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.

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.

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.

A Substituent Effect on Two-Photon Absorption of Diphenylacetylene Derivatives with an Electron-Donating/Withdrawing Group.

Two-photon absorption for diphenylacetylene derivatives with an electron-donating (ED) or electron-withdrawing (EW) group (DPA-Rs) was investigated by high-sensitivity optical-probing photoacoustic spectroscopy. Two-photon absorption spectra and two-photon absorption cross sections σ(2) for DPA-Rs were successfully obtained. Two-photon absorption spectra of DPA-Rs with stronger ED or EW groups display more significant red-shifts and larger σ(2) values. Simulated two-photon absorption spectra, using time-dependent density functional theory within the Tamm-Dancoff approximation, compared well with the experimental spectra. Based on the three-state model, the substituent effect on the two-photon absorption for DPA-Rs was expected to manifest in the transition dipole moments and detuning energies. Information obtained from investigating the monosubstituent effect on two-photon absorption of DPA is critical for an improved understanding of two-photon absorption.

Two-photon absorption property of Cl-substituted diphenylacetylenes by optical-probing photoacoustic spectroscopy.

Two-photon absorption spectra and two-photon absorption cross sections of Cl-substituted diphenylacetylenes (ClDPAs) were investigated by optical-probing photoacoustic spectroscopy and quantum chemical calculations for the first time. The two-photon absorption spectra of ClDPAs exhibited intense two-photon absorption bands at around 480 nm, which are forbidden by one-photon absorption. The two-photon absorption cross sections σ(2) of o-, m-, and p-ClDPAs at 476 nm were determined to be 22 ± 1, 23 ± 1, and 38 ± 2 GM, respectively. Compared with diphenylacetylene (DPA) (27 GM at 472 nm), the σ(2) values of o- and m-ClDPAs were lower, while that of p-ClDPA was higher. Simulated two-photon absorption spectra using the TD-B3LYP/6-311+G(d,p) level of calculations within the Tamm-Dancoff approximation, based on the three-state model, well agreed with the experimental results. The difference in the σ(2) values of DPA and ClDPAs was responsible for those in the transition dipole moments between the intermediate and the final states.

Mechanism of Rapid Nuclear Factor-E2-Related Factor 2 (Nrf2) Activation via Membrane-Associated Estrogen Receptors: Roles of NADPH Oxidase 1, Neutral Sphingomyelinase 2 and Epidermal Growth Factor Receptor (EGFR).

Membrane-associated estrogen receptors (ER)-α36 and G protein-coupled estrogen receptor (GPER) play important roles in the estrogen's rapid non-genomic actions including stimulation of cell proliferation. Estrogen via these receptors induces rapid activation of transcription factor nuclear factor-E2-related factor 2 (Nrf2), a master regulator of detoxification and antioxidant systems, playing a key role in the metabolic reprogramming to support cell proliferation. This review highlights the possible mechanism underlying rapid Nrf2 activation via membrane-associated estrogen receptors by estrogen and phytoestrogens. Stimulation of ER-α36-GPER signaling complex rapidly induces Src-mediated transactivation of epidermal growth factor receptor (EGFR) leading to a kinase-mediated signaling cascade. We propose a novel hypothesis that ER-α36-GPER signaling initially induces rapid and temporal activation of NADPH oxidase 1 to generate superoxide, which subsequently activates redox-sensitive neutral sphingomyelinase 2 generating the lipid signaling mediator ceramide. Generation of ceramide is required for Ras activation and ceramide-protein kinase C ζ-casein kinase 2 (CK2) signaling. Notably, CK2 enhances chaperone activity of the Cdc37-Hsp90 complex supporting activation of various signaling kinases including Src, Raf and Akt (protein kinase B). Activation of Nrf2 may be induced by cooperation of two signaling pathways, (i) Nrf2 stabilization by direct phosphorylation by CK2 and (ii) EGFR-Ras-PI 3 kinase (PI3K)-Akt axis which inhibits glycogen synthase kinase 3ß leading to enhanced nuclear transport and stability of Nrf2.

Circadian control of BDNF-mediated Nrf2 activation in astrocytes protects dopaminergic neurons from ferroptosis.

Astrocyte-neuron interactions protect neurons from iron-mediated toxicity. As dopamine can be metabolized to reactive quinones, dopaminergic neurons are susceptible to oxidative damage and ferroptosis-like induced cell death. Detoxification enzymes are required to protect neurons. Brain-derived neurotrophic factor (BDNF) plays a key role in the regulation of redox sensitive transcription factor Nrf2 in astrocytes and metabolic cooperation between astrocytes and neurons. This article reviews the importance of BDNF and astrocyte-neuron interactions in the protection of neurons against oxidative damages in rodent brains. We previously proposed that BDNF activates Nrf2 via the truncated TrkB.T1 and p75NTR receptor complex in astrocytes. Stimulation by BDNF generates the signaling molecule ceramide, which activates PKCζ leading to induction of the CK2-Nrf2 signaling axis. As a cell clock regulates p75NTR expression, we suggested that BDNF effectively activates Nrf2 in astrocytes during the rest phase. In contrast, neurons express both TrkB.FL and TrkB.T1, and TrkB.FL tyrosine kinase activity inhibits p75NTR-dependent ceramide generation and internalizes p75NTR. Therefore, BDNF may not effectively activate Nrf2 in neurons. Notably, neurons only weakly activate detoxification and antioxidant enzymes/proteins via the Nrf2-ARE signaling axis. Thus, astrocytes may provide relevant transcripts and/or proteins to neurons via microparticles/exosomes increasing neuronal resistance to oxidative stress. Circadian increases in the levels of circulating glucocorticoids may further facilitate material transfer from astrocytes to neurons via the stimulation of pannexin 1 channels-P2X7R signaling pathway in astrocytes at the beginning of the active phase. Dysregulation of astrocyte-neuron interactions could therefore contribute to the pathogenesis of neurodegenerative diseases including Parkinson's disease.

Circadian control of p75 neurotrophin receptor leads to alternate activation of Nrf2 and c-Rel to reset energy metabolism in astrocytes via brain-derived neurotrophic factor.

Circadian clock genes regulate energy metabolism partly through neurotrophins in the body. The low affinity neurotrophin receptor p75NTR is a clock component directly regulated by the transcriptional factor Clock:Bmal1 complex. Brain-derived neurotrophic factor (BDNF) is expressed in the brain and plays a key role in coordinating metabolic interactions between neurons and astrocytes. BDNF transduces signals through TrkB and p75NTR receptors. This review highlights a novel molecular mechanism by which BDNF via circadian control of p75NTR leads to daily resetting of glucose and glycogen metabolism in brain astrocytes to accommodate their functional interaction with neurons. Astrocytes store glycogen as an energy reservoir to provide active neurons with the glycolytic metabolite lactate. Astrocytes predominantly express the truncated receptor TrkB.T1 which lacks an intracellular receptor tyrosine kinase domain. TrkB.T1 retains the capacity to regulate cell morphology through regulation of Rho GTPases. In contrast, p75NTR mediates generation of the bioactive lipid ceramide upon stimulation with BDNF and inhibits PKA activation. As ceramide directly activates PKCζ, we discuss the importance of the TrkB.T1-p75NTR-ceramide-PKCζ signaling axis in the stimulation of glycogen and lipid synthesis and activation of RhoA. Ceramide-PKCζ-casein kinase 2 signaling activates Nrf2 to support oxidative phosphorylation via upregulation of antioxidant enzymes. In the absence of p75NTR, TrkB.T1 functionally interacts with adenosine A2AR and dopamine D1R receptors to enhance cAMP-PKA signaling and activate Rac1 and NF-κB c-Rel, favoring glycogen hydrolysis, gluconeogenesis and aerobic glycolysis. Thus, diurnal changes in p75NTR levels in astrocytes resets energy metabolism via BDNF to accommodate their metabolic interaction with neurons.

Deletion of both p62 and Nrf2 spontaneously results in the development of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is one of the leading causes of chronic liver disease worldwide. However, details of pathogenetic mechanisms remain unknown. Deletion of both p62/Sqstm1 and Nrf2 genes spontaneously led to the development of NASH in mice fed a normal chow and was associated with liver tumorigenesis. The pathogenetic mechanism (s) underlying the NASH development was investigated in p62:Nrf2 double-knockout (DKO) mice. DKO mice showed massive hepatomegaly and steatohepatitis with fat accumulation and had hyperphagia-induced obesity coupled with insulin resistance and adipokine imbalance. They also showed dysbiosis associated with an increased proportion of gram-negative bacteria species and an increased lipopolysaccharide (LPS) level in feces. Intestinal permeability was elevated in association with both epithelial damage and decreased expression levels of tight junction protein zona occludens-1, and thereby LPS levels were increased in serum. For Kupffer cells, the foreign body phagocytic capacity was decreased in magnetic resonance imaging, and the proportion of M1 cells was increased in DKO mice. In vitro experiments showed that the inflammatory response was accelerated in the p62:Nrf2 double-deficient Kupffer cells when challenged with a low dose of LPS. Diet restriction improved the hepatic conditions of NASH in association with improved dysbiosis and decreased LPS levels. The results suggest that in DKO mice, activation of innate immunity by excessive LPS flux from the intestines, occurring both within and outside the liver, is central to the development of hepatic damage in the form of NASH.

Peroxiredoxin 1 Contributes to Host Defenses against Mycobacterium tuberculosis.

Peroxiredoxin (PRDX)1 is an antioxidant that detoxifies hydrogen peroxide and peroxinitrite. Compared with wild-type (WT) mice, Prdx1-deficient (Prdx1-/-) mice showed increased susceptibility to Mycobacterium tuberculosis and lower levels of IFN-γ and IFN-γ-producing CD4+ T cells in the lungs after M. tuberculosis infection. IL-12 production, c-Rel induction, and p38 MAPK activation levels were lower in Prdx1-/- than in WT bone marrow-derived macrophages (BMDMs). IFN-γ-activated Prdx1-/- BMDMs did not kill M. tubercuosis effectively. NO production levels were lower, and arginase activity and arginase 1 (Arg1) expression levels were higher, in IFN-γ-activated Prdx1-/- than in WT BMDMs after M. tuberculosis infection. An arginase inhibitor, Nω-hydroxy-nor-arginine, restored antimicrobial activity and NO production in IFN-γ-activated Prdx1-/- BMDMs after M. tuberculosis infection. These results suggest that PRDX1 contributes to host defenses against M. tuberculosis PRDX1 positively regulates IL-12 production by inducing c-Rel and activating p38 MAPK, and it positively regulates NO production by suppressing Arg1 expression in macrophages infected with M. tuberculosis.

Functional links between SQSTM1 and ALS2 in the pathogenesis of ALS: cumulative impact on the protection against mutant SOD1-mediated motor dysfunction in mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons in the brain and spinal cord. Multiple toxicity pathways, such as oxidative stress, misfolded protein accumulation, and dysfunctional autophagy, are implicated in the pathogenesis of ALS. However, the molecular basis of the interplay between such multiple factors in vivo remains unclear. Here, we report that two independent ALS-linked autophagy-associated gene products; SQSTM1/p62 and ALS2/alsin, but not antioxidant-related factor; NFE2L2/Nrf2, are implicated in the pathogenesis in mutant SOD1 transgenic ALS models. We generated SOD1H46R mice either on a Nfe2l2-null, Sqstm1-null, or Sqstm1/Als2-double null background. Loss of SQSTM1 but not NFE2L2 exacerbated disease symptoms. A simultaneous inactivation of SQSTM1 and ALS2 further accelerated the onset of disease. Biochemical analyses revealed that loss of SQSTM1 increased the level of insoluble SOD1 at the intermediate stage of the disease, whereas no further elevation occurred at the end-stage. Notably, absence of SQSTM1 rather suppressed the mutant SOD1-dependent accumulation of insoluble polyubiquitinated proteins, while ALS2 loss enhanced it. Histopathological examinations demonstrated that loss of SQSTM1 accelerated motor neuron degeneration with accompanying the preferential accumulation of ubiquitin-positive aggregates in spinal neurons. Since SQSTM1 loss is more detrimental to SOD1H46R mice than lack of ALS2, the selective accumulation of such aggregates in neurons might be more insulting than the biochemically-detectable insoluble proteins. Collectively, two ALS-linked factors, SQSTM1 and ALS2, have distinct but additive protective roles against mutant SOD1-mediated toxicity by modulating neuronal proteostasis possibly through the autophagy-endolysosomal system.

Close teamwork between Nrf2 and peroxiredoxins 1 and 6 for the regulation of prostaglandin D2 and E2 production in macrophages in acute inflammation.

Inflammation is a complex biological self-defense reaction triggered by tissue damage or infection by pathogens. Acute inflammation is regulated by the time- and cell type-dependent production of cytokines and small signaling molecules including reactive oxygen species and prostaglandins. Recent studies have unveiled the important role of the transcription factor Nrf2 in the regulation of prostaglandin production through transcriptional regulation of peroxiredoxins 1 and 6 (Prx1 and Prx6) and lipocalin-type prostaglandin D synthase (L-PGDS). Prx1 and Prx6 are multifunctional proteins important for cell protection against oxidative stress, but also work together to facilitate production of prostaglandins E2 and D2 (PGE2 and PGD2). Prx1 secreted from cells under mild oxidative stress binds Toll-like receptor 4 and induces NF-κB activation, important for the expression of cyclooxygenase-2 and microsomal PGE synthase-1 (mPGES-1) expression. The activated MAPKs p38 and ERK phosphorylate Prx6, leading to NADPH oxidase-2 activation, which contributes to production of PGD2 by hematopoietic prostaglandin D synthase (H-PGDS). PGD2 and its end product 15-deoxy-∆(12,14)-prostaglandin J2 (15d-PGJ2) activate Nrf2 thereby forming a positive feedback loop for further production of PGD2 by L-PGDS. Maintenance of cellular glutathione levels is an important role of Nrf2 not only for cell protection but also for the synthesis of prostaglandins, as mPGES-1 and H-PGDS require glutathione for their activities. This review is aimed at describing the functions of Prx1 and Prx6 in the regulation of PGD2 and PGE2 production in acute inflammation in macrophages and the importance of 15d-PGJ2 as an intrinsic Nrf2 activator.

Redox status in mammalian cells and stem cells during culture in vitro: critical roles of Nrf2 and cystine transporter activity in the maintenance of redox balance.

Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms regulating redox signaling in cells with implications for medicine. However, standard culture techniques maintain mammalian cells in vitro under an artificial physicochemical environment such as ambient air and 5% CO2. Oxidative stress is caused by the rapid oxidation of cysteine to cystine in culture media catalyzed by transition metals, leading to diminished intracellular cysteine and glutathione (GSH) pools. Some cells, such as fibroblasts and macrophages, express cystine transport activity, designated as system [Formula: see text], which enables cells to maintain these pools to counteract oxidative stress. Additionally, many cells have the ability to activate the redox sensitive transcription factor Nrf2, a master regulator of cellular defenses against oxidative stress, and to upregulate xCT, the subunit of the [Formula: see text] transport system leading to increases in cellular GSH. In contrast, some cells, including lymphoid cells, embryonic stem cells and iPS cells, express relatively low levels of xCT and cannot maintain cellular cysteine and GSH pools. Thus, fibroblasts have been used as feeder cells for the latter cell types based on their ability to supply cysteine. Other key Nrf2 regulated gene products include heme oxygenase 1, peroxiredoxin 1 and sequestosome1. In macrophages, oxidized LDL activates Nrf2 and upregulates the scavenger receptor CD36 forming a positive feedback loop to facilitate removal of the oxidant from the vascular microenvironment. This review describes cell type specific responses to oxygen derived stress, and the key roles that activation of Nrf2 and membrane transport of cystine and cysteine play in the maintenance and proliferation of mammalian cells in culture.

Peroxiredoxin I plays a protective role against UVA irradiation through reduction of oxidative stress.

BACKGROUND: Exposure of skin to long-wave UV radiation (UVA) increases the cellular levels of reactive oxygen species (ROS), which have been linked to apoptosis induction through the damage of lipids, proteins, and nucleic acids. Peroxiredoxin I (Prx I) is one of a family of antioxidant proteins that plays a protective role against oxidative damage; however the role of Prx I in UVA-induced damage remains to be clarified. OBJECTIVE: Here we investigated the protective role of Prx I against UVA-induced changes using mouse embryonic fibroblasts (MEFs) derived from Prx I homozygous knockout (Prx I (-/-)) mice. METHODS: Prx I (-/-) and wild-type (Prx I (+/+)) MEFs were subjected to UVA irradiation, and the resulting apoptosis was analyzed using flow cytometry, quantitative real-time PCR, and western blotting. RESULTS: Prx I (-/-) MEFs showed enhanced sensitivity to UVA treatment, exhibiting increased apoptosis and ROS production compared to Prx I (+/+) MEFs. Consistent with the increase in apoptosis, p53 expression was significantly higher, while Bcl-2, Bcl-xL, and Nrf2 expressions were all lower in Prx I (-/-) versus (+/+) MEFs. The UVA-induced inflammatory response was upregulated in Prx I (-/-) MEFs, as indicated by increased expressions of IκB, TNFα, and IL-6. Evidence was presented indicating that Prx I impacts these pathways by modifying critical signaling intermediates including p53, IκB, and Nrf2. CONCLUSION: Our results indicate that Prx I plays a protective role against UVA-induced oxidative damage by controlling ROS accumulation. Both the UVA-induced apoptotic and inflammatory signals were found to be modulated by Prx I.

Deficiency of p62/Sequestosome 1 causes hyperphagia due to leptin resistance in the brain.

The cytoplasmic regulatory protein p62 (Sequestosome 1/A170) is known to modulate various receptor-mediated intracellular signaling pathways. p62 deficiency was shown to result in mature-onset obesity in mice, but the mechanisms underlying this abnormality remained unclear. Here we report that hyperphagia due to central leptin resistance is the cause of obesity in p62(-/-) mice. We found that these mice show hyperphagia. Restriction of food to the amount eaten by wild-type mice prevented excess body weight gain and fat accumulation, suggesting that overfeeding is the primary cause of obesity in p62(-/-) mice. Brain-specific p62 deficiency caused mature-onset obesity to the same extent as in p62(-/-) mice, further supporting a neuronal mechanism as the major cause of obesity in these mice. Immunohistochemical analysis revealed that p62 is highly expressed in hypothalamic neurons, including POMC neurons in the arcuate nucleus. Central leptin resistance was observed even in young preobese p62(-/-) mice. We found a defect in intracellular distribution of the transcription factor Stat3, which is essential for the action of leptin, in p62(-/-) mice. These results indicate that brain p62 plays an important role in bodyweight control by modulating the central leptin-signaling pathway and that lack of p62 in the brain causes leptin resistance, leading to hyperphagia. Thus, p62 could be a clinical target for treating obesity and metabolic syndrome.

Sequestosome1/p62: a regulator of redox-sensitive voltage-activated potassium channels, arterial remodeling, inflammation, and neurite outgrowth.

Sequestosome1/p62 (SQSTM1) is an oxidative stress-inducible protein regulated by the redox-sensitive transcription factor Nrf2. It is not an antioxidant but known as a multifunctional regulator of cell signaling with an ability to modulate targeted or selective degradation of proteins through autophagy. SQSTM1 implements these functions through physical interactions with different types of proteins including atypical PKCs, nonreceptor-type tyrosine kinase p56(Lck) (Lck), polyubiquitin, and autophagosomal factor LC3. One of the notable physiological functions of SQSTM1 is the regulation of redox-sensitive voltage-gated potassium (Kv) channels which are composed of α and ß subunits: (Kvα)4 (Kvß)4. Previous studies have established that SQSTM1 scaffolds PKCζ, enhancing phosphorylation of Kvß which induces inhibition of pulmonary arterial Kv1.5 channels under acute hypoxia. Recent studies reveal that Lck indirectly interacts with Kv1.3 α subunits and plays a key role in acute hypoxia-induced Kv1.3 channel inhibition in T lymphocytes. Kv1.3 channels provide a signaling platform to modulate the migration and proliferation of arterial smooth muscle cells and activation of T lymphocytes, and hence have been recognized as a therapeutic target for treatment of restenosis and autoimmune diseases. In this review, we focus on the functional interactions of SQSTM1 with Kv channels through two key partners aPKCs and Lck. Furthermore, we provide molecular insights into the functions of SQSTM1 in suppression of proliferation of arterial smooth muscle cells and neointimal hyperplasia following carotid artery ligation, in T lymphocyte differentiation and activation, and in NGF-induced neurite outgrowth in PC12 cells.

p62/SQSTM1 differentially removes the toxic mutant androgen receptor via autophagy and inclusion formation in a spinal and bulbar muscular atrophy mouse model.

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders that are caused by the expansion of trinucleotide CAG repeats in the causative genes. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease that is caused by the expansion of a polyQ tract within the androgen receptor (AR). p62 is a ubiquitin- and light-chain 3-binding protein that is known to regulate the degradation of targeted proteins via autophagy and inclusion formation. In this study, we examined the effects of p62 depletion and overexpression on cultured cells and in a transgenic mouse model that overexpressed the mutant AR. Here, we demonstrate that depletion of p62 significantly exacerbated motor phenotypes and the neuropathological outcome, whereas overexpression of p62 protected against mutant AR toxicity in SBMA mice. Depletion of p62 significantly increased the levels of monomeric mutant AR and mutant AR protein complexes in an SBMA mouse model via the impairment of autophagic degradation. In addition, p62 overexpression improved SBMA mouse phenotypes by inducing cytoprotective inclusion formation. Our results demonstrate that p62 provides two different therapeutic targets in SBMA pathogenesis: (1) autophagy-dependent degradation and (2) benevolent inclusion formation of the mutant AR.

Amelioration of cisplatin-induced nephrotoxicity in peroxiredoxin I-deficient mice.

PURPOSE: Cisplatin is one of the most potent chemotherapeutic agents used to treat cancer. However, cisplatin-induced nephrotoxicity, which is partly caused by oxidative damage, is a serious problem. We previously showed that murine embryonic fibroblasts deficient in Peroxiredoxin I (Prx I), a major Nrf2-linked anti-oxidant enzyme, are susceptible to cisplatin-induced cytotoxicity. In the present study, we examined the role of Prx I against cisplatin-induced renal injury in vivo using Prx I-null mice. METHODS: Prx I-null mice and wild-type (WT) mice were given an intraperitoneal injection of cisplatin, and tissues were removed and evaluated histopathologically. In addition, gene and protein expression of efflux transporters was analyzed. RESULTS: In contrast to an in vitro cell study, Prx I-null mice exhibited less cisplatin-induced renal damage than WT mice in histological and blood biochemical analyses. Moreover, Prx I-null mice showed a higher clearance rate of cisplatin than WT mice following intraperitoneal cisplatin injection. Consistent with these results, Prx I-null mice exhibited higher expression of renal efflux transporters Mrp2 and Mrp4 compared with WT mice under both basal and the cisplatin-induced conditions. We suggest the enhanced transcriptional activity of c-Myc in Prx I-null mice may partly contribute the enhanced expression of renal efflux transporters. CONCLUSION: In summary, the enhanced clearance rate of cisplatin significantly attenuates nephrotoxicity in Prx I-null mice.

Deletion of Nrf2 leads to rapid progression of steatohepatitis in mice fed atherogenic plus high-fat diet.

BACKGROUND: The transcription factor nuclear factor-E2-related factor-2 (Nrf2) inhibits lipid accumulation and oxidative stress in the liver by interfering with lipogenic pathways and inducing antioxidative stress genes. METHODS: The involvement of Nrf2 in defense against the development of steatohepatitis was studied in an experimental model induced by an atherogenic plus high-fat (Ath + HF) diet. Wild-type (WT) and Nrf2-null mice were fed the diet. Their specimens were analyzed for pathology as well as for the expression levels of genes involved in fatty acid metabolism and those involved via the Nrf2 transcriptional pathway. RESULTS: In Nrf2-null mice fed the diet, steatohepatitis developed rapidly, leading to precirrhosis. The Ath + HF diet increased hepatic triglyceride levels and changed fatty acid composition in both mouse groups. However, oleic acid (C18:1 n-9) predominated in the livers of Nrf2-null mice. Correlating well with the pathology, the mRNA levels of the factors involved in fatty acid metabolism (Lxr, Srebp-1a, 1c, Acc-1, Fas, Scd-1, and Fatty acid transporting peptides 1, 3, 4), the inflammatory cytokine genes (Tnf-α and IL-1ß), and the fibrogenesis-related genes (Tgf-ß1 and α-Sma) were significantly increased in the livers of Nrf2-null mice fed the diet, compared with the levels of these factors in matched WT mice. Oxidative stress was significantly increased in the livers of Nrf2-null mice fed the diet. This change was closely associated with the decreased levels of antioxidative stress genes. CONCLUSIONS: Nrf2 deletion leads to the rapid onset and progression of steatohepatitis induced by an Ath + HF diet, through both up-regulation of co-regulators of fatty acid metabolism and down-regulation of oxidative metabolism regulators in the liver.