatic fibrosis, was reduced. Conclusion: Rescue of muscle-specific p62 in the whole-body p62 knock-out mice ameliorates the insulin resistance and retards the progression of NASH caused by systemic p62 ablation.

Antioxidative Self-Assembling Nanoparticles Attenuate the Development of Steatohepatitis and Inhibit Hepatocarcinogenesis in Mice.

Oxidative stress (OS) contributes to nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. We investigated whether antioxidative self-assembling nanoparticles (SMAPoTN) could reduce the development of NASH and hepatocellular carcinoma (HCC) in p62/Sqstm1 and Nrf2 double knockout (DKO) mice and studied protective mechanisms. We measured disease development in male DKO mice fed a normal chow (NASH model) or a 60% high-fat diet (HFD; HCC model) with or without SMAPoTN administration for 26 weeks. SMAPoTN inhibited liver fibrosis in both groups and prevented HCC development (0% vs. 33%, p < 0.05) in the HFD group. SMAPoTN reduced OS, inflammatory cytokine signaling, and liver fibrosis. RNA-sequencing revealed that SMAPoTN decreased endoplasmic reticulum stress signaling genes in both groups, HCC driver genes, and cancer pathway genes, especially PI3K-AKT in the HFD groups. In the SMAPoTN treatment HFD group, serum lipopolysaccharide levels and liver lipopolysaccharide-binding protein expression were significantly lower compared with those in the nontreatment group. SMAPoTN improved the α-diversity of gut microbiota, and changed the microbiota composition. Oral SMAPoTN administration attenuated NASH development and suppressed hepatocarcinogenesis in DKO mice by improving endoplasmic reticulum stress in the liver and intestinal microbiota. SMAPoTN may be a new therapeutic option for NASH subjects and those with a high HCC risk.

Cystine-dependent antiporters buffer against excess intracellular reactive sulfur species-induced stress.

Reactive sulfur species (RSS) play a role in redox homeostasis; however, adaptive cell responses to excessive intracellular RSS are not well understood. Therefore, in this study, we generated transgenic (Tg) mice overexpressing cystathionine gamma-lyase (CSE) to produce excessive RSS. Contrary to expectations, tissue concentrations of RSS, such as cysteine persulfide (CysSSH), were comparable in both wild-type and CSE Tg mice, but the plasma concentrations of CysSSH were significantly higher in CSE Tg mice than in wild-type mice. This export of surplus intracellular RSS was also observed in primary hepatocytes of CSE Tg mice. Exposure of primary hepatocytes to the RSS generator sodium tetrasulfide (Na2S4) resulted in an initial increase in the intracellular concentration of RSS, which later returned to basal levels after export into the extracellular space. Interestingly, among all amino acids, cystine (CysSSCys) was found to be essential for CysSSH export from primary mouse hepatocytes, HepG2 cells, and HEK293 cells during Na2S4 exposure, suggesting that the cystine/glutamate transporter (SLC7A11) contributes, at least partially, to CysSSH export. We established HepG2 cell lines with knockout and overexpression of SLC7A11 and used them to confirm SLC7A11 as the predominant antiporter of CysSSCys and CysSSH. We observed that the poor efflux of excess CysSSH from the cell enhanced cellular stresses induced by Na2S4 exposure, such as polysulfidation of intracellular proteins, mitochondrial damage, and cytotoxicity. These results suggest the presence of a cellular response to excess intracellular RSS that involves the extracellular efflux of excess CysSSH by a cystine-dependent transporter to maintain intracellular redox homeostasis.

Mechanisms underlying Nrf2 nuclear translocation by non-lethal levels of hydrogen peroxide: p38 MAPK-dependent neutral sphingomyelinase2 membrane trafficking and ceramide/PKCζ/CK2 signaling.

Hydrogen peroxide is an aerobic metabolite playing a central role in redox signaling and oxidative stress. H2O2 could activate redox sensitive transcription factors, such as Nrf2, AP-1 and NF-κB by different manners. In some cells, treatment with non-lethal levels of H2O2 induces rapid activation of Nrf2, which upregulates expression of a set of genes involved in glutathione (GSH) synthesis and defenses against oxidative damage. It depends on two steps, the rapid translational activation of Nrf2 and facilitation of Nrf2 nuclear translocation. We review the molecular mechanisms by which H2O2 induces nuclear translocation of Nrf2 in cultured cells by highlighting the role of neutral sphingomyelinase 2 (nSMase2), a GSH sensor. H2O2 enters cells through aquaporin channels in the plasma membrane and is rapidly reduced to H2O by GSH peroxidases to consume cellular GSH, resulting in nSMase2 activation to generate ceramide. H2O2 also activates p38 MAP kinase, which enhances transfer of nSMase2 from perinuclear regions to plasma membrane lipid rafts to accelerate ceramide generation. Low levels of ceramide activate PKCζ, which then activates casein kinase 2 (CK2). These protein kinases are able to phosphorylate Nrf2 to stabilize and activate it. Notably, Nrf2 also binds to caveolin-1 (Cav1), which protects Nrf2 from Keap1-mediated degradation and limits Nrf2 nuclear translocation. We propose that Cav1serves as a signaling hub for the control of H2O2-mediated phosphorylation of Nrf2 by kinases, which results in release of Nrf2 from Cav1 to facilitate nuclear translocation. In summary, H2O2 induces GSH depletion which is recovered by Nrf2 activation dependent on p38/nSMase2/ceramide signaling.

In vitro assessment of the inhibitory effect of goreisan extract and its ingredients on the P-glycoprotein drug transporter and cytochrome P-450 metabolic enzymes.

Kampo medicines are widely used in Japan; however, their potential to cause drug interactions still remains unclear and needs to be further investigated. The effects of goreisan on the P-glycoprotein (P-gp) and the cytochrome P-450 (CYP), which are associated with drug interactions, were investigated.The inhibitory effect of goreisan extract on P-gp was evaluated using a Caco-2 cell permeability assay. The results indicated that it inhibited P-gp function in a concentration-dependent manner.The inhibitory effect of three goreisan ingredients (alisol A, tumulosic acid, and (E)-cinnamic acid) on seven CYP isoforms was evaluated using human liver microsomes (HLM). Of these, tumulosic acid and (E)-cinnamic acid exhibited less than 16% inhibition at concentrations of 10 µmol/L against any of the CYP isoforms tested. Alisol A inhibited only CYP3A but showed no inhibitory effect with pre-incubation.These results indicate that goreisan extract has inhibitory activity against P-gp and that alisol A, a goreisan ingredient, exhibits an inhibitory effect on CYP3A. However, these are thought to be minor or negligible in vivo. Overall, these findings will be useful to evaluate possible drug interactions and provide support for the interpretation of future clinical drug-drug interaction studies involving goreisan.

Central role for p62/SQSTM1 in the elimination of toxic tau species in a mouse model of tauopathy.

Intracellular accumulation of filamentous tau aggregates with progressive neuronal loss is a common characteristic of tauopathies. Although the neurodegenerative mechanism of tau-associated pathology remains unclear, molecular elements capable of degrading and/or sequestering neurotoxic tau species may suppress neurodegenerative progression. Here, we provide evidence that p62/SQSTM1, a ubiquitinated cargo receptor for selective autophagy, acts protectively against neuronal death and neuroinflammation provoked by abnormal tau accumulation. P301S mutant tau transgenic mice (line PS19) exhibited accumulation of neurofibrillary tangles with localization of p62 mostly in the brainstem, but neuronal loss with few neurofibrillary tangles in the hippocampus. In the hippocampus of PS19 mice, the p62 level was lower compared to the brainstem, and punctate accumulation of phosphorylated tau unaccompanied by co-localization of p62 was observed. In PS19 mice deficient in p62 (PS19/p62-KO), increased accumulation of phosphorylated tau, acceleration of neuronal loss, and exacerbation of neuroinflammation were observed in the hippocampus as compared with PS19 mice. In addition, increase of abnormal tau and neuroinflammation were observed in the brainstem of PS19/p62-KO. Immunostaining and dot-blot analysis with an antibody selectively recognizing tau dimers and higher-order oligomers revealed that oligomeric tau species in PS19/p62-KO mice were significantly accumulated as compared to PS19 mice, suggesting the requirement of p62 to eliminate disease-related oligomeric tau species. Our findings indicated that p62 exerts neuroprotection against tau pathologies by eliminating neurotoxic tau species, suggesting that the manipulative p62 and selective autophagy may provide an intrinsic therapy for the treatment of tauopathy.

SQSTM1, a protective factor of SOD1-linked motor neuron disease, regulates the accumulation and distribution of ubiquitinated protein aggregates in neuron.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motor neurons in the brain and spinal cord. Recent studies have shown that mutations in SQSTM1 are linked to ALS. It has also been demonstrated that a systemic loss of SQSTM1 exacerbates disease phenotypes in an ALS mouse model. However, it is still unclear whether and how SQSTM1 in the central nervous system (CNS) specifically regulates ALS-associated disease phenotypes. To address this issue, we generated CNS-specific Sqstm1 deficient SOD1H46R transgenic mice, and conducted gross phenotype analyses as well as the immunohistochemical and biochemical examinations of spinal cord tissues using these mice. CNS-specific SQSTM1 deficiency accelerated the disease onset and shortened the lifespan of SOD1H46R mice. The CNS-specific SQSTM1 ablation also resulted in increased number of ubiquitin-positive aggregates, while their size rather became much smaller. Remarkably, ubiquitin-positive aggregates, which were usually present in extracellular space and/or neuropil in SOD1H46R mice, were preferentially localized to soma and neurites of spinal neurons in CNS-specific SQSTM1 deficient SOD1H46R mice. Next, to further clarify the function of SQSTM1 in neurons, we investigated the contribution of SQSTM1 to the accumulation of polyubiquitinated proteins in relation to the ubiquitin proteasome system (UPS) and the autophagy-endolysosomal system (APELS) in primary cultured motor neurons (PMNs). Loss of SQSTM1 in PMNs resulted in decreased accumulation of insoluble polyubiquitinated proteins, which was induced by simultaneous treatment with proteasome and lysosome inhibitors, suggesting a pivotal role of SQSTM1 in the formation of insoluble protein aggregates. However, SQSTM1 silencing had a limited impact on the susceptibility to proteasome and/or lysosome inhibitor-induced apoptosis in PMNs. Taken together, neuronal SQSTM1, whose functions are associated with both the UPS and APELS, might primarily regulate the distribution and accumulation of misfolded protein aggregates in the CNS, thereby protecting neurons from degeneration in mice.

Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility.

Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

Mitochondrial reactive oxygen species trigger metformin-dependent antitumor immunity via activation of Nrf2/mTORC1/p62 axis in tumor-infiltrating CD8T lymphocytes.

BACKGROUND: Metformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However, the underlying mechanism of Met-dependent antitumor immunity remains to be elucidated. METHODS: MitoTEMPO, a scavenger of mitochondrial superoxide, abolished the antitumor effect of Met, but not antiprogrammed cell death (PD-1) antibody (Ab) treatment. Consequently, we studied the mechanism of the Met-induced antitumor effect. Expressions of glucose transporter (Glut)-1, mitochondrial reactive oxygen species (mtROS), interferon (IFN)-γ, Ki67, autophagy markers, activation markers for NF-E2-related factor 2 (Nrf2), and mammalian target of rapamaycin complex 1 (mTORC1) in CD8+ tumor-infiltrating T lymphocytes (CD8TILs) were examined by flow cytometry analysis. In addition, conditional knockout mice for Nrf2 and p62 were used to detect these markers, together with the monitoring of in vivo tumor growth. RNA sequencing was performed for CD8TILs and tumor cells. Melanoma cells containing an IFN-γ receptor (IFNγR) cytoplasmic domain deletion mutant was overexpressed and used for characterization of the metabolic profile of those tumor cells using a Seahorse Flux Analyzer. RESULTS: Met administration elevates mtROS and cell surface Glut-1, resulting in the production of IFN-γ in CD8TILs. mtROS activates Nrf2 in a glycolysis-dependent manner, inducing activation of autophagy, glutaminolysis, mTORC1, and p62/SQSTM1. mTORC1-dependent phosphorylation of p62 at serine 351 (p-p62(S351)) is also involved in activation of Nrf2. Conditional deletion of Nrf2 in CD8TILs abrogates mTORC1 activation and antitumor immunity by Met. In synergy with the effect of anti-PD-1 Ab, Met boosts CD8TIL proliferation and IFN-γ secretion, resulting in decreased glycolysis and oxidative phosphorylation in tumor cells. Consequently, Glut-1 is elevated in CD8TILs, together with the expansion of activated dendritic cells. Moreover, tumor cells lacking in IFNγR signaling abolish IFN-γ production and proliferation of CD8TILs. CONCLUSIONS: We found that Met stimulates production of mtROS, which triggers Glut-1 elevation and Nrf2 activation in CD8TILs. Nrf2 activates mTORC1, whereas mTORC1 activates Nrf2 in a p-p62(S351)-dependent manner, thus creating a feedback loop that ensures CD8TILs' proliferation. In combination with anti-PD-1 Ab, Met stimulates robust proliferation of CD8TILs and IFN-γ secretion, resulting in an IFN-γ-dependent reprogramming of the tumor microenvironment.

Mechanisms underlying unidirectional laminar shear stress-mediated Nrf2 activation in endothelial cells: Amplification of low shear stress signaling by primary cilia.

Endothelial cells are sensitive to mechanical stress and respond differently to oscillatory flow versus unidirectional flow. This review highlights the mechanisms by which a wide range of unidirectional laminar shear stress induces activation of the redox sensitive antioxidant transcription factor nuclear factor-E2-related factor 2 (Nrf2) in cultured endothelial cells. We propose that fibroblast growth factor-2 (FGF-2), brain-derived neurotrophic factor (BDNF) and 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) are potential Nrf2 activators induced by laminar shear stress. Shear stress-dependent secretion of FGF-2 and its receptor-mediated signaling is tightly controlled, requiring neutrophil elastase released by shear stress, αvß3 integrin and the cell surface glycocalyx. We speculate that primary cilia respond to low laminar shear stress (<10 dyn/cm2), resulting in secretion of insulin-like growth factor 1 (IGF-1), which facilitates αvß3 integrin-dependent FGF-2 secretion. Shear stress induces generation of heparan-binding epidermal growth factor-like growth factor (HB-EGF), which contributes to FGF-2 secretion and gene expression. Furthermore, HB-EGF signaling modulates FGF-2-mediated NADPH oxidase 1 activation that favors casein kinase 2 (CK2)-mediated phosphorylation/activation of Nrf2 associated with caveolin 1 in caveolae. Higher shear stress (>15 dyn/cm2) induces vesicular exocytosis of BDNF from endothelial cells, and we propose that BDNF via the p75NTR receptor could induce CK2-mediated Nrf2 activation. Unidirectional laminar shear stress upregulates gene expression of FGF-2 and BDNF and generation of 15d-PGJ2, which cooperate in sustaining Nrf2 activation to protect endothelial cells against oxidative damage.

Nuclear factor E2-related factor 2 (NRF2) deficiency accelerates fast fibre type transition in soleus muscle during space flight.

Microgravity induces skeletal muscle atrophy, particularly in the soleus muscle, which is predominantly composed of slow-twitch myofibre (type I) and is sensitive to disuse. Muscle atrophy is commonly known to be associated with increased production of reactive oxygen species. However, the role of NRF2, a master regulator of antioxidative response, in skeletal muscle plasticity during microgravity-induced atrophy, is not known. To investigate the role of NRF2 in skeletal muscle within a microgravity environment, wild-type and Nrf2-knockout (KO) mice were housed in the International Space Station for 31 days. Gene expression and histological analyses demonstrated that, under microgravity conditions, the transition of type I (oxidative) muscle fibres to type IIa (glycolytic) was accelerated in Nrf2-KO mice without affecting skeletal muscle mass. Therefore, our results suggest that NRF2 affects myofibre type transition during space flight.

Prevention of non-alcoholic steatohepatitis by long-term exercise via the induction of phenotypic changes in Kupffer cells of hyperphagic obese mice.

Exercise ameliorates nonalcoholic fatty liver disease (NAFLD) by inducing phenotypic changes in Kupffer cells (KCs). p62/Sqstm1-knockout (p62-KO) mice develop NAFLD alongside hyperphagia-induced obesity. We evaluated (1) the effects of long-term exercise on the foreign-body phagocytic capacity of KCs, their surface marker expression, and the production of steroid hormones in p62-KO mice; and (2) whether long-term exercise prevented the development of non-alcoholic steatohepatitis (NASH) in p62-KO mice fed a high-fat diet (HFD). In experiment 1, 30-week-old male p62-KO mice were allocated to resting (p62-KO-Rest) or exercise (p62-KO-Ex) groups, and the latter performed long-term exercise over 4 weeks. Then, the phenotype of their KCs was compared to that of p62-KO-Rest and wild-type (WT) mice. In experiment 2, 5-week-old male p62-KO mice that were fed a HFD performed long-term exercise over 12 weeks. In experiment 1, the phagocytic capacity of KCs and the proportion of CD68-positive cells were lower in the p62-KO-Rest group than in the WT group, but they increased with long-term exercise. The percentage of CD11b-positive KCs was higher in the p62-KO-Rest group than in the WT group, but lower in the p62-KO-Ex group. The circulating dehydroepiandrosterone (DHEA) concentration was higher in p62-KO-Ex mice than in p62-KO-Rest mice. In experiment 2, the body mass and composition of the p62-KO-Rest and p62-KO-Ex groups were similar, but the hepatomegaly, hepatic inflammation, and fibrosis were less marked in p62-KO-Ex mice. The DHEA concentration was higher in p62-KO-Ex mice than in WT or p62-KO-Rest mice. Thus, long-term exercise restores the impaired phagocytic capacity of KCs in NAFLD obese mice, potentially through greater DHEA production, and prevents the development of NASH by ameliorating hepatic inflammation and fibrogenesis. These results suggest a molecular mechanism for the beneficial effect of exercise in the management of patients with NAFLD.

Redox cycling of 9,10-phenanthrenequinone activates epidermal growth factor receptor signaling through S-oxidation of protein tyrosine phosphatase 1B.

[Page 358, Figure 5C] The label of Western blot data in Figure 5C in the paper was erroneously placed.

Redox cycling of 9,10-phenanthrenequinone activates epidermal growth factor receptor signaling through S-oxidation of protein tyrosine phosphatase 1B.

9,10-Phenanthrenequinone (9,10-PQ) is a polycyclic aromatic hydrocarbon quinone contaminated in diesel exhaust particles and particulate matter 2.5. It is an efficient electron acceptor that induces redox cycling with electron donors, resulting in excessive reactive oxygen species and oxidized protein production in cells. The current study examined whether 9,10-PQ could activate epidermal growth factor receptor (EGFR) signaling in A431 cells through S-oxidation of its negative regulators such as protein tyrosine phosphatase (PTP) 1B. 9,10-PQ oxidized recombinant human PTP1B at Cys215 and inhibited its catalytic activity, an effect that was blocked by catalase (CAT), whereas cis-9,10-dihydroxy-9,10-dihydrophenanthrene (DDP), which lacks redox cycling activity, had no effect on PTP1B activity. Exposure of A431 cells to 9,10-PQ, but not DDP, activated signaling through EGFR and its downstream extracellular signal-regulated kinase 1/2 (ERK1/2), coupled with a decrease of cellular PTP activity. Immunoprecipitation and UPLC-MSE revealed that PTP1B easily undergoes oxidation during exposure of A431 cells to 9,10-PQ. Pretreatment with polyethylene glycol conjugated with CAT (PEG-CAT) abolished 9,10-PQ-generated H2O2 production and significantly blocked the activation of EGFR-ERK1/2 signaling by 9,10-PQ, indicating the involvement of H2O2 in the activation because scavenging agents for hydroxyl radicals had no effect on the redox signal activation. These results suggest that such an air pollutant producing H2O2, activates EGFR-ERK1/2 signaling, presumably through the S-oxidation of PTPs such as PTP1B, and activation of the signal cascade may contribute, at least in part, to cellular responses in A431 cells.

Gender difference in development of steatohepatitis in p62/Sqstm1 and Nrf2 double-knockout mice.

Gender and menopause influence the severity and development manner of nonalcoholic steatohepatitis (NASH). Male p62/Sqstm1 and nuclear factor E2-related factor-2 (p62 and Nrf2) double-knockout (DKO) mice exhibit severe steatohepatitis caused by hyperphagia-induced obesity, overload of lipopolysaccharide (LPS) into the liver, and potentiation of the inflammatory response in Kupffer cells. However, the pathogenetic phenotype of steatohepatitis in female DKO mice remains unknown. Phenotypic changes of steatohepatitis in DKO mice were compared in terms of gender differences. Compared with DKO male mice, DKO female mice exhibited later onset of steatohepatitis with obesity after 30 weeks of age, as well as milder severity of hepatic inflammation and fibrosis. Serum estradiol was higher in female than male mice, with levels increasing up to 30 weeks of age before decreasing until 50 weeks of age (corresponding to the post-menopausal period). Fecal and serum LPS were lower in female mice than male mice, and inflammatory signaling in the liver was attenuated in female compared with male mice. Correlating with LPS levels, the composition of intestinal microbiota in female mice was different from male mice. Gender differences were observed for the development of steatohepatitis in DKO mice. Low-grade inflammatory hit in the liver under in vivo conditions of high estradiol may be attributable to the milder pathological features of steatohepatitis in female mice.