The aryl hydrocarbon receptor (AhR) is a ligand-sensitive transcription factor which is responsible for most 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities. Without ligand, the AhR complex is cytoplasmic and contains p23. Our objective was to investigate whether the wild type p23 levels are important for the AhR function. We generated eight p23-specific knockdown stable cell lines via either electroporation or lentiviral infection. Five of these stable cell lines were generated from a mouse hepatoma cell line (Hepa1c1c7) and three were from human hepatoma and cervical cell lines (Hep3B and HeLa). All of them expressed lower AhR protein levels, leading to reduced ligand-induced, DRE-driven downstream activity. The AhR protein levels in p23-specific knockdown stable cells were reversed back to wild type levels after exogenous p23 was introduced. Reduction of the AhR protein levels in these stable cells was caused by a decrease in the AhR message levels and an increase of the AhR protein degradation in the absence of ligand. This ligand-independent degradation of AhR was insensitive to MG132, suggesting that the 26S proteasome was not responsible for the degradation. In addition, MG132 could not protect AhR from the ligand-induced degradation in both mouse and human p23-knockdown stable cells.
p23 co-chaperone; aryl hydrocarbon receptor; AhR; protein degradation; dioxin
The objective of this study was to examine the influence of persistence of the MetS (MetS) and its individual components over a 3-year period on carotid intima media thickness (CIMT) in overweight Latino children.
Ninety-seven healthy male and female overweight Latino children (mean age at baseline: 11.0±1.8 yrs) were assessed for MetS on four annual evaluations and classified according to the persistence of MetS: NEVER (0 annual visits with the MetS, n=53), INTERMITTENT (1 or 2 visits with the MetS, n=28), and PERSISTENT (3 or 4 visits with the MetS, n=16). CIMT was measured with high-resolution B-mode ultrasound (7.9±0.7 months after the most recent MetS assessment; mean age: 14.6±1.8 yr).
PERSISTENT MetS was associated with significantly higher CIMT (0.647mm±0.018 compared to (0.600mm±0.007 in those who NEVER had MetS, p<0.01). This difference remained significant after controlling for gender, baseline age, total fat mass, total lean tissue mass and insulin sensitivity. PERSISTENT high waist circumference and PERSISTENT high blood pressure were also significantly associated with higher mean CIMT, but these differences were no longer significant after controlling for total fat and lean tissue mass. Baseline systolic blood pressure and 2-hour glucose were significantly related to CIMT independent of all other MetS components (p<0.05).
Persistence of the MetS over a 3-year period was uniquely associated with increased CIMT during childhood. Children with hypertension, persistent abdominal adiposity and impaired glucose tolerance may also be at higher risk for elevated CIMT.
CIMT; obesity; children; MetS
To assess carotid artery intima media thickness (CIMT) change over two years in overweight Latino adolescents and examine its relationship to cardiometabolic risk.
72 healthy overweight male and female Latino adolescents (mean age: 14.5±1.7 yrs; mean BMI: 31.5±6.9 kg/m2) were evaluated at baseline and 2 years later for: CIMT by high resolution B-mode ultrasound, the metabolic syndrome and its features, body composition by DEXA and MRI, and glucose/insulin measures by fasting blood, and oral and intravenous glucose tolerance tests.
Baseline CIMT did not differ from 2-year follow-up; however 38 participants increased CIMT (0.017±0.003mm; +2.8%) and 34 decreased (-0.019±0.002mm; −3.1%). ANCOVA analyses showed that participants with CIMT progression had higher baseline LDL-cholesterol and total cholesterol (91.3±3.4 and 150.3±3.9mg/dL) compared with those with CIMT regression (78.1±3.6 and 135.6±4.2mg/dL, p<0.05), independent of sex, baseline CIMT, age, and height. In multivariate regression, LDL-cholesterol was the sole predictor of CIMT progression, but the effect was small (odds of CIMT progression increased by 3% for each 1 mg/dL higher baseline LDL-cholesterol [95% CI: 1.004-1.006, p=0.03].
These results indicate a high variability in the magnitude of CIMT change in growing overweight Latino youth and support the use of LDL-cholesterol to assess sub-clinical atherosclerosis risk in this population.
Obesity; Cardiovascular disease risk; Ultrasound imaging
Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)-mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2-activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that modulate the Nrf2-Keap1 signaling pathway. A series of cinnamoyl-based electrophilic Michael acceptors including cinnamic aldehyde and methyl-1-cinnamoyl-5-oxo-2-pyrrolidine-carboxylate was identified as potent Nrf2-activators. Hit confirmation was performed in a secondary screen, based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts, HaCaT keratinocytes, and primary skin keratinocytes. Bioefficacy profiling of positive test compounds in skin cells demonstrated compound-induced upregulation of hemeoxygenase I and NAD(P)H-quinone oxidoreductase, two Nrf2 target genes involved in the cellular antioxidant response. Pretreatment with cinnamoyl-based Nrf2-activators suppressed intracellular oxidative stress and protected against photo-oxidative induction of apoptosis in skin cells exposed to high doses of singlet oxygen. Our pilot studies suggest feasibility of developing cinnamoyl-based Nrf2-activators as novel photo-chemopreventive agents targeting skin cell photo-oxidative stress.
Nrf2; skin cancer; photo-oxidative stress; photo-chemoprevention; Michael acceptor; cinnamic aldehyde; singlet oxygen
The aryl hydrocarbon receptor nuclear translocator (ARNT) heterodimerizes with hypoxia inducible factor-1α (HIF-1α), followed by upregulation of genes that are essential for carcinogenesis. We utilized a novel peptide (Ainp1) to address whether the HIF-1α signaling could be suppressed by an ARNT-mediated mechanism. Ainp1 suppresses the HIF-1α-dependent luciferase expression in Hep3B cells and this suppression can be reversed by ARNT. Ainp1 reduces the interaction between ARNT and HIF-1α, suppresses the formation of the HIF-1 gel shift complex, and suppresses the ARNT recruitment to the vegf promoter. These effects are partly mediated by redistribution of the nuclear ARNT contents to the cytoplasm.
ARNT-interacting peptide; HIF-1α; ARNT; AhR; anticancer; Hep3B
The E2F1 transcription factor is post-translationally modified and stabilized in response to various forms of DNA damage to regulate the expression of cell cycle and pro-apoptotic genes. E2F1 also forms foci at DNA double-strand breaks (DSBs) but the function of E2F1 at sites of damage is unknown. Here we demonstrate that the absence of E2F1 leads to spontaneous DNA breaks and impaired recovery following exposure to ionizing radiation. E2F1 deficiency results in defective NBS1 phosphorylation and foci formation in response to DSBs but does not affect NBS1 expression levels. Moreover, an increased association between NBS1 and E2F1 is observed in response to DNA damage, suggesting that E2F1 may promote NBS1 foci formation through a direct or indirect interaction at sites of DNA breaks. E2F1 deficiency also impairs RPA and Rad51 foci formation indicating that E2F1 is important for DNA end resection and the formation of single-stranded DNA at DSBs. These findings establish new roles for E2F1 in the DNA damage response, which may directly contribute to DNA repair and genome maintenance.
E2F; NBS1; RPA; RAD51; DNA damage response
Adverse environmental conditions produce ER stress and elicit the unfolded protein response (UPR) in plants. Plants are reported to have two "arms" of the ER stress signaling pathway-one arm involving membrane-bound transcription factors and the other involving a membrane-associated RNA splicing factor, IRE1. IRE1 in yeast to mammals recognizes a conserved twin loop structure in the target RNA.
A segment of the mRNA encoding ZmbZIP60 in maize can be folded into a twin loop structure, and in response to ER stress this mRNA is spliced, excising a 20b intron. Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus. Splicing of ZmbZIP60 can be elicited in maize seedlings by ER stress agents such as dithiothreitol (DTT) or tunicamycin (TM) or by heat treatment. Younger, rather than older seedlings display a more robust splicing response as do younger parts of leaf, along a developmental gradient in a leaf. The molecular signature of an ER stress response in plants includes the upregulation of Binding Protein (BIP) genes. Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb.
The splicing of ZmbZIP60 mRNA is an indicator of ER stress in maize seedlings resulting from adverse environmental conditions such as heat stress. ZmbZIP60 mRNA splicing in maize leads predictively to the formation of active bZIP transcription factor targeted to the nucleus to upregulate stress response genes. Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.
IRE1; bZIP transcription factor; mRNA splicing; Unfolded protein response; Binding protein; Chaperone; Heat stress; Corn
We explored whether modulation of the estrogen receptor (ER) signaling is possible through an aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent mechanism. We utilized the Arnt-interacting protein 2 (Ainp2) to examine whether the presence of Ainp2 in MCF-7 cells would interfere with the Arnt-mediated ER signaling. We found that Arnt increased the 17 beta-estradiol (E2)-dependent luciferase activity and Ainp2 significantly suppressed this Arnt-mediated luciferase activity. Ainp2 significantly suppressed 25% of the E2- and Arnt-dependent upregulation of the GREB1 message. No suppression of the ER target gene expression by Ainp2 was detected in Arnt-knockdown MCF-7 cells and in Arnt-independent ER signaling. Although Ainp2 did not interact with ER alpha and ER beta, it suppressed the ER alpha::Arnt interaction and reduced the E2-driven recruitment of Arnt to the GREB1 promoter. We concluded that Ainp2 suppresses the ER signaling by not allowing Arnt to participate in the ER-dependent, Arnt-mediated activation of gene transcription.
estrogen receptor; Arnt; Ainp2; Arnt-interacting protein
To examine a relationship between alterations of structure and function of the arterial wall in response to glucose-lowering therapy in type 2 diabetes mellitus (DM) after a 1-year follow-up (FU).
Methods and results
In DM (n = 22) and in healthy controls (n = 17), coronary artery calcification (CAC) was assessed with electron beam tomography and carotid intima–media thickness (IMT) with ultrasound, whereas coronary function was determined with positron emission tomography-measured myocardial blood flow (MBF) at rest, during cold pressor testing (CPT), and during adenosine stimulation at baseline and after FU. The decrease in plasma glucose in DM after a mean FU of 14 ± 1.9 months correlated with a lower progression of CAC and carotid IMT (r = 0.48, P ≤ 0.036 and r = 0.46, P ≤ 0.055) and with an improvement in endothelium-related ΔMBF to CPT and to adenosine (r = 0.46, P ≤ 0.038 and r = 0.36, P ≤ 0.056). After adjusting for metabolic parameters by multivariate analysis, the increases in ΔMBF to CPT after glucose-lowering treatment remained a statistically significant independent predictor of the progression of CAC (P ≤ 0.001 by one-way analysis of variance).
In DM, glucose-lowering treatment may beneficially affect structure and function of the vascular wall, whereas the observed improvement in endothelium-related coronary artery function may also mediate direct preventive effects on the progression of CAC.
Cardiovascular disease prevention; Carotid IMT; Coronary artery calcification; Coronary circulation; Endothelium; Diabetes mellitus; Positron emission tomography
We used a xenograft model to investigate whether the aryl hydrocarbon receptor deletion construct CΔ553 suppresses tumor growth. HeLa cells that were infected with CΔ553 expressing adenovirus (Ad553) formed very small tumors whereas the control adenovirus-infected cells formed large tumors at day 15. CΔ553 inhibited the formation of the HIF-1 DNA complex and suppressed the induction of the HIF-1α target proteins CAIX and GLUT1. The Ad553 tumors had less HIF-1 function since they showed reduced microvessel formation and lesser amounts of HIF-1α, Arnt, phospho-Akt, CAIX, and GLUT1. Proteasome-mediated Arnt degradation was enhanced in Ad553-infected HeLa cells and tumors.
Drinking water contaminated with arsenic, a human carcinogen, is a worldwide health issue. An understanding of cellular signaling events in response to arsenic exposure and rational designing of strategies to reduce arsenic damages by modulating signaling events are important to fight against arsenic-induced diseases. Previously, we reported that activation of the Nrf2-mediated cellular defense pathway confers protection against toxic effects induced by sodium arsenite [As(III)] or monomethylarsonous acid [MMA(III)]. Paradoxically, arsenic has been reported to induce the Nrf2-dependent signaling pathway. Here, we report the unique mechanism of Nrf2 induction by arsenic. Similar to tert-butylhydroquinone (tBHQ) or sulforaphane (SF), arsenic induced the Nrf2-dependent response through enhancing Nrf2 protein levels by inhibiting Nrf2 ubiquitination and degradation. However, the detailed action of arsenic in Nrf2 induction is different from that of tBHQ or SF. Arsenic markedly enhanced the interaction between Keap1 and Cul3, subunits of the E3 ubiquitin ligase for Nrf2, which led to impaired dynamic assembly/disassembly of the E3 ubiquitin ligase and thus decreased its ligase activity. Furthermore, induction of Nrf2 by arsenic is independent of the previously identified C151 residue in Keap1 that is required for Nrf2 activation by tBHQ or SF. Distinct mechanisms of Nrf2 activation by seemingly harmful and beneficial reagents provide a molecular basis to design Nrf2-activating agents for therapeutic intervention.
Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers. Here, we report that inhibition of NF-E2-related factor 2 (Nrf2) may be a promising strategy to combat chemoresistance. Nrf2 is a critical transcription factor regulating a cellular protective response that defends cells against toxic insults from a broad spectrum of chemicals. Under normal conditions, the low constitutive amount of Nrf2 protein is maintained by the Kelch-like ECH-associated protein1 (Keap1)-mediated ubiquitination and proteasomal degradation system. Upon activation, this Keap1-dependent Nrf2 degradation mechanism is quickly inactivated, resulting in accumulation and activation of the antioxidant response element (ARE)-dependent cytoprotective genes. Since its discovery, Nrf2 has been viewed as a ‘good’ transcription factor that protects us from many diseases. In this study, we demonstrate the dark side of Nrf2: stable overexpression of Nrf2 resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin and etoposide. Inversely, downregulation of the Nrf2-dependent response by overexpression of Keap1 or transient transfection of Nrf2–small interfering RNA (siRNA) rendered cancer cells more susceptible to these drugs. Upregulation of Nrf2 by the small chemical tert-butylhydroquinone (tBHQ) also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, we provide evidence that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not limited to certain cancer types or anticancer drugs and thus can be applied during the course of chemotherapy to treat many cancer types.
The impact of HIV infection and exposure to antiretroviral therapy on the development of subclinical atherosclerosis is incompletely understood.
To compare intima–media thickness (IMT) of the carotid artery between HIV-infected subjects receiving protease inhibitor-containing regimens and subjects not receiving these regimens and to compare differences in the IMT of the carotid artery between HIV-infected subjects and HIV-uninfected subjects.
A prospective matched cohort study in university-based outpatient clinics. Groups of three individuals (triads) matched on the following characteristics were enrolled: age, sex, race/ethnicity, smoking status, blood pressure and menopausal status. Group 1, HIV-infected subjects with continuous use of protease inhibitor (PI) therapy for ≥ 2 years; group 2, HIV-infected subjects without prior PI use; and group 3: HIV-uninfected. Ultrasonographers at six sites sent standardized ultrasound images to a central reading site for carotid IMT measurements. Carotid IMT was compared within the HIV-infected groups (1 and 2) and between the HIV-infected and uninfected groups in a matched analysis.
One hundred and thirty-four individuals were enrolled in 45 triads. The median IMT in groups 1, 2 and 3 was 0.690, 0.712 and 0.698 mm, respectively. There were no statistically significant differences in IMT between groups 1 and 2, or in the combined HIV groups compared with the HIV uninfected group. Significant predictors of carotid IMT in a multivariate model included high-density lipoprotein (HDL) cholesterol, the interaction of HDL cholesterol and triglycerides, age and body mass index.
We found no association between PI inhibitor exposure or HIV infection and carotid IMT.
atherosclerosis; HIV infection; carotid intima; media thickness; protease inhibitors