Misalignment of Circadian Rhythms in Diet-Induced Obesity.

The biological clocks of the circadian timing system coordinate cellular and physiological processes and synchronize them with daily cycles. While the central clock in the suprachiasmatic nucleus (SCN) is mainly synchronized by the light/dark cycles, the peripheral clocks react to other stimuli, including the feeding/fasting state, nutrients, sleep-wake cycles, and physical activity. During the disruption of circadian rhythms due to genetic mutations or social and occupational obligations, incorrect arrangement between the internal clock system and environmental rhythms leads to the development of obesity. Desynchronization between the central and peripheral clocks by altered timing of food intake and diet composition leads to uncoupling of the peripheral clocks from the central pacemaker and to the development of metabolic disorders. The strong coupling of the SCN to the light-dark cycle creates a situation of misalignment when food is ingested during the "wrong" time of day. Food-anticipatory activity is mediated by a self-sustained circadian timing, and its principal component is a food-entrainable oscillator. Modifying the time of feeding alone greatly affects body weight, whereas ketogenic diet (KD) influences circadian biology, through the modulation of clock gene expression. Night-eating behavior is one of the causes of circadian disruption, and night eaters have compulsive and uncontrolled eating with severe obesity. By contrast, time-restricted eating (TRE) restores circadian rhythms through maintaining an appropriate daily rhythm of the eating-fasting cycle. The hypothalamus has a crucial role in the regulation of energy balance rather than food intake. While circadian locomotor output cycles kaput (CLOCK) expression levels increase with high-fat diet-induced obesity, peroxisome proliferator-activated receptor-alpha (PPARα) increases the transcriptional level of brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1) in obese subjects. In this context, effective timing of chronotherapies aiming to correct SCN-driven rhythms depends on an accurate assessment of the SCN phase. In fact, in a multi-oscillator system, local rhythmicity and its disruption reflects the disruption of either local clocks or central clocks, thus imposing rhythmicity on those local tissues, whereas misalignment of peripheral oscillators is due to exosome-based intercellular communication.Consequently, disruption of clock genes results in dyslipidemia, insulin resistance, and obesity, while light exposure during the daytime, food intake during the daytime, and sleeping during the biological night promote circadian alignment between the central and peripheral clocks. Thus, shift work is associated with an increased risk of obesity, diabetes, and cardiovascular diseases because of unusual eating times as well as unusual light exposure and disruption of the circadian rhythm.

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-ß activation. Activated PKC-ß induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

The Regulation Pathway of VEGF Gene Expression Is Different between 2D Cells and 3D Spheroids in Human Lung Cancer Cells.

Angiogenesis is involved in the malignant transformation of cancers. Vascular endothelial growth factor (VEGF) is important in inducing angiogenesis. Cultured cells play an important role in analyzing the regulation of VEGF expression, and it is revealed that VEGF expression is induced under hypoxia. However, it has been shown that there are differences in the pathway for gene expression between two-dimensional (2D) cells and in vivo cells. Three-dimensional (3D) spheroids constructed in 3D culture with a gene expression pattern more similar to that of in vivo cells than 2D cells have been used to solve this problem. This study analyzed the VEGF gene expression pathway in 3D spheroids of human lung cancer cells, A549 and H1703. Hypoxia-inducible factor-1α (HIF-1α) and aryl hydrocarbon receptor nuclear translocator (ARNT) regulated VEGF gene expression in 3D spheroids. However, VEGF gene expression was not regulated by HIF-1α in 2D cells. To conclude, we found that the regulatory pathway of VEGF gene expression is different between 2D cells and 3D spheroids in human lung cancer cells. These results suggest the possibility of a new VEGF gene expression regulation pathway in vivo. In addition, they show useful knowledge for the analysis of angiogenesis induction mechanisms and also demonstrate the usefulness of 3D spheroids.

New insight into methamphetamine-associated heart failure revealed by transcriptomic analyses: Circadian rhythm disorder.

Methamphetamine (METH) abuse is a significant public health concern globally. Cardiac toxicity is one of the important characteristics of METH, in addition to its effects on the nervous system. However, to date, research on the cardiotoxic injury induced by METH consumption has been insufficient. To systematically analyze the potential molecular mechanism of cardiac toxicity in METH-associated heart failure (HF), a rat model was constructed with a dose of 10 mg/kg of METH consumption. Cardiac function was evaluated by echocardiography, and HE staining was used to clarify the myocardial histopathological changes. Integrated analyses, including mRNA, miRNA and lncRNA, was performed to analyze the RNA expression profile and the potential molecular mechanisms involved in METH-associated HF. The results showed that METH caused decreased myocardial contractility, with a decreased percent ejection fraction (%EF). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses of the RNAs with expression changes revealed abnormal circadian rhythm regulation in the METH groups, with circadian rhythm-related genes and their downstream effectors expressed differentially, especially the aryl hydrocarbon receptor nuclear translocator-like (Arntl). Competing endogenous RNA (ceRNA) networks associated with circadian rhythm, including Arntl, was also observed. Therefore, this study revealed that long-term METH consumption was associated with the HF in a rat model by decreasing the %EF, and that the abnormal circadian rhythm could provide new directions for investigating the METH-associated HF, and that the differentially expressed genes in this model could provide candidate genes for the identification and assessment of cardiac toxicity in METH-associated HF, which is fundamental for further understanding of the disease.

Role of Different Doses of Ketamine in Postoperative Neurocognitive Function in Aged Mice Undergoing Partial Hepatectomy by Regulating the Bmal1/NMDA/NF-Κb Axis.

BACKGROUND: The current study set out to probe the function of different doses of ketamine in postoperative neurocognitive disorder (PND) in aged mice undergoing partial hepatectomy (PH) with the involvement of the brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1)/n-methyl-D-aspartate (NMDA)/nuclear factor-kappa B (NF-κB) axis. METHODS: First, aged mice were intraperitoneally injected with different doses of ketamine prior to surgery, followed by hepatic lobectomy. Afterward, mice cognitive function was assessed. In addition, Bmal1 mRNA expression patterns were quantified, while NMDA 2B receptor, NF-κB p65, synapsin 1, and postsynaptic density 95 (PSD95) levels were determined; the release of inflammatory factors was detected, and ionized calcium-binding adapter molecule-1 expression was measured to quantify microglia activation. In addition, Bmal1-knockout (Bmal1-KO) mice were intraperitoneally injected with a subanesthetic dose of ketamine to verify the mechanism of Bmal1 in regulating the NMDA 2B subunit (NR2B)/NF-κB axis to affect PH in aged patients. RESULTS: After PH, hippocampal-dependent memory was impaired, and synapsin 1 and PSD95 levels were downregulated. On the other hand, PH diminished Bmal1 expression but elevated NR2B and NF-κB p65 levels and anesthetic doses of ketamine further regulated the Bmal1/NMDA/NF-κB axis. In Bmal1-KO mice, the NMDA/NF-κB axis was activated, the release of inflammatory cytokines was promoted, and hippocampus-dependent memory was impaired, which were reversed by a subanesthetic dose of ketamine. CONCLUSION: Altogether, findings obtained in our study indicated that a subanesthetic dose of ketamine activated Bmal1, downregulated the NMDA/NF-κB axis, and reduced inflammation and microglia activation to alleviate PND in aged mice undergoing PH.

Downregulation of Arntl mRNA Expression in Women with Hypertension: A Case-Control Study.

BACKGROUND: Previous studies have reported that disturbance of endogenous circadian rhythms enhances the chance of hypertension and suggested that circadian clock genes could have a crucial function in the onset of the disease. This case-control study was aimed to investigate the association of the mRNA expression of aryl hydrocarbon receptor nuclear translocator like (Arntl), clock circadian regulator (Clock), and period circadian regulators 1 and 2 (Per1 and Per2) with hypertension and blood pressure levels. METHODS: A total of 172 subjects were recruited in this study, including 86 hypertension and 86 nonhypertension controls. The mRNA expression levels in peripheral blood mononuclear cells were determined by real-time quantitative polymerase chain reaction. The differences in Arntl, Clock, Per1, and Per2 mRNA expression were compared between the 2 groups, and the relationship between mRNA expression and cardiometabolic risk profiles was also assessed. RESULTS: We found that the mRNA expression of Arntl was downregulated in the hypertension cases compared with controls in women (1.10 [0.66, 1.71] vs. 1.30 [0.99, 2.06], p = 0.031). There was a significant negative correlation between the Arntl mRNA expression and SBP (r = -0.301, p = 0.004) and DBP (r = -0.222, p = 0.034) in women. In men, a negative correlation between the Per1 mRNA expression and SBP (r = -0.247, p = 0.026) was found. CONCLUSIONS: The Arntl mRNA expression may play an important role in progression of hypertension in women.

The AHR1-ARNT1 dimerization pair is a major regulator of the response to natural ligands, but not to TCDD, in the chicken.

The activation of the aryl hydrocarbon receptor (AHR) occurs through the binding of dioxin-like compounds (DLCs) or natural ligands. In this pathway, the AHR-ARNT (AHR nuclear translocator) heterodimer serves to regulate critical physiological functions, such as immune responses and the metabolism of xenobiotics. Birds have three AHR isoforms (AHR1, AHR1ß, and AHR2) and two ARNT isoforms (ARNT1 and ARNT2). However, how AHR and ARNT dimerization pair in birds regulates the AHR signaling pathway in an isoform-specific manner remains unknown. In this study, we initially sought to clarify the major chicken AHR-ARNT (ckAHR-ckARNT) pairs by estimating the mRNA tissue distributions of various ckAHR and ckARNT isoforms. Our results indicated that the ckAHR1-ckARNT1 represented the major dimerization pair in most tissues except the brain. We then measured the transactivation potencies of various ckAHR-ckARNT pairs by natural ligands and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in in vitro reporter gene assays using COS-7 and LMH cell lines. Our results from the in vitro assays demonstrated that the ckAHR1-ckARNT1 pair was strongly activated by the five natural ligands, namely, 6-formylindolo [3,2-b]carbazole, L-kynurenin, kynurenic acid, indoxyl-3-sulfate, and 1,3,7-tribromodibenzo-p-dioxin, but not by TCDD. In in silico ligand docking simulations with ckAHR1 homology models, all the natural ligands showed a interaction pattern that was distinct from that observed with anthropogenic DLCs, including TCDD. In conclusion, our findings indicate that the ckAHR1-ckARNT1 may be the most important dimerization pair in most tissues for regulating the physiological functions driven by natural ligands, although it was less reactive to TCDD.

Transitional States in Ligand-Dependent Transformation of the Aryl Hydrocarbon Receptor into Its DNA-Binding Form.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.

Overexpression of ARNT2 is associated with decreased cell proliferation and better prognosis in gastric cancer.

Aryl hydrocarbon-receptor nuclear translocator (ARNT2) is a member of the bHLH PAS (basic helix-loop-helix Period/ARNT/Single-minded) family of transcription factors. Recently, some studies indicate that ARNT2 is associated with the occurrence and development of carcinoma. However, its roles in gastric cancer (GC) remain unclear. In the present study, we found that ARNT2 expression level is lower in GC tissues compared with adjacent non-tumor tissues, and negatively correlated with depth of invasion of the tumor, differentiated degree, and poor survival of GC patients. Overexpression of ARNT2 inhibits cell proliferation. Furthermore, AKT pathway contributed to ARNT2 -mediated PC proliferation. Taken together, our results provide the first evidence that high expression of ARNT2 inhibited proliferation of GC cells and affected tumor aggressiveness in GC patients.

Aryl Hydrocarbon Receptor in Atopic Dermatitis and Psoriasis.

The aryl hydrocarbon receptor (AHR)/AHR-nuclear translocator (ARNT) system is a sensitive sensor for small molecular, xenobiotic chemicals of exogenous and endogenous origin, including dioxins, phytochemicals, microbial bioproducts, and tryptophan photoproducts. AHR/ARNT are abundantly expressed in the skin. Once activated, the AHR/ARNT axis strengthens skin barrier functions and accelerates epidermal terminal differentiation by upregulating filaggrin expression. In addition, AHR activation induces oxidative stress. However, some AHR ligands simultaneously activate the nuclear factor-erythroid 2-related factor-2 (NRF2) transcription factor, which is a master switch of antioxidative enzymes that neutralizes oxidative stress. The immunoregulatory system governing T-helper 17/22 (Th17/22) and T regulatory cells (Treg) is also regulated by the AHR system. Notably, AHR agonists, such as tapinarof, are currently used as therapeutic agents in psoriasis and atopic dermatitis. In this review, we summarize recent topics on AHR related to atopic dermatitis and psoriasis.

Expression of the neuroprotective protein aryl hydrocarbon receptor nuclear translocator 2 correlates with neuronal stress and disability in models of multiple sclerosis.

BACKGROUND: Axonal degeneration and neuronal loss have been described as the major causes of irreversible clinical disability in multiple sclerosis (MS). The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) protein has been associated with neuroprotection in models of ischemia and neuronal responses to stressors. METHODS: To characterize its potential to influence inflammatory neurodegeneration, we examined ARNT2 expression in the experimental autoimmune encephalomyelitis (EAE) model of MS and characterized mediators that influence ARNT2 expression as well as plausible partners and targets. RESULTS: Arnt2 message and protein levels dropped significantly in EAE spinal cords as disease developed and were lowest at peak disability. ARNT2 expression is prominent in neuronal cell bodies within the gray matter with some staining in glial fibrillary acidic protein (GFAP)+ astrocytes in healthy animals. At peak disease, ARNT2 expression is reduced by 20-50% in gray matter neurons compared to healthy controls. ARNT2 intensity in neurons throughout the EAE spinal cord correlated inversely with the degree of immune cell infiltration (r = - 0.5085, p < 0.01) and axonal damage identified with SMI32 staining (r = - 0.376, p = 0.032). To understand the relationship between ARNT2 expression and neuronal health, we exposed enriched cortical cultures of neurons to hydrogen peroxide (H2O2) to mimic oxidative stress. H2O2 at lower doses rapidly increased ARNT2 protein levels which returned to baseline within 3-4 h. Exposure to higher doses of H2O2) dropped ARNT2 levels below baseline, preceding cytotoxicity measured by morphological changes and lactate dehydrogenase release from cells. Decreases in ARNT2 secondary to staurosporine and H2O2 preceded increases in cleaved caspase 3 and associated apoptosis. We also examined expression of neuronal pas 4 (Npas4), whose heterodimerization with ARNT2 drives expression of the neurotrophic factor brain-derived neurotrophic factor (Bdnf). Like ARNT2, Npas4 levels also decline at the onset of EAE and are linked to decreases in Bdnf. In vitro, H2O2 exposure drives Npas4 expression that is tied to increases in Bdnf. CONCLUSION: Our data support ARNT2 as a neuronal transcription factor whose sustained expression is linked to neuronal and axonal health, protection that may primarily be driven through its partnering with Npas4 to influence BDNF expression.

Effects of curcumin on hypoxia-inducible factor as a new therapeutic target.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that consists of two subunits, the HIF-1α and HIF-1ß (ARNT). Under hypoxic conditions, HIF-1 is an adaptive system that regulates the transcription of multiple genes associated with growth, angiogenesis, proliferation, glucose transport, metabolism, pH regulation and cell death. However, aberrant HIF-1 activation contributes to the pathophysiology of several human diseases such as cancer, ischemic cardiovascular disorders, and pulmonary and kidney diseases. A growing body of evidence indicates that curcumin, a natural bioactive compound of turmeric root, significantly targets both HIF-1 subunits, but is more potent against HIF-1α. In this review, we have summarized the knowledge about the pharmacological effects of curcumin on HIF-1 and the related molecular mechanisms that may be effective candidates for the development of multi-targeted therapy for several human diseases.

ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells.

BACKGROUND: The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT) participates in the hypoxia-inducible factor (HIF) pathway which senses a decline in cellular oxygen tension. In hypoxia, HIF-1α and ARNT form the transcriptional active complex HIF-1 followed by the expression of target genes. ARNT is considered as constitutively expressed and unaffected by hypoxia. However, certain tumour cell lines derived from different entities are capable to elevate ARNT expression under hypoxic conditions which implies a survival benefit. It was demonstrated that high ARNT protein levels mediate radioresistance in tumour cells. Furthermore, a HIF-1α-driven feed-forward loop leading to augmented HIF signalling was discovered in Hep3B cells. Herein HIF-1α elevates the mRNA and protein expression of its binding partner ARNT in hypoxia. However, the detailed mechanism remained unclear. The objective of this study was to test whether HIF-1α might directly regulate ARNT expression by recruitment to the ARNT promoter. METHODS: Chromatin immunoprecipitation (ChIP), CRISPR/Cas9 genome editing, Western blotting, quantitative RT-PCR and reporter gene assays were applied. The unpaired t test was used for statistical analysis. RESULTS: ChIP assays revealed the binding of both HIF-1α and ARNT to the ARNT promoter in hypoxia. The relevance of this particular region for hypoxic ARNT induction was confirmed by CRISPR/Cas9 genome editing. ARNT normoxic basal expression and hypoxic inducibility was reduced in genome-edited Hep3B cells. This phenotype was accompanied with impaired HIF signalling and was rescued by ARNT overexpression. CONCLUSIONS: The results indicate ARNT to be a putative HIF-1 target gene and a limiting factor in this model.

Hepatoprotective and antioxidant effects of dietary Glycyrrhiza polysaccharide against TCDD-induced hepatic injury and RT-PCR quantification of AHR2, ARNT2, CYP1A mRNA in Jian Carp (Cyprinus carpio var. Jian).

To evaluate the protective effects of Glycyrrhiza polysaccharide (GPS) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatotoxicity in Jian carp, the fish were fed diets containing GPS at doses of 0.1, 0.5 and 1.0g/kg for 60days before an intraperitoneal injection of 0.6µg/kg TCDD at a volume of 0.05mL/10g body weight. At 72hr post-injection, blood and liver samples were taken for biochemical analysis and the fish liver samples were used for the preparation of pathological slices. The results showed that increases in alanine aminotransferase (GPT), aspartate aminotransferase (GOT), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP) in serum induced by TCDD were significantly inhibited by pre-treatment with 1.0g/kg GPS. Following the 1.0g/kg GPS pre-treatment, total protein (TP), albumin (Alb), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activities in liver tissue increased significantly, malondialdehyde (MDA) formation (P<0.05 or P<0.01) was significantly inhibited, and the expression of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor 2 (AHR2) and aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) mRNA (P<0.05) was significantly enhanced. Histological observations on fish liver were obtained by preparing paraffin tissue sections via HE staining, and the results showed that histological changes were obviously reduced by 0.5 and 1.0g/kg GPS. GPS significantly reduced liver tissue damage caused by TCDD. Overall, these results proved the hepatoprotective effect of GPS in protecting against fish liver injury induced by TCDD, and supported the use of GPS (1.0g/kg) as a hepatoprotective and antioxidant agent in fish.

Modulation of Dcytb (Cybrd 1) expression and function by iron, dehydroascorbate and Hif-2α in cultured cells.

BACKGROUND: Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines. METHODS: Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and (59)Fe uptake assays. RESULTS: Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake. CONCLUSIONS: Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α. GENERAL SIGNIFICANCE: Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes.

Reduction of ARNT in myeloid cells causes immune suppression and delayed wound healing.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing.

GSK3ß negatively regulates HIF1α mRNA stability via nucleolin in the MG63 osteosarcoma cell line.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor involved in the growth, invasion and metastasis of malignant tumors. Glycogen synthase kinase 3 beta (GSK3ß) is a protein kinase involved in a variety of signaling pathways, such as the Wnt and NF-κB pathways; this kinase can affect tumor progress through the regulation of transcription factor expression and apoptosis. Recent studies showed that GSK3ß was involved in the expression of HIF1α. However, the effect of GSK3ß on HIF1α expression in osteosarcoma cells remains unknown. To understand the relationship between GSK3ß and HIF1α comprehensively, small RNA interference techniques, Western blot analyses, quantitative real-time PCR analyses and luciferase assays were used in our study. Experimental data revealed that inhibition of GSK3ß could increase HIF1α protein levels and expression of its target genes by increasing the stability of the HIF1α mRNA, not by affecting the HIF1α protein stability, and that this process could be mediated by nucleolin.

Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines.

The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Exposure to 1% O2 prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity.

Familial isolated pituitary adenoma is independent of Ahr genotype in a novel mouse model of disease.

Human familial isolated pituitary adenoma (FIPA) has been linked to germline heterozygous mutations in the gene encoding the aryl hydrocarbon receptor-interacting protein (AIP, also known as ARA9, XAP2, FKBP16, or FKBP37). To investigate the hypothesis that AIP is a pituitary adenoma tumor suppressor via its role in aryl hydrocarbon receptor (AHR) signaling, we have compared the pituitary phenotype of our global null Aip (AipΔC) mouse model with that of a conditional null Aip model (Aipfx/fx) carrying the same deletion, as well as pituitary phenotypes of Ahr global null and Arnt conditional null animals. We demonstrate that germline AipΔC heterozygosity results in a high incidence of pituitary tumors in both sexes, primarily somatotropinomas, at 16 months of age. Biallelic deletion of Aip in Pit-1 cells (Aipfx/fx:rGHRHRcre) increased pituitary tumor incidence and also accelerated tumor progression, supporting a loss-of-function/loss-of-heterozygosity model of tumorigenesis. Tumor development exhibited sexual dimorphism in wildtype and Aipfx/fx:rGHRHRcre animals. Despite the role of AHR as a tumor suppressor in other cancers, the observation that animals lacking AHR in all tissues, or ARNT in Pit-1 cells, do not develop somatotropinomas argues against the hypothesis that pituitary tumorigenesis in AIP-associated FIPA is related to decreased activities of either the Ahr or Arnt gene products.

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 µg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.