Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.
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ABSTRACT:
SUBMITTER: Park JY
PROVIDER: S-EPMC8055680 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transcriptional factor EB (TFEB) and nuclear factor E2-related factor 2 (Nrf2) play crucial roles in the biological response against cellular stressors; however, their relationship has not yet been investigated. Here, we constructed human neuroglioma cell lines stably expressing TFEB. The expression of Nrf2-response genes, including heme oxygenase (HO)-1, glutathione-s-transferase-mu1 (GSTM1), and p62, was induced in the cell line, independent of oxidative stress. Of note, the protein level of Nrf2 was significantly increased, and its ubiquitinated fraction was reduced in stable cells compared to that in the control cells. Among E3 ubiquitin ligases known to be involved in the ubiquitination of Nrf2, DDB1 and Cullin4 associated factor 11 (DCAF11) was down-regulated at both protein and mRNA levels in stable cells, indicating that the repression of DCAF11 by TFEB may be mainly involved in the stabilization of Nrf2. In addition, the level of phosphorylated p62 at S349 was highly increased in stable cells compared to that in control cells, which could allow it to interfere with the association of Keap1 and Nrf2, thus stabilizing Nrf2. We suggest for the first time that TFEB could activate Nrf2 by increasing its stability under conditions devoid of oxidative stress.
| S-EPMC6778067 | biostudies-literature
Project description:Tolvaptan, a vasopressin type 2 receptor antagonist initially developed to increase free-water diuresis, has been approved for the treatment of autosomal dominant polycystic kidney disease in multiple countries. Furthermore, tolvaptan has been shown to improve the renal functions in rodent models of chronic kidney disease (CKD); however, the underlying molecular mechanisms remain unknown. CKD is characterized by increased levels of oxidative stress, and an antioxidant transcription factor-nuclear factor erythroid 2-related factor 2 (Nrf2)-has been gaining attention as a therapeutic target. Therefore, we investigated the effects of tolvaptan and a well-known Nrf2 activator, bardoxolone methyl (BARD) on Nrf2. To determine the role of tolvaptan, we used a renal cortical collecting duct (mpkCCD) cell line and mouse kidneys. Tolvaptan activated Nrf2 and increased mRNA and protein expression of antioxidant enzyme heme oxygenase-1 (HO-1) in mpkCCD cells and the outer medulla of mouse kidneys. In contrast to BARD, tolvaptan regulated the antioxidant systems via a unique mechanism. Tolvaptan activated the Nrf2/HO-1 antioxidant pathway through phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, tolvaptan and BARD could successfully generate synergistic activating effects on Nrf2/HO-1 antioxidant pathway, suggesting that this combination therapy can contribute to the treatment of CKD.
| S-EPMC6592894 | biostudies-literature
Project description:Lipotoxicity, induced by saturated fatty acid (SFA)-mediated cell death, plays an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). The KEAP1 (kelch like ECH associated protein 1)-NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) pathway is a pivotal defense mechanism against lipotoxicity. We previously reported that SQSTM1/p62 has a cytoprotective role against lipotoxicity through activation of the noncanonical KEAP1- NFE2L2 pathway in hepatocytes. However, the underlying mechanisms and physiological relevance of this pathway have not been clearly defined. Here, we demonstrate that NFE2L2-mediated induction of SQSTM1 activates the noncanonical KEAP1-NFE2L2 pathway under lipotoxic conditions. Furthermore, we identified that SQSTM1 induces ULK1 (unc-51 like autophagy activating kinase 1) phosphorylation by facilitating the interaction between AMPK (AMP-activated protein kinase) and ULK1, leading to macroautophagy/autophagy induction, followed by KEAP1 degradation and NFE2L2 activation. Accordingly, the activity of this SQSTM1-mediated noncanonical KEAP1-NFE2L2 pathway conferred hepatoprotection against lipotoxicity in the livers of conventional sqstm1- and liver-specific sqstm1-knockout mice. Moreover, this pathway activity was evident in the livers of patients with nonalcoholic fatty liver. This axis could thus represent a novel target for NAFLD treatment. Abbreviations: ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; BafA1: bafilomycin A1; CM-H2DCFDA:5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; FASN: fatty acid synthase; GSTA1: glutathione S-transferase A1; HA: hemagglutinin; Hepa1c1c7: mouse hepatoma cells; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch like ECH associated protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC: N-acetyl-L-cysteine; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PA: palmitic acid; PARP: poly (ADP-ribose) polymerase 1; PRKAA1/2: protein kinase AMP-activated catalytic subunits alpha1/2; RBX1: ring-box 1; ROS: reactive oxygen species; SESN2: sestrin 2; SFA: saturated fatty acid; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; TBK1: TANK binding kinase 1; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; ULK1: unc-51 like autophagy activating kinase.
| S-EPMC7595589 | biostudies-literature
Project description:BACKGROUND AND PURPOSE Sauchinone, an antioxidant lignan, protects hepatocytes from iron-induced toxicity. This study investigated the protective effects of sauchinone against acetaminophen (APAP)-induced toxicity in the liver and the role of nuclear factor erythroid-2-related factor-2 (Nrf2) in this effect. EXPERIMENTAL APPROACH Blood biochemistry and histopathology were assessed in mice treated with APAP or APAP + sauchinone. The levels of mRNA and protein were measured using real-time PCR assays and immunoblottings. KEY RESULTS Sauchinone ameliorated liver injury caused by a high dose of APAP. This effect was prevented by a deficiency of Nrf2. Sauchinone treatment induced modifier subunit of glutamate-cysteine ligase, NAD(P)H:quinone oxidoreductase-1 (NQO1) and heat shock protein 32 in the liver, which was abolished by Nrf2 deficiency. In a hepatocyte model, sauchinone activated Nrf2, as evidenced by the increased nuclear accumulation of Nrf2, the induction of NQO1-antioxidant response element reporter gene, and glutamate-cysteine ligase and NQO1 protein induction, which contributed to the restoration of hepatic glutathione content. Consistently, treatment of sauchinone enhanced Nrf2 phosphorylation with a reciprocal decrease in its interaction with Kelch-like ECH-associated protein-1. Intriguingly, sauchinone activated protein kinase C-δ (PKCδ), which led to Nrf2 phosphorylation. In addition, it increased the inhibitory phosphorylation of glycogen synthase kinase-3β (GSK3β), derepressing Nrf2 activity, which was supported by the reversal of sauchinone's activation of Nrf2 by an activated mutant of GSK3β. Moreover, phosphorylation of GSK3β by sauchinone depended on PKCδ activation. CONCLUSION AND IMPLICATIONS Our results demonstrate that sauchinone protects the liver from APAP-induced toxicity by activating Nrf2, and this effect is mediated by PKCδ activation, which induces inhibitory phosphorylation of GSK3β.
| S-EPMC3166693 | biostudies-literature
Project description:p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of Neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which trigegrs p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.
| S-SCDT-EMBOR-2019-48902V1 | biostudies-other
Project description:p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which triggers p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.
| S-EPMC7054683 | biostudies-literature
Project description:Nuclear factor erythroid 2-related factor 2 (Nrf2), which is linked to autophagy regulation and melanogenesis regulation, is activated by marliolide. In this study, we investigated the effect of a marliolide derivative on melanosome degradation through the autophagy pathway. The effect of the marliolide derivative on melanosome degradation was investigated in α-melanocyte stimulating hormone (α-MSH)-treated melanocytes, melanosome-incorporated keratinocyte, and ultraviolet (UV)B-exposed HRM-2 mice (melanin-possessing hairless mice). The marliolide derivative, 5-methyl-3-tetradecylidene-dihydro-furan-2-one (DMF02), decreased melanin pigmentation by melanosome degradation in α-MSH-treated melanocytes and melanosome-incorporated keratinocytes, evidenced by premelanosome protein (PMEL) expression, but did not affect melanogenesis-associated proteins. The UVB-induced hyperpigmentation in HRM-2 mice was also reduced by a topical application of DMF02. DMF02 activated Nrf2 and induced autophagy in vivo, evidenced by decreased PMEL in microtubule-associated proteins 1A/1B light chain 3B (LC3)-II-expressed areas. DMF02 also induced melanosome degradation via autophagy in vitro, and DMF02-induced melanosome degradation was recovered by chloroquine (CQ), which is a lysosomal inhibitor. In addition, Nrf2 silencing by siRNA attenuated the DMF02-induced melanosome degradation via the suppression of p62. DMF02 induced melanosome degradation in melanocytes and keratinocytes by regulating autophagy via Nrf2-p62 activation. Therefore, Nrf2 activator could be a promising therapeutic agent for reducing hyperpigmentation.
| S-EPMC8070456 | biostudies-literature
Project description:Methylmercury (MeHg) is a ubiquitous environmental toxicant that leads to long-lasting neurological deficits in animals and humans. Curcumin, a polyphenol obtained from the rhizome of turmeric, has well-known antioxidant functions. Here, we evaluated curcumin's efficacy in mitigating MeHg-induced cytotoxicity and further investigated the underlying mechanism of this neuroprotection in primary rat astrocytes. Pretreatment with curcumin (2, 5, 10 and 20 μM for 3, 6, 12 or 24 h) protected against MeHg-induced (5 μM for 6 h) cell death in a time and dose-dependent manner. Curcumin (2, 5, 10 or 20 μM) pretreatment for 12 h significantly ameliorated the MeHg-induced astrocyte injury and oxidative stress, as evidenced by morphological alterations, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, and glutathione (GSH) and catalase (CAT) levels. Moreover, curcumin pretreatment increased Nrf2 nuclear translocation and downstream enzyme expression, heme oxygenase-1 (HO-1) and NADPH quinone reductase-1 (NQO1). Knockdown of Nrf2 with siRNA attenuated the protective effect of curcumin against MeHg-induced cell death. However, both the pan-protein kinase C (PKC) inhibitor, Ro 31-8220, and the selective PKCδ inhibitor, rottlerin, failed to suppress the curcumin-activated Nrf2/Antioxidant Response Element(ARE) pathway and attenuate the protection exerted by curcumin. Taken together, these findings confirm that curcumin protects against MeHg-induced neurotoxicity by activating the Nrf2/ARE pathway and this protection is independent of PKCδ activation. More studies are needed to understand the mechanisms of curcumin cytoprotection.
| S-EPMC6710134 | biostudies-literature
Project description:Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause nonalcoholic steatohepatitis (NASH). NASH, in turn, is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). Formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here, we show that, upon saturated fatty acid-induced lipotoxicity, TANK binding kinase 1 (TBK1) is activated and phosphorylates p62. TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of ubiquitin-p62 aggregates not only in cultured hepatocytes, but also in mouse models of obesity and NASH. CONCLUSION:These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. (Hepatology 2018).
| S-EPMC6005718 | biostudies-literature
Project description:BackgroundCDGSH iron sulfur domain 2 (CISD2) is an iron-sulfur protein with a [2Fe-2S] cluster, which is critical for cell proliferation and iron homeostasis. It has been demonstrated that aberrant expression of CISD2 is associated with the progression of multiple cancers. However, the underlying mechanism of CISD2 in regulating tumorigenesis remains obscure.MethodsBioinformatics strategies were used to investigate the protein interaction network and functional annotation of CISD2. In the functional experiment, cell viability was measured by CCK-8 kit. The levels of cellular reactive oxygen species (ROS), intracellular free iron, lipid peroxides, and lysosomal activity were determined by DCF-DA, RPA, C11-BODIPY, and cathepsin B staining, respectively. The glutathione (GSH) content was determined using a GSH assay kit.ResultsWe showed that knockdown of CISD2 significantly accelerated the Erastin-induced ferroptotic cell death with excess lipid peroxidation, GSH exhaustion, and iron accumulation, while overexpression of CISD2 hindered the sensitivity to Erastin. Further assays via confocal microscopy and western blot exhibited that CISD2 knockdown markedly enhanced the lysosomal activity, and activated ferritinophagy under the exposure of Erastin. Pharmacological inhibition of lysosomal function could inhibit the degradation of ferritin heavy chain (FTH), and attenuate the phenotypes of ferroptosis, such as accelerated iron accumulation and lipid peroxidation. Notably, we found that Erastin-induced compensatory elevation of nuclear factor erythroid 2-related factor 2 (NRF2) could be eliminated in CISD2 depletion cells. Mechanically, CISD2 knockdown promoted the degradation of autophagy adaptor p62 and resulted in an increased binding affinity of Keap1 with NRF2, thus leading to the increased ubiquitination and subsequent degradation of NRF2. Enforced expression of NRF2 reversed the sensitivity of shCISD2 cells to ferroptosis both in vitro and in vivo. Conversely, enforced expression of Keap1 exacerbated the degradation of NRF2, reduced the transcriptional expression of FTH and heme oxygenase 1 (HO-1), increased the oxidative damage, and thus further facilitated ferroptosis.ConclusionTaken together, our current results illustrated two parallel mechanisms involved in the shCISD2-mediated ferroptosis. One was that shCISD2 enhanced the accumulation of free iron via ferritinophagy-dependent ferritin turnover; the other was that CISD2 depletion induced the inhibition of the p62-Keap1-NRF2 pathway, which resulted in oxidative stress and ferroptosis.
| S-EPMC9523958 | biostudies-literature