Maternal DHA intake in mice increased DHA metabolites in the pup brain and ameliorated MeHg-induced behavioral disorder.

Although pregnant women's fish consumption is beneficial for the brain development of the fetus due to the DHA in fish, seafood also contains methylmercury (MeHg), which adversely affects fetal brain development. Epidemiological studies suggest that high DHA levels in pregnant women's sera may protect the fetal brain from MeHg-induced neurotoxicity, but the underlying mechanism is unknown. Our earlier study revealed that DHA and its metabolite 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) produced by cytochrome P450s (P450s) and soluble epoxide hydrolase (sEH) can suppress MeHg-induced cytotoxicity in mouse primary neuronal cells. In the present study, DHA supplementation to pregnant mice suppressed MeHg-induced impairments of pups' body weight, grip strength, motor function, and short-term memory. DHA supplementation also suppressed MeHg-induced oxidative stress and the decrease in the number of subplate neurons in the cerebral cortex of the pups. DHA supplementation to dams significantly increased the DHA metabolites 19,20-epoxydocosapentaenoic acid (19,20-EDP) and 19,20-DHDP as well as DHA itself in the fetal and infant brains, although the expression levels of P450s and sEH were low in the fetal brain and liver. DHA metabolites were detected in the mouse breast milk and in human umbilical cord blood, indicating the active transfer of DHA metabolites from dams to pups. These results demonstrate that DHA supplementation increased DHA and its metabolites in the mouse pup brain and alleviated the effects of MeHg on fetal brain development. Pregnant women's intake of fish containing high levels of DHA (or DHA supplementation) may help prevent MeHg-induced neurotoxicity in the fetus.

Brain Pericytes Acquire Stemness via the Nrf2-Dependent Antioxidant System.

Pericytes (PCs) are a mural support cell population elongated at intervals along the walls of capillaries. Recent studies reported that PCs are multipotent cells that are activated in response to tissue injury and contribute to the regenerative process. Using a C.B-17 mouse model of ischemic stroke, it has been proposed that normal brain pericytes (nPCs) are converted to ischemic pericytes (iPCs), some of which function as multipotent stem cells. Furthermore, oxygen-glucose deprivation (OGD) promoted mesenchymal-epithelial transition in nPCs; however, nestin was not induced under OGD conditions. Therefore, further studies are needed to elucidate the PC reprogramming phenomenon. We herein isolated nPCs from the cortex of C.B-17 mice, and compared the traits of iPCs and nPCs. The results obtained showed that nPCs and iPCs shared common pericytic markers. Furthermore, intercellular levels of reactive oxygen species and the nuclear accumulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a key player in antioxidant defenses, were higher in iPCs than in nPCs. OGD/reoxygenation and a treatment with tBHQ, an Nrf2 inducer, increased nestin levels in nPCs. Moreover, epithelial marker levels, including nestin, Sox2, and CDH1 (E-cadherin) mRNAs, were elevated in Nrf2-overexpressing PCs, which formed neurosphere-like cell clusters that differentiated into Tuj1-positive neurons. The present results demonstrate that oxidative stress and Nrf2 are required for the generation of stem cells after stroke and will contribute to the development of novel therapeutic strategies for ischemic stroke.

Yeast ß-glucan Increases Etoposide Sensitivity in Lung Cancer Cell Line A549 by Suppressing Nuclear Factor Erythroid 2-Related Factor 2 via the Noncanonical Nuclear Factor Kappa B Pathway.

Etoposide is regarded as one of the main standard cytotoxic drugs for lung cancer. However, mutations in Kelch-like ECH-associated protein 1 (Keap1), the main regulator of nuclear factor erythroid 2-related factor 2 (Nrf2), are often detected in lung cancer and lead to chemoresistance. Since the aberrant activation of Nrf2 enhances drug resistance, the suppression of the Nrf2 pathway is a promising therapeutic strategy for lung cancer. We herein used the human lung adenocarcinoma cell line A549 because it harbors a Keap1 loss-of-function mutation. A treatment with ß-glucan, a major component of the fungal cell wall, reduced Nrf2 protein levels; downregulated the expression of cytochrome P450 3A5, UDP glucuronosyltransferase 1A1, and multidrug resistance protein 1; and increased etoposide sensitivity in A549 cells. Furthermore, the ephrin type-A receptor 2 (EphA2) receptor was important for the recognition and biologic activity of ß-glucan in A549 cells. EphA2 signaling includes nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). However, treatment of cells with stattic (STAT3 inhibitor) or SB203580 (p38 MAPK inhibitor) did not diminish the effects of ß-glucan. In contrast, knockdown of v-rel reticuloendotheliosis viral oncogene homolog B (RelB) abolished the effects of ß-glucan, suggesting the involvement of the noncanonical NF-κB pathway. The ß-glucan effects were also attenuated by the knockdown of WD40 Repeat protein 23 (WDR23). The ß-glucan treatment and RelB overexpression induced the expression of Cullin-4A (CUL4A), which increased WDR23 ligase activity and promoted the subsequent depletion of Nrf2. These results revealed a novel property of ß-glucan as a resistance-modifying agent in addition to its widely reported immunomodulatory effects for lung cancer therapy via the EphA2-RelB-CUL4A-Nrf2 axis. SIGNIFICANCE STATEMENT: Chemotherapeutic resistance remains a major obstacle in cancer therapy despite extensive efforts to elucidate the underlying molecular mechanisms and overcome multidrug resistance. The present study revealed a novel resistance-modifying property of ß-glucan, thereby expanding our knowledge on the beneficial roles of ß-glucan and providing an alternative strategy to prevent drug resistance by cancer. The present results provide evidence for the involvement of a novel mode of NF-κB and Nrf2 crosstalk in the drug resistance phenotype.

Sp1 is a substrate of Keap1 and regulates the activity of CRL4AWDR23 ubiquitin ligase toward Nrf2.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical transcription factor that orchestrates cellular responses to oxidative stress. Because the dysregulation of Nrf2 has been implicated in many diseases, precise regulation of its protein level is crucial for maintaining homeostasis. Kelch-like-ECH-associated protein 1 (Keap1) and WD40 repeat protein 23 (WDR23) directly regulate Nrf2 levels via similar but distinct proteasome-dependent pathways. WDR23 forms a part of the WDR23-Cullin 4A-RING ubiquitin ligase complex (CRL4AWDR23), whereas Keap1 serves as a substrate adaptor for the Cullin 3-containing ubiquitin ligase complex. However, the mechanisms underlying crosstalk between these Keap1 and WDR23 pathways for the regulation of Nrf2 levels have not been investigated. Here, we showed that knockdown (KD) of Keap1 upregulated the expression of Cullin4A (CUL4A) in a specificity protein 1 (Sp1)-dependent manner. We also revealed that Sp1 interacted with Keap1, leading to ubiquitination of Sp1. Increases in Sp1 by Keap1 KD triggered Sp1 binding to the fourth Sp1 binding site (Sp1_M4) within the -230/+50 region of the CUL4A gene. We also demonstrated that the overexpression and KD of Sp1 reduced and increased Nrf2 protein levels, respectively. These effects were abrogated by the WDR23 KD, suggesting that Sp1 also regulates Nrf2 levels via the ubiquitin ligase complex CRL4AWDR23. In conclusion, we discovered Sp1 as a novel substrate of Keap1 and provided evidence that Sp1 regulates the expression of CUL4A. We revealed a novel role for Sp1 in mediating crosstalk between two independent regulators of Nrf2 protein levels.

Bisphenol A and Its Derivatives Induce Degradation of HIF-1alpha via the Lysosomal Pathway in Human Hepatocarcinoma Cell Line, Hep3B.

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane), one of the phenolic compounds widely used in the manufacture of plastic and epoxy resins, is known as an endocrine disruptor. In a previous study, we found that BPA induced hypoxia inducible factor-1alpha (HIF-1alpha) degradation by dissociation from heat shock protein 90 (Hsp90). In this study, to investigate the structural requirements for degradation of HIF-1alpha, we estimated the effect of BPA derivatives (BPE, BPF, BPB, Dimethyl butylidene diphenol (DMBDP), Ethyl hexylidene diphenol (EHDP), Bishydroxyphenyl cyclohexane (BHCH), and Methyl benzylidene bisphenol (MBBP)) on HIF-1alpha protein degradation, using human hepatocarcinoma cell line, Hep3B. BPB, DMBDP, BHCH, and MBBP decreased HIF-1alpha protein levels more efficiently than BPA, but BPE, BPF, and EHDP did not affect HIF-1alpha protein levels. BPA degraded HIF-1alpha even in the presence of MG132, a proteasome inhibitor. In this study, we found that ammonium chloride (NH4Cl), a lysosomal enzyme inhibitor, efficiently restored the decrease in HIF-1alpha protein levels by BPA. Recent studies indicated that HIF-1alpha is degraded by the lysosomal pathway as well as the proteasomal pathway. Therefore, we investigated the levels of heat shock cognate 70 kDa protein (HSC70) protein after treatment with BPA. We found that BPA induced HSC70 protein and overexpression of HSC70 enhanced HIF-1alpha degradation in Hep3B cells. These results suggested that BPA causes the degradation of HIF-1alpha by induction of HSC70, leading lysosomal degradation of HIF-1alpha.

Down-regulation of EPHX2 gene transcription by Sp1 under high-glucose conditions.

sEH (soluble epoxide hydrolase), which is encoded by the EPHX2 gene, regulates the actions of bioactive lipids, EETs (epoxyeicosatrienoic acids). Previously, we found that high-glucose-induced oxidative stress suppressed sEH levels in a hepatocarcinoma cell line (Hep3B) and sEH was decreased in streptozotocin-induced diabetic mice in vivo. In the present study, we investigated the regulatory mechanisms underlying EPHX2 transcriptional suppression under high-glucose conditions. The decrease in sEH was prevented by an Sp1 (specificity protein 1) inhibitor, mithramycin A, and overexpression or knockdown of Sp1 revealed that Sp1 suppressively regulated sEH expression, in contrast with the general role of Sp1 on transcriptional activation. In addition, we found that AP2α (activating protein 2α) promoted EPHX2 transcription. The nuclear transport of Sp1, but not that of AP2α, was increased under high glucose concomitantly with the decrease in sEH. Within the EPHX2 promoter -56/+32, five Sp1-binding sites were identified, and the mutation of each of these sites showed that the first one (SP1_1) was important in both suppression by Sp1 and activation by AP2α. Furthermore, overexpression of Sp1 diminished the binding of AP2α by DNA-affinity precipitation assay and ChIP, suggesting competition between Sp1 and AP2α on the EPHX2 promoter. These findings provide novel insights into the role of Sp1 in transcriptional suppression, which may be applicable to the transcriptional regulation of other genes.

Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding ß-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.

Isolation and characterization of Xenopus soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

Protein-disulfide isomerase regulates the thyroid hormone receptor-mediated gene expression via redox factor-1 through thiol reduction-oxidation.

Protein-disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase that regulates the redox state of proteins. We previously found that overexpression of PDI in rat pituitary tumor (GH3) cells suppresses 3,3',5-triiodothyronine (T(3))-stimulated growth hormone (GH) expression, suggesting the contribution of PDI to the T(3)-mediated gene expression via thyroid hormone receptor (TR). In the present study, we have clarified the mechanism of regulation by which TR function is regulated by PDI. Overexpression of wild-type but not redox-inactive mutant PDI suppressed the T(3)-induced GH expression, suggesting that the redox activity of PDI contributes to the suppression of GH. We considered that PDI regulates the redox state of the TR and focused on redox factor-1 (Ref-1) as a mediator of the redox regulation of TR by PDI. Interaction between Ref-1 and TRß1 was detected. Overexpression of wild-type but not C64S Ref-1 facilitated the GH expression, suggesting that redox activity of Cys-64 in Ref-1 is involved in the TR-mediated gene expression. Moreover, PDI interacted with Ref-1 and changed the redox state of Ref-1, suggesting that PDI controls the redox state of Ref-1. Our studies suggested that Ref-1 contributes to TR-mediated gene expression and that the redox state of Ref-1 is regulated by PDI. Redox regulation of PDI via Ref-1 is a new aspect of PDI function.

Seven in absentia homolog 2 (Siah2) protein is a regulator of NF-E2-related factor 2 (Nrf2).

Under pathological conditions such as ischemia-reperfusion, Nrf2 acts as a key regulator of cellular oxidative response. Provided that Nrf2 is sensitive to hypoxia during ischemia, Nrf2 may affect reactive oxygen species metabolism during reoxygenation. In this study, hypoxia suppressed Nrf2 protein, and its hypoxic suppression was not recovered with knockdown of the Nrf2 repressor Keap1. Moreover, an Nrf2 mutant lacking the Keap1 binding domain was suppressed under hypoxia, suggesting that Keap1 does not contribute to hypoxic Nrf2 suppression. HIF-1α and Siah2 are both key regulators of hypoxic responses. Hypoxia induced the Siah2 protein. Although inhibition or knockdown of Siah2 prevented the suppression of Nrf2, knockdown of HIF-1α did not. Moreover, Siah2 interacted with Nrf2 through a binding motif, suggesting that Siah2 contributes to the suppression of Nrf2. Some cytosolic kinases also play important roles in Nrf2 regulation. In this study, PKC phosphorylates serine residues of Nrf2 during hypoxia. Knockdown of Siah2 rescued hypoxic decreases in an Nrf2 mutant that mimicked phosphorylation at serine 40 or lacked this phosphorylation site, suggesting that Siah2 contributes to the degradation of Nrf2 irrespective of its phosphorylation status. Moreover, knockdown of Siah2 attenuated ubiquitination of the Nrf2 mutant, suggesting that association of Siah2 with Nrf2 causes proteasome-mediated degradation of Nrf2.

A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response.

Endoplasmic reticulum protein (ERp) 29 binds as strongly as protein disulfide isomerase (PDI) to bisphenol A.

Bisphenol A (BPA), which is used in polycarbonate and epoxy resins, affects the development or function of the central nervous system. Previously, we isolated a BPA-binding protein from rat brain, identified it as protein disulfide isomerase (PDI), and found that BPA binds to the b' domain of PDI and inhibits its activity. There are 20 kinds of PDI family proteins in mammalian endoplasmic reticulum. The member proteins each have a different length and domain arrangement. Here we investigated the binding of BPA and T3 to ERp29, ERp57, and ERp72, which each have the b or b' domain. BPA/T3 binding of ERp57 and that of ERp72 were lower than that of PDI, and BPA did not inhibit the oxidase or reductase activity of these proteins. On the other hand, BPA and T3 bound to ERp29 as strongly as to PDI. The CD spectrum of PDI was changed in the presence of BPA in a dose-dependent manner, while that of ERp29 was not, suggesting that BPA did not affect the conformation of ERp29. We found that PDI suppresses GH expression in rat GH3 cells stimulated by thyroid hormone (T3) overexpression of PDI and that ERp57 reduced the GH level, but overexpression of ERp29 did not change GH expression. These results suggested that affinity to T3 does not involve the reduction of the T3 response. In this study, ERp29 was first identified as a BPA-binding protein but is not involved in the T3 response of GH3 cells.

Hydrogen peroxide activates APE1/Ref-1 via NF-κB and Parkin: a role in liver cancer resistance to oxidative stress.

Cancer cells exhibit an altered redox balance and aberrant redox signaling due to genetic, metabolic, and microenvironment-associated reprogramming. Persistently elevated levels of reactive oxygen species (ROS) contribute to many aspects of tumor development and progression. Emerging studies demonstrated the vital role of apurinic/apyrimidinic endonuclease 1 or reduction/oxidation (redox) factor 1(APE1/Ref-1) in the oxidative stress response and survival of cancer cells. APE1/Ref-1 is a multifunctional enzyme involved in the DNA damage response and functions as a redox regulator of transcription factors. We herein demonstrated that basal hydrogen peroxide (H2O2) and APE1/Ref-1 expression levels were markedly higher in cancer cell lines than in non-cancerous cells. Elevated APE1/Ref-1 levels were associated with shorter survival in liver cancer patients. Mechanistically, we showed that H2O2 activated nuclear factor-κB (NF-κB). RelA/p65 inhibited the expression of the E3 ubiquitin ligase Parkin, possibly by interfering with ATF4 activity. Parkin was responsible for the ubiquitination and proteasomal degradation of APE1/Ref-1; therefore, the H2O2-induced suppression of Parkin expression increased APE1/Ref-1 levels. The probability of survival was lower in liver cancer patients with low Parkin and high RelA expression levels. Additionally, Parkin and RelA expression levels negatively and positively correlated with APE1/Ref-1 levels, respectively, in the TCGA liver cancer cohort. We concluded that increases in APE1/Ref-1 via the NF-κB and Parkin pathways are critical for cancer cell survival under oxidative stress. The present results show the potential of the NF-κB-Parkin-APE1/Ref-1 axis as a prognostic factor and therapeutic strategy to eradicate liver cancer.

Nrf2 Regulates the Expression of CYP2D6 by Inhibiting the Activity of Krüppel-Like Factor 9 (KLF9).

AIMS: The aim of the present study is to gain insight into the biology of Parkinson's disease (PD) and cancer to drive translational advances enabling more effective prevention and/or potential treatments. BACKGROUND: The expression of Cytochrome P450 2D6 (CYP2D6) is correlated with various diseases such as PD and cancer; therefore, exploring its regulatory mechanism at transcriptional levels is of interest. NF-E2-related factor 2 (Nrf2) has been known to be responsible for regulating phase II and phase III drug-metabolizing genes. OBJECTIVES: The objectives of this study are to investigate the transcriptional regulation of CYP2D6 by Nrf2 and to analyze its role in PD and cancer. METHODS: Nrf2 was transiently expressed in human hepatoma Hep3B cells, and the expression of CYP2D6 was examined by RT-qPCR. The promoter activity of CYP2D6 and the DNA binding of Nrf2 were examined by luciferase and ChIP assay, respectively. We then investigated the expression and correlation of Nrf2 and CYP2D6 in the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. RESULTS: In the present study, we demonstrated that Nrf2 down-regulated CYP2D6 mRNA expression in hepatoma Hep3B cells. Mechanistically, Nrf2 binds to the antioxidant responsive element (ARE) in the proximity of krüppel- like factor 9 (KLF9)-binding site within the -550/+51 of CYP2D6 promoter. The inhibition and activation of Nrf2 enhanced and suppressed KLF9 effects on CYP2D6 expression, respectively. The expression levels of Nrf2 and CYP2D6 were upregulated and downregulated in the PD patient GEO datasets compared to the healthy control tissues, and Nrf2 was negatively correlated with CYP2D6. In liver cancer patients, decreased CYP2D6 levels were apparent and associated with a lower probability of survival. CONCLUSION: Our work revealed the inhibitory role of Nrf2 in regulating CYP2D6 expression. Moreover, Nrf2- dependent regulation of CYP2D6 can be used as a prognostic factor and therapeutic strategy in PD and liver cancer.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 µM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions.

Crystallization and preliminary crystallographic analysis of the complex between triiodothyronine and the bb' fragment of rat protein disulfide isomerase.

Protein disulfide isomerase (PDI) is a multifunctional protein that catalyzes the formation of a disulfide bond in nascent and misfolded proteins and is also known to bind to the thyroid hormone triiodothyronine (T3). When T3 is bound to PDI its catalytic activity is inhibited, but the biological function of this binding is not well understood. In previous studies, it was found that T3 binds to the bb' fragment of PDI. Therefore, to clarify the structure of the complex consisting of PDI bound to T3, a crystallographic analysis of the three-dimensional structure of the T3-rat PDI bb' complex was performed. Native bb' crystals and T3-bb' complex crystals were both obtained using the hanging-drop vapour-diffusion technique with 1.6 M trisodium citrate pH 6.2 as a precipitant. The space group of the native bb' crystals was found to be C222, with unit-cell parameters a = 94.8, b = 114.9, c = 182.9 Å, while the space group of the T3-bb' complex crystals was P2(1)2(1)2(1), with unit-cell parameters a = 99.9, b = 184.5, c = 232.2 Å. Diffraction data for the native and complex crystals were collected to resolutions of 3.06 and 3.00 Å, respectively.

Chlorogenic Acid Activates Nrf2/SKN-1 and Prolongs the Lifespan of Caenorhabditis elegans via the Akt-FOXO3/DAF16a-DDB1 Pathway and Activation of DAF16f.

Chlorogenic acid (CGA) is the most abundant polyphenol in coffee. It has been widely reported to exhibit antioxidant activity by activating nuclear factor erythroid 2-related factor 2 (Nrf2) potentially via the canonical Kelch-like-ECH-associated protein 1 (Keap1)-Nrf2 pathway. We herein demonstrated that the knockdown of WD40 repeat protein 23 (WDR23), but not Keap1, abolished the effects of CGA on the activation of Nrf2. CGA decreased the expression of DDB1, an adaptor for WDR23-Cullin 4A-RING ligase (CRL4AWDR23). FOXO3, a major target for inactivation by the PI3K/Akt pathway, was identified as the transcription factor responsible for the basal and CGA-inhibited expression of the DDB1 gene. CGA blocked FOXO3 binding to importin-7 (IPO7), thereby inhibiting the nuclear accumulation of FOXO3, down-regulating the expression of DDB1, inhibiting the activity of CRL4WDR23, and ultimately increasing that of Nrf2. This pathway was conserved in Caenorhabditis elegans, and CGA extended the lifespan partly through this pathway. We found that in C. elegans, the isoform DAF-16a, but not DAF-16f, regulated the expression levels of ddb-1 mRNA and SKN-1 protein. CGA prolonged the mean lifespan of DAF-16a- and DAF-16f-rescued worms by 24% and 9%, respectively, suggesting that both isoforms involve in lifespan-extending effects of CGA, with DAF-16a being more important than DAF-16f. Based on these results, we established a novel Akt-FOXO3/DAF16a-DDB1 axis that links nutrient sensing and oxidative stress response pathways. Our results also provide a novel molecular mechanism for Nrf2/SKN-1 activation by CGA and the increased lifespan of C. elegans by CGA via this pathway.

Nrf2 and Parkin-Hsc70 regulate the expression and protein stability of p62/SQSTM1 under hypoxia.

Solid tumors often contain regions with very low oxygen concentrations or hypoxia resulting from altered metabolism, uncontrolled proliferation, and abnormal tumor blood vessels. Hypoxia leads to resistance to both radio- and chemotherapy and a predisposition to tumor metastases. Under hypoxia, sequestosome 1 (SQSTM1/p62), a multifunctional stress-inducible protein involved in various cellular processes, such as autophagy, is down-regulated. The hypoxic depletion of p62 is mediated by autophagic degradation. We herein demonstrated that hypoxia down-regulated p62 in the hepatoma cell line Hep3B at the transcriptional and post-translational levels. At the transcriptional level, hypoxia down-regulated p62 mRNA by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2). The overexpression of Nrf2 and knockdown of Siah2, a negative regulator of Nrf2 under hypoxia, diminished the effects of hypoxia on p62 mRNA. At the post-translational level, the proteasome inhibitor MG132, but not the lysosomal inhibitors ammonium chloride and bafilomycin, prevented the hypoxic depletion of p62, suggesting the involvement of the proteasome pathway. Under hypoxia, the expression of the E3 ubiquitin ligase Parkin was up-regulated in a hypoxia-inducible factor 1α-dependent manner. Parkin ubiquitinated p62 and led to its proteasomal degradation, ensuring low levels of p62 under hypoxia. We demonstrated that the effects of Parkin on p62 required heat shock cognate 71 kDa protein (Hsc70). We also showed that the overexpression of Nrf2 and knockdown of Parkin or Hsc70 induced the accumulation of p62 and reduced the viability of cells under hypoxia. We concluded that a decrease in p62, which involves regulation at the transcriptional and post-translational levels, is critical for cell survival under hypoxia. The present results show the potential of targeting Nrf2/Parkin-Hsc70-p62 as a novel strategy to eradicate hypoxic solid tumors.

Feedback of hypoxia-inducible factor-1alpha (HIF-1alpha) transcriptional activity via redox factor-1 (Ref-1) induction by reactive oxygen species (ROS).

Hypoxia-inducible factor-1alpha (HIF-1alpha) is important for adaptation to hypoxia. Hypoxia is a common feature of cancer and inflammation, by which HIF-1alpha increases. However, prolonged hypoxia decreases HIF-1alpha, and the underlying mechanisms currently remain unclear. Cellular reactive oxygen species (ROS) increases in cancer and inflammation. In the present study, we demonstrated that prolonged hypoxia increased ROS, which induced prolyl hydroxylase domain-containing protein 2 (PHD2) and factor inhibiting HIF-1 (FIH-1), major regulators of HIF-1alpha. Cellular stress response (CSR) increased HIF-1alpha transcriptional activity by scavenging endogenous ROS. PHD2 and FIH-1 were induced by external hydrogen peroxide (H2O2) but were suppressed by ROS-scavenging catalase. We investigated the mechanisms by which PHD2 and FIH-1 are regulated by ROS. The knockdown of HIF-1alpha decreased PHD2 and FIH-1 mRNA levels, suggesting their regulation by HIF-1alpha. We then focused on redox factor-1 (Ref-1), which is a regulator of HIF-1alpha transcriptional activity. The knockdown of Ref-1 decreased PHD2 and FIH-1. Ref-1 was regulated by ROS. Prolonged hypoxia and the addition of H2O2 induced the expression of Ref-1. Furthermore, the knockdown of p65, a component of kappa-light-chain enhancer of activated B cells (NF-κB), efficiently inhibited the induction of Ref-1 by ROS. Collectively, the present results showed that prolonged hypoxia or increased ROS levels induced Ref-1, leading to the activation of HIF-1alpha transcriptional activity, while the activation of HIF-1alpha via Ref-1 induced PHD2 and FIH-1, causing the feedback of HIF-1alpha. To the best of our knowledge, this is the first study to demonstrate the regulation of HIF-1alpha via Ref-1 by ROS.