Enzyme Genes In Response Research Articles

PI3K/Akt/CncC signaling pathway mediates the response to EPN-Bt infection in Holotrichia parallela larvae.

Combining the entomopathogenic nematode (EPN), Heterorhabditis beicherriana LF strain, and Bacillus thuringiensis (Bt) HBF-18 strain is a practical strategy to manage the larvae of Holotrichia parallela Motschulsky (white grubs). However, the mechanisms underlying the larval defense response to this combined biocontrol strategy are unknown. The activities of some antioxidant enzymes (SOD, POD, CAT) and some detoxifying enzymes (AChE, P-450, CarE, GST) in grubs showed an activation-inhibition trend throughout the EPN-Bt exposure time course. Eight potentially key antioxidant and detoxifying enzyme genes in response to EPN-Bt infection were identified from the midgut of grubs through RNA sequencing. After silencing CAT, CarE18, and GSTs1, the enzyme activities were significantly decreased by 30.29%, 68.80%, and 34.63%, respectively. Meanwhile, the mortality of grubs was increased by 18.40%, 46.30%, and 42.59% after exposure to EPN-Bt for 1day. Interestingly, the PI3K/Akt signaling pathway was significantly enriched in KEGG enrichment analysis, and the expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), cap 'n' collar isoform-C (CncC), kelch-like ECH-associated protein 1 (Keap1), and CarE18 were all up-regulated when exposed to EPN-Bt for 1day. Furthermore, RNAi-mediated PI3K silencing showed a similar down-regulated trend between PI3K/Akt/CncC and CarE18. Moreover, silencing PI3K rendered grubs more susceptible to EPN-Bt and accelerated symbiotic bacteria multiplication in grubs. These results suggest that the PI3K/Akt/CncC pathway mediates the expression of CarE18 and participates in the defense response of H. parallela larvae against EPN-Bt infection. Our data provide valuable insights into the design of appropriate management strategies for this well-known agricultural pest. © 2022 Society of Chemical Industry.

Alterations in the Expression of Antioxidant Enzyme Genes in Response to Pesticide Exposure During Embryonic Development in the Native Reptile Species Caiman latirostris

The aim of this study was to quantify the expression levels of Catalase (cat) and copper, zinc Superoxide dismutase (Cu, Zn-sod) genes involved in the antioxidant response in Caiman latirostris (broad-snouted caiman) blood, after embryonic exposure to the formulations cypermethrin (CYP), chlorpyrifos (CPF), glyphosate (GLY), and their binary and ternary mixtures. Experimental groups were: negative control (NC-distilled water), vehicle control (VC-ethanol), GLY-2%, CYP- 0.12%, CPF- 0.8%, a ternary mixture of them (TM), and three binary mixtures. The applications were made on the nest material in contact with the eggs at the beginning of the incubation period. After hatching, RNA was isolated from blood and expression levels analyzed through qPCR. The results showed downregulation in the expression of sod and cat genes in the three binary mixtures studied, compared to the controls. In addition, we found a possible antagonistic effect between different pesticides in the TM on the expression of both genes.

Screening and identification of key enzyme genes for steroidal saponin biosynthesis in Dioscorea zingiberensis under low phosphorus stress

To investigate the responses of key enzymes involved in steroidal saponin biosynthesis of Dioscorea zingiberensis to low phosphorus stress, we designed three treatments of severe phosphorus stress, moderate phosphorus stress, and normal phosphorus level. The D. zingiberensis plants were collected at the early, middle, and late stages of treatment. The content of total steroidal saponins in different tissues of D. zingiberensis was determined by spectrophotometry for the identification of the critical stage in response to low phosphorus stress. BGI 500 sequencing platform was employed to obtain the transcript information of D. zingiberensis samples at the critical stage of low phosphorus stress, and then a transcriptome library was constructed. The correlation between the expression of genes involved in steroidal saponin biosynthesis and the content of total steroidal saponins was analyzed for the screening of the key enzyme genes in response to low phosphorus stress. Further, the expression patterns of these genes were analyzed by real-time fluorescence PCR(qRT-PCR). The content of total steroidal saponins in D. zingiberensis had obvious tissue specificity under low phosphorus stress, and the early stage of stress was particularly important for D. zingiberensis to respond to low phosphorus stress. A total of 101 593 unigenes were obtained by transcriptome sequencing, of which 77.35% were annotated in NT, NR, SwissProt, KOG, GO, and KEGG. A total of 256 transcripts of known key enzyme genes in the biosynthetic pathway of steroidal saponins were identified. The expression levels of 69 transcripts encoding 18 catalytic enzymes were significantly correlated with the content of total steroidal saponins. The qRT-PCR results showed that several key enzyme genes presented different expression patterns in four tissues under low phosphorus stress. The results indicated that the content of total steroidal saponins and the expression of key enzyme genes regulating steroidal saponin biosynthesis in D. zingensis changed under low phosphorus stress. This study provides the biological information for elucidating the molecular mechanism of steroidal saponin biosynthesis in D. zingensis exposed to low phosphorus stress.

Serine-arginine protein kinase-like protein, SrpkF, stimulates both cellobiose-responsive and D-xylose-responsive signaling pathways in Aspergillus aculeatus.

Aspergillus aculeatus produces cellulolytic enzymes in the presence of their substrates. We screened a library of 12,000 A. aculeatus T-DNA-inserted mutants to identify a regulatory factor involved in the expression of their enzyme genes in response to inducers. We found one mutant that reduced the expression of FIII-avicelase (chbI) in response to cellulose. T-DNA was inserted into a putative protein kinase gene similar to AN10082 in A. nidulans, serine-arginine protein kinase F, SrpkF. Fold increases in srpkF gene expression in response to various carbon sources were 2.3 (D-xylose), 44 (Avicel®), 59 (Bacto™ Tryptone), and 98 (no carbon) compared with D-glucose. Deletion of srpkF in A. aculeatus resulted in a significant reduction in cellulose-responsive expression of chbI, hydrocellulase (cel7b), and FIb-xylanase (xynIb) genes at an early induction phase. Further, the srpkF-overexpressing strain showed upregulation of the srpkF gene from four- to nine-fold higher than in the control strain. srpkF overexpression upregulated cbhI and cel7b in response to cellobiose and the FI-carboxymethyl cellulase gene (cmc1) and xynIb in response to D-xylose. However, the srpkF deletion did not affect the expression of xynIb in response to D-xylose due to the less expression of srpkF under the D-xylose condition. Our data demonstrate that SrpkF is primarily involved in cellulose-responsive expression, though it has a potential to stimulate gene expression in response to both cellobiose and D-xylose in A. aculeatus.

Artificial AmyR::XlnR transcription factor induces α-amylase production in response to non-edible xylan-containing hemicellulosic biomass

Filamentous fungi belonging to the Aspergillus genus are one of the most favored microorganisms for industrial enzyme production because they can secrete large amounts of proteins into the culture medium. α-Amylase, an enzyme produced by Aspergillus species, is important for food and industrial applications. The production of α-amylase is induced by starch, mainly obtained from the edible biomass; however, the increasing demand for foods is limiting the application of the latter. Therefore, it is expected that using the non-edible biomass, such as rice straw, could improve the competition for industrial application starch containing resources. The transcription factor AmyR activates the transcription of amylolytic enzyme genes, while the transcription factor XlnR activates the transcription of xylanolytic enzyme genes in response to xylose. In this study, we aimed to construct an artificial AmyR::XlnR transcription factor (AXTF) by replacing the DNA-binding domain (1–159 amino acids) of XlnR with that (1–68 aa) of AmyR, which is capable of inducing amylolytic enzyme production in response to xylan-containing hemicellulosic biomass. The chimeric transcription factor AXTF was constructed and expressed using the gapA promoter in the amyR-deficient mutant strain SA1. When the AXTF strain was cultured in the minimal medium containing xylose as the carbon source, the amyB, amyF, agdB, and agdE transcription levels were 41.1-, 11.3-, 37.9-, and 23.7-fold higher, respectively, than those of the wild-type strain. The α-amylase and α-glucosidase activities in the culture supernatant of the AXTF strain grown with xylose for 48 h were 696.6 and 536.1 U/mL, respectively, while these activities were not detected in the culture supernatant of the wild-type and SA1 strains. When rice straw hydrolysate was used as a carbon source, the α-amylase and α-glucosidase activities were 590.2 and 362.7 U/mL, respectively. Thus, we successfully generated an Aspergillus nidulans strain showing amylolytic enzyme production in response to non-edible xylan-containing hemicellulosic biomass by transforming it with the chimeric transcription factor AXTF. Furthermore, the use of genes encoding engineered transcription factors is advantageous because introducing such genes into an industrial Aspergillus strain has similar simultaneous effects on multiple amylase genes controlled by AmyR.

Homologs of vacuolar invertase inhibitor INH2 in tuber-bearing wild potato species and Solanum tuberosum: gene polymorphism and co-expression with saccharolytic enzyme genes in response to cold stress

Wild tuber-bearing species are commonly used as a source of valuable traits to develop new potato cultivars. One of the trends in potato breeding is elimination of cold-induced sweetening, which depends on the balance between activities of saccharolytic enzymes (vacuolar invertase PAIN-1 and sucrose synthase SUS4 that hydrolyze sucrose) and vacuolar invertase inhibitor INH2 that regulates the PAIN-1 activity. To clarify the mechanisms underlying cold resistance, we investigated INH2 polymorphism and tissue-specific expression, and co-expression profiles of INH2, PAIN-1, and SUS4 in wild tuber-bearing potatoes and Solanum tuberosum cultivars. Novel INH2 homologs were identified in eight wild species and four cultivars. Structural analysis predicted an N-terminal signal peptide and showed that nucleotide and amino acid polymorphisms (13.09% and 18.91%, respectively) did not affect the INH2 tertiary structure, suggesting that the INH2 function may be conserved in potato both at the intra- and inter-specific levels. INH2 and PAIN-1 mRNA were detected in buds, flowers, leaves, stems, roots, and tubers of S. tuberosum cv. Nadezhda, showing the highest (INH2) and lowest (PAIN-1) levels in the roots. During cold storage, tubers of four cultivars accumulated glucose and fructose, while the sucrose content changed insignificantly; INH2, PAIN-1, and SUS4 co-expression patterns in freshly harvested and cold-stored tubers differed among the cultivars. Cold exposure also caused distinct effects on INH2, PAIN-1, and SUS4 co-expression patterns in the leaves of four wild species differing in cold resistance. These data indicate that cold response in potato may depend on specific combinations of INH2, PAIN-1, and SUS4 activities.

Response mechanism in Populus × euramericana cv. ‘74/76’ revealed by RNA-seq under salt stress

Populus spp., had the characteristics of rapid growth and high biological yield, is one of the major tree species in the world’s afforestation and shelterbelt. But the growing soil salinization has become an important limited factor in the application of poplar planting. High-throughput sequencing technology was used to analyze the transcriptome of Populus × euramericana cv. ‘74/76’ under treatments with different NaCl concentrations to determine the formation pathway of the poplar response to salt stress and further reveal the mechanism of resistance to salt stress, so providing theoretical guidance for the salt tolerant breeding of poplar. Sequencing obtained 31.5G sequencing data and generated a total of 263 million reads, which were mapped to 32,840 non-repeatable genes with a sequencing quality value of Q30 > 96.70%. After filtering, 292 and 3284 differentially expressed genes (DEGs) were obtained from 3 and 6‰ NaCl treatments, respectively. There were a large number of stress-induced genes differentially expressed under the 6‰ NaCl treatment. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results showed that the salt resistance mechanism of poplar 107 was different under different NaCl treatments. Poplar 107, under a high NaCl concentration treatment had a more salt resistant metabolic pathway, which included the citric acid cycle [tricarboxylic acid (TCA) cycle], terpenoid backbone biosynthesis, flavone and flavonol biosynthesis, carotenoid biosynthesis, zeatin biosynthesis, calcium signaling pathway, and other important salt-related pathways. We excavated the genes that affected the electron transport involved in the process of photosynthesis under salt stress and the key enzyme genes in response to salt stress involved in glutathione and terpenoid backbone metabolism. A total of 52 transcription factors (TFs), including 24 new TFs closely related to the salt stress response mechanism, were identified in the co-expression genes. This result provided important theoretical guidance for the establishment of the poplar salt resistance database, excavation of salt tolerant genes, and breeding of new species of poplar by means of molecular biology.

A Nuclear Factor Involved in Transcriptional Regulation of the AREBP Gene.

The AICAR responsive element binding protein (AREBP) suppresses transcription of the gluconeogenic enzyme genes in response to AICAR treatment. Moreover, overexpression of AREBP also suppresses gluconeogenic gene expressions in animals, indicating that AREBP plays an important role in gluconeogenesis. Through a combination of systematic analyses of the AREBP gene promoter and assays for DNA-protein interaction, we identified a nuclear factor involved in tissue-specific expression of AREBP. By targeting this nuclear factor, pharmacological or nutraceutical induction of AREBP gene expression is expected to reduce blood glucose levels in patient with insulin resistance.

17β-estradiol ameliorates light-induced retinal damage in Sprague-Dawley rats by reducing oxidative stress.

Oxidative stress is considered as a major cause of light-induced retinal neurodegeneration. The protective role of 17β-estradiol (βE2) in neurodegenerative disorders is well known, but its underlying mechanism remains unclear. Here, we utilized a light-induced retinal damage model to explore the mechanism by which βE2 exerts its neuroprotective effect. Adult male and female ovariectomized (OVX) rats were exposed to 8,000 lx white light for 12 h to induce retinal light damage. Electroretinogram (ERG) assays and hematoxylin and eosin (H&E) staining revealed that exposure to light for 12 h resulted in functional damage to the rat retina, histological changes, and retinal neuron loss. However, intravitreal injection (IVI) of βE2 significantly rescued this impaired retinal function in both female and male rats. Based on the level of malondialdehyde (MDA) production (a biomarker of oxidative stress), an increase in retinal oxidative stress followed light exposure, and βE2 administration reduced this light-induced oxidative stress. Quantitative reverse-transcriptase (qRT)-PCR indicated that the messenger RNA (mRNA) levels of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (Gpx) were downregulated in female OVX rats but were upregulated in male rats after light exposure, suggesting a gender difference in the regulation of these antioxidant enzyme genes in response to light. However, βE2 administration restored or enhanced the SOD and Gpx expression levels following light exposure. Although the catalase (CAT) expression level was insensitive to light stimulation, βE2 also increased the CAT gene expression level in both female OVX and male rats. Further examination indicated that the antioxidant proteins thioredoxin (Trx) and nuclear factor erythroid 2-related factor 2 (Nrf2) are also involved in βE2-mediated antioxidation and that the cytoprotective protein heme oxygenase-1 (HO-1) plays a key role in the endogenous defense mechanism against light exposure in a βE2-independent manner. Taken together, we provide evidence that βE2 protects against light-induced retinal damage via its antioxidative effect, and its underlying mechanism involves the regulation of the gene expression levels of antioxidant enzymes (SOD, CAT, and Gpx) and proteins (Trx and Nrf2). Our study provides conceptual evidence in support of estrogen replacement therapy for postmenopausal women to reduce the risk of age-related macular degeneration.

Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains

Keap1 is a substrate adaptor of a Cullin 3-based E3 ubiquitin ligase complex that recognizes Nrf2, and also acts as a cellular sensor for xenobiotics and oxidative stresses. Nrf2 is a transcriptional factor regulating the expression of cytoprotective enzyme genes in response to such stresses. Under unstressed conditions Keap1 binds Nrf2 and results in rapid degradation of Nrf2 through the proteasome pathway. In contrast, upon exposure to oxidative and electrophilic stress, reactive cysteine residues in intervening region (IVR) and Broad complex, Tramtrack, and Bric-à-Brac domains of Keap1 are modified by electrophiles. This modification prevents Nrf2 from rapid degradation and induces Nrf2 activity by repression of Keap1. Here we report the structure of mouse Keap1 homodimer by single particle electron microscopy. Three-dimensional reconstruction at 24-A resolution revealed two large spheres attached by short linker arms to the sides of a small forked-stem structure, resembling a cherry-bob. Each sphere has a tunnel corresponding to the central hole of the beta-propeller domain, as determined by x-ray crystallography. The IVR domain appears to surround the core of the beta-propeller domain. The unexpected proximity of IVR to the beta-propeller domain suggests that any distortions generated during modification of reactive cysteine residues in the IVR domain may send a derepression signal to the beta-propeller domain and thereby stabilize Nrf2. This study thus provides a structural basis for the two-site binding and hinge-latch model of stress sensing by the Nrf2-Keap1 system.

Identification of cis-regulatory elements and trans-acting proteins of the rat carbohydrate response element binding protein gene

Carbohydrate response element binding protein (ChREBP) is a transcription factor that activates liver glycolytic and lipogenetic enzyme genes in response to high carbohydrate diet. Here we report the transcriptional regulatory mechanisms for the rat ChREBP gene. Firstly, we determined the transcription initiation site and the nucleotide sequences of the rat ChREBP promoter region encompassing approximately 900 bp from the ATG initiation codon. Reporter gene assays demonstrated that the major positive regulatory region exists in the nucleotide sequence between −163 and −32 of the ChREBP gene. This region contains a cluster of putative transcription factor binding elements that consist of two specificity protein 1 (Sp1) binding sites (−66 to −50 and −93 to −78), a sterol regulatory element (−101 to −110), and two nuclear factor-Y (NF-Y) binding sites (−23 to −19 and −131 to −127). Mutations introduced into these sites caused marked reduction of ChREBP promoter activities. Functional synergisms were observed between Sp1/NF-Y and Sp1/sterol regulatory element-binding protein. Additionally, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that these factors bound to these elements. Thus, we conclude that functional synergisms between these transcription factors are critical for ChREBP gene transcription.

The Role of SREBP-1c in Nutritional Regulation of Lipogenic Enzyme Gene Expression

A high carbohydrate diet up-regulates the transcription of enzymes of triglyceride biosynthesis (lipogenesis) in the mammalian liver. This treatment stimulates hepatic insulin signaling, leading to transcription of sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c has been implicated as a major factor that up-regulates lipogenic genes in response to carbohydrate feeding. However, we presented evidence for another factor, carbohydrate response factor, which is also involved in this response, and we proposed a model wherein SREBP-1c and carbohydrate response factor are independent transcription factors that act in response to insulin and glucose, respectively. In this study, we examined the contribution of SREBP-1c to the expression of lipogenic genes in glucose- and insulin-treated primary rat hepatocytes using an inducible adenovirus system. We found that SREBP-1c overexpression leads to a modest induction of fatty acid synthase, S(14), and acetyl-CoA carboxylase mRNAs to 20% (fatty acid synthase), 10% (S(14)), and 5% (acetyl-CoA carboxylase) of the induction seen by high glucose and insulin treatment. Restoring insulin to cells overexpressing SREBP-1c did not further increase these mRNA levels. In contrast, adenovirus-expressed SREBP-1c did not induce pyruvate kinase mRNA, suggesting that induction of this gene is SREBP-1c-independent. SREBP-1c does indeed play a role in the induction of lipogenic enzyme genes in response to insulin treatment, but it is not sufficient for the induction seen when hepatocytes are treated with insulin and high glucose.

Carbohydrate responsive element-binding protein (ChREBP): a key regulator of glucose metabolism and fat storage

Feeding a high carbohydrate diet induces transcription of more than 15 genes involved in the metabolic conversion of glucose to fat. A new transcription factor binding to a glucose response element of the pyruvate kinase and lipogenesis enzyme genes was discovered recently. This factor, termed carbohydrate responsive element-binding protein (ChREBP), is activated in response to high glucose and up-regulates these genes. Cyclic AMP and a high fat diet inhibit ChREBP and slow down glucose utilization. ChREBP is able to control transcription of lipogenic enzyme genes in response to nutritional and hormonal inputs, and may play an important role in disease states such as diabetes, obesity, and hypertension.

Functional characterization and role of INrf2 in antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene.

Antioxidant response element (ARE) and nuclear transcription factor Nrf2 are known to regulate expression and coordinated induction of NQO1 and other detoxifying enzyme genes in response to antioxidants and xenobiotics. A cytosolic inhibitor of Nrf2, INrf2, that retains Nrf2 in the cytoplasm, was cloned and sequenced. Treatment of cells with antioxidants and xenobiotics results in the release of Nrf2 from INrf2. Nrf2 then moves in the nucleus. This leads to the induction of ARE-mediated NQO1 and other detoxifying enzyme genes expression. INrf2 after dissociation from Nrf2 remains in the cytosol. Overexpression of INrf2 repressed ARE-mediated NQO1 gene expression. Deletion mapping of INrf2 revealed the requirement of KELCH domain (amino acid residues 361-597) and C-terminal region (amino acid residues 598-624) in retention of Nrf2 in the cytosol. Both these regions of INrf2 independently retained Nrf2 in the cytosol leading to the repression of ARE-mediated NQO1 gene expression. These results may indicate that two different regions of INrf2 interact with a single molecule of Nrf2 or two or more molecules of Nrf2 interact with a single molecule of INrf2. The transcription of Nrf2 and INrf2 did not change in response to antioxidants and xenobiotics. This indicated that INrf2 and/or Nrf2 might be post-transcriptionally modified in response to antioxidants and xenobiotics leading to the release of Nrf2 from INrf2 and induction of ARE-mediated NQO1 and other detoxifying enzyme genes expression.

Small Maf (MafG and MafK) Proteins Negatively Regulate Antioxidant Response Element-mediated Expression and Antioxidant Induction of the NAD(P)H:Quinone Oxidoreductase1 Gene

The antioxidant response element (ARE) is known to regulate expression and induction of NQO1, GST Ya, and other detoxifying enzyme genes in response to antioxidants and xenobiotics. The nuclear transcription factor Nrf2 and Nrf1 bind to the ARE and positively regulate expression and induction of the NQO1 and GST Ya genes. In this study, we demonstrate that overexpression of small Maf (MafG and MafK) proteins negatively regulate ARE-mediated expression and tert-butyl hydroquinone induction of the NQO1 and GST Ya genes in transfected Hep-G2 cells. In similar experiments, overexpression of small Maf proteins also repressed Nrf2-mediated up-regulation of ARE-mediated NQO1 and GST Ya genes expression in Hep-G2 cells co-transfected with Nrf2 and small Maf proteins. Band and supershift assays with the NQO1 gene ARE and nuclear proteins demonstrate that small MafG and MafK bind to the ARE as Maf-Maf homodimers and Maf-Nrf2 heterodimers. Therefore, Maf-Maf homodimers and possibly Maf-Nrf2 heterodimers play a role in negative regulation of ARE-mediated transcription and antioxidant induction of NQO1 and other detoxifying enzyme genes. In contrast to Maf-Nrf2, the Maf-Nrf1 heterodimers failed to bind with the NQO1 gene ARE and did not demonstrate the repressive effect in transfection assays.

Regulation of genes encoding NAD(P)H:quinone oxidoreductases

NAD(P)H:quinone oxidoreductase (NQO1) and NRH:quinone oxidoreductase (NQO2) are flavoproteins that catalyze two-electron reduction and detoxification of quinones and its derivatives. This leads to the protection of cells against redox cycling, oxidative stress, and neoplasia. NQO1 is expressed ubiquitously in all the tissues. However, the level of expression varied among the human tissues. NQO1 gene is expressed at higher levels in several tumor tissue types, including liver and colon, as compared to normal tissues of similar origin. NQO1 gene expression is coordinately induced with other detoxifying enzyme genes in response to xenobiotics, antioxidants, oxidants, heavy metals, and radiations. Deletion mutagenesis in the NQO1 gene promoter identified several cis-elements including antioxidant response element (ARE), a basal element, and AP-2 element. ARE elements have also been found in the promoter regions of other detoxifying enzyme genes including glutathione S-transferases. ARE is essentially required for expression and coordinated induction of NQO1 and other detoxifying enzyme genes. Nuclear transcription factors Nrf2 and c-Jun bind to the ARE and activate the gene expression. The binding of Nrf2 + c-Jun to the ARE required unknown cytosolic factor(s). In addition to Nrf2 and c-Jun, other nuclear transcription factors including Nrf1, Jun-B, and Jun-D also bind to the ARE and regulate expression and induction of NQO1 gene. A hypothetical model is presented based on the available information on ARE-mediated regulation of detoxifying enzyme genes. Briefly, the Nrf2 is retained in the cytosplasm by a repressor protein Keap1 in untreated normal cells. The treatment of cells with xenobiotics and antioxidants leads to the activation of unknown cytosolic factor(s) that catalyze modification of Nrf2 and/or Keap1. The modification follows dissociation of Nrf2 and Keap1. The free Nrf2 translocates in the nucleus. Nrf2 in the nucleus heterodimerizes with c-Jun and binds to the ARE resulting in the induction of NQO1 and other ARE-regulated genes expression. The identity of cytosolic factor(s) remains unknown.

Coordinated induction of the c- jun gene with genes encoding quinone oxidoreductases in response to xenobiotics and antioxidants

Xenobiotics and antioxidants induce expression of detoxifying enzymes including NAD(P)H:quinone oxidoreductase (NQO1), NRH:quinone oxidoreductase (NQO2), and glutathione S-transferase Ya (GST Ya), presumably to provide protection to cells against electrophilic and oxidative stress. Antioxidant response elements (AREs) have been found in the promoter regions of the various detoxifying enzyme genes. An ARE is required for basal expression and induction of the various detoxifying enzyme genes in response to xenobiotics and antioxidants. In this study, we demonstrated that exposure of cells to xenobiotics [e.g. β-naphthoflavone (β-NF)] and antioxidants [e.g. tert-butyl hydroquinone ( t-BHQ)] also induced the expression of the proto-oncogene c- jun. The induction of c- jun gene expression followed kinetics similar to the induction of NQO1 and NQO2 genes with respect to the level and time of exposure. Sequence analysis of the c- jun gene promoter revealed the presence of an ARE between nucleotides -538 and -514. The c- jun ARE was highly homologous to the AREs from genes encoding NQO1, NQO2, and GST Ya. Constructs containing the c- jun ARE and 1.7 and 4.5 kb of the c- jun promoter ligated to the chloramphenicol acetyltransferase (CAT) gene, upon transfection in human hepatoblastoma (Hep-G2) cells, expressed the CAT gene, which was inducible with β-NF and t-BHQ. Band shift assays indicated binding of two specific nuclear protein complexes with the c- jun gene ARE. The faster running c- jun gene ARE–nuclear protein complex was specifically competed out by unlabeled NQO1 and GST Ya gene AREs. These results suggest that c- jun gene expression is coordinately induced and regulated with detoxifying enzyme genes in response to xenobiotics and antioxidants. The results also suggest involvement of an ARE-mediated mechanism of induction of c- jun gene expression. However, a comparison of fold induction of endogenous c- jun gene and transfected c- jun promoter/ARE-CAT constructs indicated involvement of another ARE upstream of the 4.5-kb promoter and/or additional mechanisms such as stabilization of c-Jun RNA in response to exposure to xenobiotics and antioxidants.