β-caryophyllene ameliorated obesity-associated airway hyperresponsiveness through some non-conventional targets

BackgroundObesity worsens airway hyperresponsiveness (AHR) in asthmatic subjects by up-regulating macrophage polarization that leads to excessive secretion of pro-inflammatory adipokines from white adipose tissue followed by generation of oxidative stress in the respiratory system. Treatment through conventional signaling pathways proved to be inadequate in obese asthmatics, so a therapeutical approach through a non-conventional pathway may prove to be effective.PurposeThis study aimed to investigate the efficacy of a FDA-approved food additive, β-caryophyllene (BCP) in obesity-associated AHR.MethodA repertoire of protein expression, cytokine and adiponectin estimation, oxidative stress assays, histopathology, and fluorescence immune-histochemistry were performed to assess the efficacy of BCP in C57BL/6 mice model of obesity-associated AHR. Additionally, human adipocyte was utilized to study the effect of BCP on macrophage polarization in Boyden chamber cell culture inserts.ResultsObesity-associated AHR is ameliorated by administration of BCP by inhibition of the macrophage polarization by activation of AMPKα, Nrf2/HO-1 and AdipoR1 and AdipoR2 signaling pathway, up-regulation of adiponectin, GLP-1, IFN-γ, SOD, catalase and down-regulation of NF-κB, leptin, IL-4, TNF, and IL-1β. Browning of eWAT by induction of thermogenesis and activation of melanocortin pathway also contributed to the amelioration of obesity-associated AHR. We conclude that BCP ameliorated the obesity-associated AHR via inhibition of macrophage polarization, activation of AMPKα, Nrf2/HO-1, and up-regulation of AdipoR1 and AdipoR2 expression and down-regulation of NFκB expression in lung of animal.ConclusionBeing an FDA-approved food additive, BCP may prove to be a safe and potential agent against obesity-associated AHR.     

Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.     

The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) binds multiple AHRs and induces multiple CYP1 genes via AHR2 in zebrafish

The tryptophan photooxidation product 6-formylindolo[3,2-b]carbazole (FICZ) has been proposed as a physiological ligand for the mammalian aryl hydrocarbon receptor (AHR), which it binds with high-affinity, inducing expression of cytochrome P450 1A1 (CYP1A1). We investigated whether the response to FICZ is evolutionarily conserved in vertebrates by measuring FICZ binding to two zebrafish AHRs (AHR1B and AHR2) and its ability to induce zebrafish CYP1 genes (CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1) in vivo. Exposure of zebrafish embryos (48 h-post-fertilization; hpf) to 10 nM FICZ for 6 h caused strong induction of CYP1A mRNA and a statistically significant but modest induction of CYP1B1 and CYP1C1. Neither CYP1C2 nor CYP1D1 expression was induced by FICZ under the conditions of dose, time or developmental stage examined here. CYP1A induction was significantly greater after 6 h than after 12 h of exposure to FICZ, suggesting a rapid degradation of inducer. The 6-h EC₅₀ values for induction of CYP1A and CYP1B1 by FICZ were 0.6 and 0.5 nM compared to 72-h EC₅₀ values of 2.3 and 2.7 nM for PCB126, indicating that in zebrafish embryos FICZ is a more potent inducer than PCB126. FICZ at 10 nM was able to completely displace binding of 2,3,7,8-tetrachloro-1,6[³H]-dibenzo-p-dioxin to in vitro-expressed zebrafish AHR2 and AHR1B. Inhibition of AHR2 translation in zebrafish embryos by an AHR2-specific morpholino antisense oligonucleotide decreased the induction of CYP1A and CYP1B1 by FICZ and by PCB126. Together, these results demonstrate that FICZ is a potent AHR agonist in zebrafish, inducing expression of multiple CYP1 genes largely through AHR2. Evolutionary conservation of the response to FICZ is consistent with a possible role as an endogenous signaling molecule acting through the AHR.     

Characterization of the aryl hydrocarbon receptor repressor gene and association of its Pro185Ala polymorphism with micropenis

BACKGROUND: Genetic background of a fetus contributes to the abnormal development after teratogen exposure. In rodents, in utero exposure to dioxins affects male external genital development. The effects of dioxins are mediated via the aryl hydrocarbon receptor (AHR) and its binding protein, aryl hydrocarbon receptor nuclear translocator (ARNT). In mice, aryl hydrocarbon receptor repressor (AHRR), which binds to ARNT in competition with AHR, plays a critical negative regulatory role in AHR signaling. We attempt to characterize the human AHRR gene and investigate the relationship between AHRR polymorphisms and the incidence of micropenis, a phenotype of undermasculinization. METHODS: We identified and characterized the human homolog of mouse AHRR, taking advantage of the publicly available draft version of the human genome sequence. After detecting an AHRR protein polymorphism by the direct sequencing of pooled human genomic DNA, we evaluated the association between the polymorphism and the presence or absence of micropenis (< -2.5 SD) in patients with micropenis and control subjects. RESULTS: The deduced sequence for human AHRR (715 residues) and the mouse AHRR protein exhibited 81% sequence homology to each other. The Pro185Ala polymorphism was identified between the PAS-A region and the highly conserved arginine/cysteine-rich RCFRCRL/VRC region. Forty-six percent (27/59) of patients with micropenis and 27% (22/80) of the controls were homozygous for 185Pro; this difference in frequencies was significant (P = 0.03). CONCLUSIONS: Homozygosity for the 185Pro allele of AHRR may increase the susceptibility of a fetus to the undermasculinizing effects of dioxin exposure in utero, presumably through the diminished inhibition of AHR-mediated signaling.     

Adipokines,Ghrelin and Obesity‐Associated Insulin Resistance in Nondiabetic Patients with Acute Coronary Syndrome

Altered glucose metabolism negatively modulates outcome in acute coronary syndromes (ACS). Insulin resistance is commonly associated with increasing BMI in the general population and these associations may involve obesity‐related changes in circulating ghrelin and adipokines. We aimed at investigating interactions between BMI, insulin resistance and ACS and their associations with plasma ghrelin and adipokine concentrations. Homeostasis model assessment of insulin resistance (HOMA_IR)‐insulin resistance index, plasma adiponectin, leptin, total (T‐Ghrelin), acylated (Acyl‐Ghrelin), and desacylated ghrelin (Desacyl‐Ghrelin) were measured in 60 nondiabetic ACS patients and 44 subjects without ACS matched for age, sex, and BMI. Compared with non‐ACS, ACS patients had similar HOMA_IR and plasma adipokines, but lower T‐ and Desacyl‐Ghrelin and higher Acyl‐Ghrelin. Obesity (BMI > 30) was associated with higher HOMA_IR, lower adiponectin, and higher leptin (P < 0.05) similarly in ACS and non‐ACS subjects. In ACS (n = 60) HOMA_IR remained associated negatively with adiponectin and positively with leptin independently of BMI and c‐reactive protein (CRP) (P < 0.05). On the other hand, low T‐ and Desacyl‐Ghrelin with high Acyl‐Ghrelin characterized both obese and non‐obese ACS patients and were not associated with HOMA_IR. In conclusion, in ACS patients, obesity and obesity‐related changes in plasma leptin and adiponectin are associated with and likely contribute to negatively modulate insulin resistance. ACS per se does not however enhance the negative impact of obesity on insulin sensitivity. High acylated and low desacylated ghrelin characterize ACS patients independently of obesity, but are not associated with insulin sensitivity.     

Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain

The aryl hydrocarbon receptor repressor (AHRR) is a negative regulator of AH receptor (AHR), which mediates most of the toxic and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has been shown to be the major reason for the exceptionally wide (ca. 1000-fold) sensitivity difference in acute toxicity of TCDD between two rat strains, sensitive Long-Evans (Turku/AB) (L-E) and resistant Han/Wistar (Kuopio) (H/W), but there is another, currently unknown contributing factor involved. In the present study, we examined AHRR structure and expression in these rat strains to find out whether AHRR could be this auxiliary factor. Molecular cloning of AHRR coding region showed that consistent with AHRR proteins in other species, the N-terminal end of rat AHRR is highly conserved, but PAS B and Q-rich domains are severely truncated or lacking. Identical structures were recorded in both strains. Next, the time-, dose-, and tissue-dependent expression of AHRR was determined using quantitative real-time RT-PCR. In liver, AHRR expression was very low in untreated rats, but it increased rapidly after TCDD exposure (100microg/kg). Testis exhibited the highest constitutive expression of AHRR, whereas kidney, spleen, and heart showed the highest induction of AHRR in response to TCDD treatment. Again, no marked differences were found between H/W and L-E rats, implying that AHRR is not the auxiliary contributing factor to the strain difference in TCDD sensitivity. However, simultaneous measurement of CYP1A1 mRNA reinforced the view that AHRR is an important determinant of tissue-specific responsiveness to TCDD.     

Changes in Circulating Satiety Hormones in Obese Children: A Randomized Controlled Physical Activity‐Based Intervention Study

The aims of this study are to examine in children: (i) obesity‐related alterations in satiety factors such as leptin, ghrelin, and obestatin; (ii) the link between satiety factors and cardiometabolic risk factors; and (iii) the impact of a physical activity‐based lifestyle intervention on the levels of these satiety factors in the obese. We studied a total of 21 adolescents (BMI percentile, 99.0 ± 0.6 for 15 obese and 56.2 ± 1.1 for 6 lean). The obese subjects underwent a 3‐month randomized controlled physical activity‐based lifestyle intervention. Leptin, soluble leptin receptor (sOB‐R), ghrelin, and obestatin levels were determined as the primary outcome measures. Other markers of cardiometabolic disease such as inflammation and insulin resistance were also determined. Body composition was measured by dual‐energy X‐ray absorptiometry. The concentrations of ghrelin, obestatin, and sOB‐R were significantly lower in the obese children compared to the lean controls, whereas that of leptin was higher (all P < 0.05). Although intervention led to a net increase in obestatin (P < 0.01) and no change in ghrelin levels, the balance between ghrelin and obestatin (ratio of ghrelin to obestatin, G/O) decreased (P < 0.02). Intervention reduced leptin and increased sOB‐R (P < 0.01 for both). Significant associations between satiety factors and other cardiometabolic risk factors were also observed. Taken together, alterations in the levels of satiety factors are evident early in the clinical course of obesity, but physical activity‐based lifestyle intervention either prevented their continued increase or normalized their levels. These beneficial effects appear to aid in the maintenance of body weight and reduction in cardiovascular risk.     

Fluorescence in situ hybridization mapping of six loci containing genes involved in the dioxin metabolism of domestic bovids

Six loci containing genes involved in the dioxin metabolism (ARNT, AHR, CYP1A1, CYP1A2, CYP1B1 and AHRR) were assigned, for the first time, to cattle (Bos taurus, 2n = 60, BTA), river buffalo (Bubalus bubalis, 2n = 50, BBU), sheep (Ovis aries, 2n = 54, OAR) and goat (Capra hircus, 2n = 60, CHI) chromosomes by comparative FISH-mapping and R-banding using bovine BAC-clones. The following chromosome locations were found: ARNT to BTA3q21, BBU6q21, OAR1p21 and CHI3q21, AHR to BTA4q15, BBU8q15, OAR4q15 and CHI4q15; CYP1A1 and CYP1A2 to BTA21q17, BBU20q17, OAR18q17 and CHI21q17; CYP1B1 to BTA11q16, BBU12q22, OAR3p16 and CHI11q16, AHRR to BTA20q24, BBU19q24, OAR16q24 and CHI20q24. All loci were mapped at the same homoeologous chromosomes and chromosome bands of the four bovid species. Comparisons with corresponding human locations were also reported.     

Resistin,Adiponectin, Ghrelin,Leptin, and Proinflammatory Cytokines: Relationships in Obesity

Objective: To evaluate interactions among leptin, adiponectin, resistin, ghrelin, and proinflammatory cytokines [tumor necrosis factor receptors (TNFRs), interleukin‐6 (IL‐6)] in nonmorbid and morbid obesity. Research Methods and Procedures: We measured these hormones by immunoenzyme or radiometric assays in 117 nonmorbid and 57 morbidly obese patients, and in a subgroup of 34 morbidly obese patients before and 6 months after gastric bypass surgery. Insulin resistance by homeostasis model assessment, lipid profile, and anthropometrical measurements were also performed in all patients. Results: Average plasma lipids in morbidly obese patients were elevated. IL‐6, leptin, adiponectin, and resistin were increased and ghrelin was decreased in morbidly obese compared with nonmorbidly obese subjects. After adjusting for age, gender, and BMI in nonmorbidly obese, adiponectin was positively associated with HDLc and gender and negatively with weight (β = ?0.38, p < 0.001). Leptin and resistin correlated positively with soluble tumor necrosis factor receptor (sTNFR) 1 (β = 0.24, p = 0.01 and β = 0.28, p = 0.007). In the morbidly obese patients, resistin and ghrelin were positively associated with sTNFR2 (β = 0.39, p = 0.008 and β = 0.39, p = 0.01). In the surgically treated morbidly obese group, body weight decreased significantly and was best predicted by resistin concentrations before surgery (β = 0.45, p = 0.024). Plasma lipids, insulin resistance, leptin, sTNFR1, and IL‐6 decreased and adiponectin and ghrelin increased significantly. Insulin resistance improved after weight loss and correlated with high adiponectin levels. Discussion: TNFα receptors were involved in the regulatory endocrine system of body adiposity independently of leptin and resistin axis in nonmorbidly obese patients. Our results suggest coordinated roles of adiponectin, resistin, and ghrelin in the modulation of the obesity proinflammatory environment and that resistin levels before surgery treatment are predictive of the extent of weight loss after bypass surgery.     

Molecules Implicated in Glucose Homeostasis are Differentially Expressed in the Trachea of Lean and Obese Zucker Rats

Recent studies indicate that the processes mediated by the (T1R2/T1R3) glucose/sugar receptor of gustatory cells in the tongue, and hormones like leptin and ghrelin contribute to the regulation of glucose homeostasis. Altered plasma levels of leptin and ghrelin are associated with obesity both in humans and rodents. In the present study, we evaluated the ultrastructure of the mucosa, and the expression of molecules implicated in the regulation of glucose homeostasis (GLUT2, SGLT1, T1R3, ghrelin and its receptor) in the trachea of an animal model of obesity (Zucker rats). We found that the tracheal epithelium of obese animals was characterized by the presence of poorly differentiated cells. Ciliated and secretory cells were the cell lineages with greatest loss of differentiation. Severe epithelial alterations were associated with marked deposit of extracellular matrix in the lamina propria. The expression pattern of GLUT2 and SGLT1 glucose transporters was similar in the trachea of both the Zucker rat genotypes, whereas that of T1R3 was reduced in ciliated cells of obese rats. A different immunolocalization for ghrelin was also found in the trachea of obese rats. In conclusion, the tracheal morphological alterations in obese animals seem to compromise the expression of molecules involved in the homeostasis of glucose.Key words: Obesity, ultrastructure, ghrelin, ghrelin receptor, sweet receptor, immunohistochemistry     

Ghrelin and body weight regulation in the obese Zucker rat in relation to feeding state and dark/light cycle

Ghrelin is a new orexigenic peptide primarily produced by the stomach but also present in the hypothalamus. It has adipogenic effects when it is chronically injected in rodents but in obese humans, its plasma concentration is decreased. It can reverse the anorectic effects of leptin when it is co-injected with this peptide in the brain ventricles. The Zucker fa/fa rat is a genetic model of obesity related to a default in the leptin receptor. It is characterized by a large dysregulation of numerous hypothalamic peptides but the ghrelin status of this rat has not yet been determined. Through several experiments, we determine in lean and obese Zucker rats its circulating form in the plasma, its tissue levels and/or expression, and studied the influence of different feeding conditions and its light/dark variations. Ghrelin expression was higher in the obese stomach and hypothalamus (P < 0.05 and P < 0.02, respectively). The ratio of [Octanoyl-Ser3]-ghrelin (active form) to [Des-Octanoyl-Ser3]-ghrelin (inactive form) was approximately 1:1 in the stomach and 2:1 in the plasma in lean and obese rats (no differences). After fasting, plasma ghrelin concentrations increased significantly in lean (+ 64%; P < 0.001) and obese (+ 60%; P < 0.02) rats. After 24 hours of refeeding, they returned to their initial ad lib levels. Ghrelin concentrations were higher in obese rats by 69% (P < 0.005), 65% (P < 0.02), and 73% (P < 0.005) in the ad libitum, fast, and refed states respectively. These results indicate that the obese Zucker rat is characterized by increases in the stomach mRNA expression and in peptide release in the circulation. They clearly support a role for ghrelin in the development of obesity in the absence of leptin signaling.     

Interplay between obesity and aging on myocardial geometry and function: Role of leptin-STAT3-stress signaling

BackgroundUncorrected obesity facilitates premature aging and cardiovascular anomalies. This study examined the interaction between obesity and aging on cardiac remodeling and contractile function. Methods: Cardiac echocardiographic geometry, function, morphology, intracellular Ca²⁺ handling, oxidative stress (DHE fluorescence), STAT3 and stress signaling were evaluated in young (3-mo) and old (12- and 18-mo) lean and leptin deficient ob/ob obese mice. Cardiomyocytes from young and old lean and ob/ob mice were treated with leptin (1 nM) for 4 h in vitro prior to assessment of mechanical and biochemical properties. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obese mice at young and old age were evaluated for comparison. Results: Our results displayed reduced survival in ob/ob mice. Obesity but less likely older age dampened echocardiographic, geometric, cardiomyocyte function and intracellular Ca²⁺ properties, elevated O₂^? and p^47phox NADPH oxidase levels with a more pronounced geometric change at older age. Immunoblot analysis revealed elevated p^47phox NADPH oxidase and dampened phosphorylation of STAT3, with a more pronounced response in old ob/ob mice, the effects were restored by leptin. Obesity and aging inhibited phosphorylation of Akt, eNOS, AMPK, and p38 while promoting phosphorylation of JNK and IκB. Leptin reconciled cardiomyocyte dysfunction, O₂^? yield, p^47phox upregulation, STAT3 dephosphorylation and stress signaling in ob/ob mice although its action on stress signaling cascades were lost at old age. High fat diet-induced and db/db obesity displayed aging-associated cardiomyocyte anomalies reminiscent of ob/ob model albeit lost leptin response.ConclusionsOur data suggest disparate age-associated obesity response in cardiac remodeling and contractile dysfunction due to phosphorylation of Akt, eNOS and stress signaling-related oxidative stress.     

Altered Subcellular Localization of Heat Shock Protein 90 Is Associated with Impaired Expression of the Aryl Hydrocarbon Receptor Pathway in Dogs

The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of AHR,AIP, ARNT, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), hypoxia inducible factor 1alpha (HIF1A), heat shock protein 90AA1 (HSP90AA1), cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1), vascular endothelial growth factor A (VEGFA), nitric oxide synthesase 3 (NOS3), and endothelin (EDN1). The observed low expression of AHR mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for AIP, ARNT2, CYP1A2, CYP1B1 and HSP90AA1 expression, whereas the expression of HIF1A was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins.     

Differential secretion of satiety hormones with progression of obesity in JCR:LA-corpulent rats

Objective: To characterize the gastrointestinal tract at the onset and in well‐established obesity. Methods and Procedures: Lean (+/?) and obese (cp/cp) male JCR:LA‐cp rats lacking a functional leptin receptor were killed at 3.5 weeks and 9 months of age and plasma concentrations of satiety hormones determined. The small intestine, colon, and stomach were measured, weighed, and mRNA levels of satiety genes quantified. Results: At the onset of obesity, obese rats had greater intestine, colon, and liver mass when adjusted for body weight compared to lean rats. Conversely, adult rats with established obesity had lower intestine and colon mass and length after adjustment for body weight. Early changes in gene expression included decreased ghrelin mRNA levels in stomach and increased peptide YY (PYY) mRNA levels in duodenum of young obese rats. After massive accumulation of adipose tissue had occurred, adult obese rats had increased proglucagon and ghrelin mRNA expression in the proximal intestine. In the distal small intestine, obese rats had lower proglucagon, ghrelin, and PYY mRNA levels. Finally, at the onset and in well‐established obesity, obese rats had higher plasma insulin, amylin, glucagon like peptide‐1 (GLP‐1), and PYY, a finding, with the exception of insulin, unique to this model. Plasma total ghrelin levels were significantly lower at the onset of obesity and established obesity compared to the lean rats. Discussion: Several defects are manifested in the obese gut early on in the disease before the accumulation of large excesses of body fat and represent potential targets for early intervention in obesity.     

Feeding response to ghrelin agonist and antagonist in lean and obese Zucker rats

Ghrelin is a new orexigenic and adipogenic peptide primarily produced by the stomach and the hypothalamus. In the present experiment, we determined the circulating ghrelin levels in 60-week old fa/fa Zucker rats with a well-established obesity (n = 12) and in their lean (FA/FA) counterparts (n = 12). We also tested the feeding response of both groups to intra-peritoneal (I.P.) injection of ghrelin agonist and antagonist. Obese rats ate significantly more than the lean rats (21.7 +/- 1.1 vs. 18.3 +/- 0.3 g/day; p < 0.01). Their plasma ghrelin concentration was 35% higher than that in the lean homozygous rats (p < 0.025). GHRP-6 (1 mg/kg I.P, a GHS-R agonist) stimulated food intake in lean but not in obese rats (p < 0.01), whereas [D-Lys)]-GHRP-6 (12 mg/kg I.P., a GHS-R antagonist) decreased food intake in both groups (p < 0.0001). These results indicate that the obese Zucker rat is characterized by an increase in plasma ghrelin concentrations and by an attenuated response to a GHS-R agonist. They support a role for ghrelin in the development of obesity in the absence of leptin signaling.     

Differential ligand-dependent activation and a role for Y322 in aryl hydrocarbon receptor-mediated regulation of gene expression

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF). Non-traditional activators, including omeprazole (Omp), are thought to regulate AHR action through phosphorylation rather than binding to the receptor. In this study, we examined the ability of these compounds to induce AHR-dependent regulation of cytochrome P450 1A1 (CYP1A1) and CYP1B1 in T-47D human breast cancer cells. The role of Y322, a residue implicated in Omp-dependent activation of AHR was also investigated. All four compounds induced CYP1A1 and CYP1B1 mRNA expression, with Omp differing from the HAHs. Chromatin immunoprecipitation assays revealed ligand- and gene-selectivity in the recruitment patterns of AHR coactivators. We also found that residue Y322 of human AHR was important for maximum activation of AHR by 2,3,7,8-TCDD and 2,3,4,7,8-PeCDF, but required for 2,3,7,8-TCDF and Omp in an AHR-deficient MCF-7 human breast cancer cell line. In summary, this study provides evidence for context- and ligand-selective differences in coactivator recruitment in AHR-regulated gene expression and reveal an important role of Y322 in AHR activation.