Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex regulating the response to ROS homeostasis.
ABSTRACT: Oxidative stress and reactive oxygen species (ROS) are associated with diseases such as cancer, cardiovascular complications, inflammation and neurodegeneration. Cellular defense systems must work constantly to control ROS levels and to prevent their accumulation. We report here that the Jun dimerization protein 2 (JDP2) has a critical role as a cofactor for transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and small Maf protein family K (MafK) in the regulation of the antioxidant-responsive element (ARE) and production of ROS. Chromatin immunoprecipitation-quantitative PCR (qPCR), electrophoresis mobility shift and ARE-driven reporter assays were carried out to examine the role of JDP2 in ROS production. JDP2 bound directly to the ARE core sequence, associated with Nrf2 and MafK (Nrf2-MafK) via basic leucine zipper domains, and increased DNA-binding activity of the Nrf2-MafK complex to the ARE and the transcription of ARE-dependent genes. In mouse embryonic fibroblasts from Jdp2-knockout (Jdp2 KO) mice, the coordinate transcriptional activation of several ARE-containing genes and the ability of Nrf2 to activate expression of target genes were impaired. Moreover, intracellular accumulation of ROS and increased thickness of the epidermis were detected in Jdp2 KO mice in response to oxidative stress-inducing reagents. These data suggest that JDP2 is required to protect against intracellular oxidation, ROS activation and DNA oxidation. qPCR demonstrated that several Nrf2 target genes such as heme oxygenase-1, glutamate-cysteine ligase catalytic and modifier subunits, the notch receptor ligand jagged 1 and NAD(P)H dehydrogenase quinone 1 are also dependent on JDP2 for full expression. Taken together, these results suggest that JDP2 is an integral component of the Nrf2-MafK complex and that it modulates antioxidant and detoxification programs by acting via the ARE.
Project description:We report here that the Jun dimerization protein 2 (JDP2) plays a critical role as a cofactor for the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and MafK in the regulation of the antioxidants and production of reactive oxygen species (ROS). JDP2 associates with Nrf2 and MafK (Nrf2-MafK) to increase the transcription of antioxidant response element-dependent genes. Oxidative-stress-inducing reagent led to an increase in the intracellular accumulation of ROS and cell proliferation in Jdp2 knock-out mouse embryonic fibroblasts. In Jdp2-Cre mice mated with reporter mice, the expression of JDP2 was restricted to granule cells in the brain cerebellum. The induced pluripotent stem cells (iPSC)-like cells were generated from DAOY medulloblastoma cell by introduction of JDP2, and the defined factor OCT4. iPSC-like cells expressed stem cell-like characteristics including alkaline phosphatase activity and some stem cell markers. However, such iPSC-like cells also proliferated rapidly, became neoplastic, and potentiated cell malignancy at a later stage in SCID mice. This study suggests that medulloblastoma cells can be reprogrammed successfully by JDP2 and OCT4 to become iPSC-like cells. These cells will be helpful for studying the generation of cancer stem cells and ROS homeostasis.
Project description:The Jun dimerization protein 2 (Jdp2) is expressed predominantly in granule cell progenitors (GCPs) in the cerebellum, as was shown in Jdp2-promoter-Cre transgenic mice. Cerebellum of Jdp2-knockout (KO) mice contains lower number of Atoh-1 positive GCPs than WT. Primary cultures of GCPs from Jdp2-KO mice at postnatal day 5 were more resistant to apoptosis than GCPs from wild-type mice. In Jdp2-KO GCPs, the levels of both the glutamate?cystine exchanger Sc7a11 and glutathione were increased; by contrast, the activity of reactive oxygen species (ROS) was decreased; these changes confer resistance to ROS-mediated apoptosis. In the absence of Jdp2, a complex of the cyclin-dependent kinase inhibitor 1 (p21Cip1) and Nrf2 bound to antioxidant response elements of the Slc7a11 promoter and provide redox control to block ROS-mediated apoptosis. These findings suggest that an interplay between Jdp2, Nrf2, and p21Cip1 regulates the GCP apoptosis, which is one of critical events for normal development of the cerebellum.
Project description:Reactive metabolites of anti-tuberculosis (anti-TB) drugs can result in excessive reactive oxygen species (ROS), which are responsible for drug-induced liver injury. The nuclear factor erythroid 2-related factor 2 (Nrf2) - antioxidant response elements (ARE) (Nrf2-ARE) signaling pathway plays a crucial role in protecting liver cells from ROS, inducing enzymes such as phase II metabolizing enzymes and antioxidant enzymes. Based on a Chinese anti-TB treatment cohort, a nested case-control study was performed to explore the association between 13 tag single-nucleotide polymorphisms (tagSNPs) in the NRF2, KEAP1, MAFF, MAFK genes in Nrf2-ARE signaling pathway and the risk of anti-TB drug-induced liver injury (ATLI) in 314 cases and 628 controls. Conditional logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) after adjusting weight and usage of hepatoprotectant. Patients carrying the TC genotype at rs4243387 or haplotype C-C (rs2001350-rs6726395) in NRF2 were at an increased risk of ATLI (adjusted OR?=?1.362, 95% CI: 1.017-1.824, P?=?0.038; adjusted OR?=?2.503, 95% CI: 1.273-4.921, P?=?0.008, respectively), whereas patients carrying TC genotype at rs2267373 or haplotype C-G-C (rs2267373-rs4444637-rs4821767) in MAFF were at a reduced risk of ATLI (adjusted OR?=?0.712, 95% CI: 0.532-0.953, P?=?0.022; adjusted OR?=?0.753, 95% CI: 0.587-0.965, P?=?0.025, respectively). Subgroup analysis also detected a significant association between multiple tagSNPs (rs4821767 and rs4444637 in MAFF, rs4720833 in MAFK) and specific clinical patterns of liver injury under different genetic models. This study shows that genetic polymorphisms of NRF2, MAFF and MAFK may contribute to the susceptibility to ATLI in the Chinese anti-TB treatment population.
Project description:Reactive oxygen species, produced by oxidative stress, initiate and promote many metabolic diseases through activation/suppression of redox-sensitive transcription factors. NF-?B and Nrf2 are important regulators of oxidation resistance and contribute to the pathogenesis of many diseases. We identified MafK, a novel transcriptional regulator that modulates NF-?B activity. MafK knockdown reduced NF-?B activation, whereas MafK overexpression enhanced NF-?B function. MafK mediated p65 acetylation by CBP upon LPS stimulation, thereby facilitating recruitment of p65 to NF-?B promoters such as IL-8 and TNF?. Consistent with these results, MafK-depleted mice showed prolonged survival with a reduced hepatic inflammatory response after LPS and D-GalN injection. Thus, our findings reveal a novel mechanism by which MafK controls NF-?B activity via CBP-mediated p65 acetylation.
Project description:Transforming growth factor-? (TGF-?) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-? suppresses the transcriptional activation of the heme oxygenase-1 (HO-1) gene, which is implicated in protection against oxidative injury and lung carcinogenesis. HO-1 is a target of the oxidative stress-responsive transcription factor Nrf2. TGF-? did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-? induced expression of transcription factors MafK and Bach1. Enhanced expression of either MafK or Bach1 was enough to suppress the electrophile-inducible expression of HO-1 even in the presence of accumulated Nrf2 in the nucleus. Knockdown of MafK and Bach1 by siRNA abolished TGF-?-dependent suppression of HO-1. Furthermore, chromatin immunoprecipitation assays revealed that Nrf2 substitutes for Bach1 at the antioxidant response elements (E1 and E2), which are responsible for the induction of HO-1 in response to oxidative stress. On the other hand, pretreatment with TGF-? suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-? is activated after tissue injury and in the process of cancer development, these findings suggest a novel mechanism by which damaged tissue becomes vulnerable to oxidative stress and xenobiotics.
Project description:The rat GST-P (placental glutathione S-transferase), a phase II detoxifying enzyme, is not expressed in normal liver cells, but is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells. Results of previous studies indicated that GST-P gene activation was mainly controlled by an enhancer element, GPE1 (GST-P enhancer 1), but the specific activation mechanism of the GST-P gene was not fully understood [Morimura, Suzuki, Hochi, Yuki, Nomura, Kitagawa, Nagatsu, Imagawa and Muramatsu (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065-2068; Suzuki, Imagawa, Hirabayashi, Yuki, Hisatake, Nomura, Kitagawa and Muramatsu (1995) Cancer Res. 55, 2651-2655]. In the present study, we investigate the transcription factor Nrf2/MafK heterodimer (where Nrf2 stands for NF-E2 p45-related factor 2) as an activator of the GST-P gene through the action of GPE1 during hepatocarcinogenesis. Electrophoretic mobility-shift assay and footprinting analysis with wild-type GPE1 and GPE1 point mutants showed that the Nrf2/MafK heterodimer specifically bound GPE1. Reporter transfection assays indicated that Nrf2 strongly stimulated GST-P gene expression in mouse F9 embryonal carcinoma cells and H4IIE rat hepatoma cells. Northern-blot analysis indicated that GST-P and Nrf2 mRNA increased in parallel with development of precancerous lesions and hepatocellular carcinoma. Keap1 (Kelch-like ECH-associated protein 1), an inhibitory factor of Nrf2, decreased the activation of GPE1 by Nrf2 and this suppression was restored after treatment with electrophilic compounds. GST-P mRNA expression in H4IIE cells was induced by electrophilic compounds, as was the expression of mRNAs of other phase II detoxifying enzymes. Chromatin immunoprecipitation analyses showed that antibodies both against Nrf2 and against MafK precipitated GPE1 from the chromatin of the pre-neoplastic hepatocytes and rat hepatoma cells (H4IIE and dRLh84), but not from normal hepatocytes. These results indicate that the Nrf2/MafK heterodimer regulates GST-P gene expression during early hepatocarcinogenesis and in hepatoma cells.
Project description:Background:Reactive oxygen species (ROS) are known to stimulate the activation of nuclear factor-erythroid 2-related factor-2 (Nrf2), the key regulator of the antioxidant and cytoprotective defense system in the body. The hypothesis underlying this study was that high dietary concentrations of vitamin E suppress Nrf2 activation, and thus could weaken the body's antioxidative and cytoprotective capacity. As the effect of vitamin E on Nrf2 pathway might be influenced by concentrations of fatty acids susceptible to oxidation in the diet, we used also diets containing either soybean oil as a reference oil or salmon oil as a source of oil rich in n-3 polyunsatuated fatty acids. Methods:Seventy-two rats were divided into 6 groups of rats which received diets with either 25, 250 or 2500 mg vitamin E/kg, with either soybean oil or salmon oil as dietary fat sources according to a bi-factorial experimental design. Electron spin resonance spectroscopy was used to determine ROS production in the liver. qPCR analysis and western blot were performed to examine the expression of Nrf2 target genes in the liver of rats. Results:Rats fed the salmon oil diet with 25 mg vitamin E/kg showed a higher production of ROS in the liver than the 5 other groups of rats which did not differ in ROS production. Relative mRNA concentrations of NFE2L2 (encoding Nrf2), KEAP1 and various Nrf2 target genes, protein concentrations of glutathione peroxidase (GPX), heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1) and activities of the antioxidant enzymes GPX, superoxide dismutase and catalase were not influenced by the dietary vitamin E concentration. The dietary fat had also less effect on Nrf2 target genes and no effect on protein concentrations of GPX, HO-1, NQO1 and activities of antioxidant enzymes. Dietary vitamin E concentration and type of fat moreover had less effect on mRNA concentrations of genes and concentrations of proteins involved in the unfolded protein response, a pathway which is closely linked with activation of Nrf2. Conclusion:We conclude that excess dietary concentrations of vitamin E do not suppress Nrf2 signaling, and thus do not weaken the endogenous antioxidant and cytoprotective capacity in the liver of rats.
Project description:HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.
Project description:The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic ?-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of ?-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced ?-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of ?-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key ?-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the ?-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous ?-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas.
Project description:BACKGROUND:Endothelial colony forming cells (ECFCs) have shown a promise in tissue engineering of vascular constructs, where they act as endothelial progenitor cells. After implantation, ECFCs are likely to be subjected to elevated reactive oxygen species (ROS). The transcription factor Nrf2 regulates the expression of antioxidant enzymes in response to ROS. METHODS:Stable knockdown of Nrf2 and Keap1 was achieved by transduction with lentiviral shRNAs; activation of Nrf2 was induced by incubation with sulforaphane (SFN). Expression of Nrf2 target genes was assessed by qPCR, oxidative stress was assessed using CM-DCFDA, and angiogenesis was quantified by scratch-wound and tubule-formation assays Results. Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H2O2 and preserved scratch-wound closure and tubule-formation. RESULTS:Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H2O2 and preserved scratch-wound closure and tubule-formation. CONCLUSION:The results of this study indicate that Nrf2 plays an important role in the angiogenic capacity of ECFCs, particularly under conditions of increased oxidative stress. Pretreatment of ECFCs with SFN prior to implantation may be a protective strategy for tissue-engineered constructs or cell therapies.