[Show abstract][Hide abstract] ABSTRACT: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Activation of AhR mediates the expression of target genes (e.g., CYP1A1) by binding to dioxin response element (DRE) sequences in their promoter region. To understand the multiple mechanisms of AhR-mediated gene regulation, a microarray analysis on liver isolated from ligand-treated transgenic mice expressing a wild-type (WT) Ahr or a DRE-binding mutant Ahr (A78D) on an ahr-null background was performed. Results revealed that AhR DRE binding is not required for the suppression of genes involved in cholesterol synthesis. Quantitative reverse-transcription polymerase chain reaction performed on both mouse liver and primary human hepatocyte RNA demonstrated a coordinated repression of genes involved in cholesterol biosynthesis, namely, HMGCR, FDFT1, SQLE, and LSS after receptor activation. An additional transgenic mouse line was established expressing a liver-specific Ahr-A78D on a Cre(Alb)/Ahr(flox/flox) background. These mice displayed a similar repression of cholesterol biosynthetic genes, compared to Ahr(flox/flox) mice, further indicating that the observed modulation is AhR specific and occurs in a DRE-independent manner. Elevated hepatic transcriptional levels of the genes of interest were noted in congenic C57BL/6J-Ah(d) allele mice, when compared to the WT C57BL/6J mice, which carry the Ah(b) allele. Down-regulation of AhR nuclear translocator levels using short interfering RNA in a human cell line revealed no effect on the expression of cholesterol biosynthetic genes. Finally, cholesterol secretion was shown to be significantly decreased in human cells after AhR activation. CONCLUSION: These data firmly establish an endogenous role for AhR as a regulator of the cholesterol biosynthesis pathway independent of its DRE-binding ability, and suggest that AhR may be a previously unrecognized therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: Repression of the nuclear factor-kappaB (NF-kappaB) pathway has been extensively researched because of its pivotal role in inflammation. We investigated the potential of the aryl hydrocarbon receptor (AHR) to suppress NF-kappaB regulated-gene expression, especially acute-phase genes, such as serum amyloid A (Saa). Using AHR mutants, it was determined that nuclear translocation and heterodimerization with AHR-nuclear translocator are essential, but DNA binding is not involved in AHR-mediated Saa repression. A number of AHR ligands were capable of repressing Saa3 expression. AHR activation leads to a decrease in RELA and C/EBP/beta recruitment to and histone acetylation at Saa3 gene promoter. A battery of acute-phase genes (eg C-reactive protein and haptoglobin) induced by cytokine exposure was repressed by AHR activation in mouse hepatocytes. Dietary exposure to an AHR ligand represses cytokine-induced acute-phase response in the liver. Use of a human liver-derived cell line revealed similar repression of Saa mRNA levels and secreted protein. Repression of AHR expression also enhanced Saa induction in response to cytokines, suggesting that AHR is capable of constitutively repressing Saa gene expression. These results establish a role for AHR in inflammatory signaling within the liver, presenting a new therapeutic opportunity, and signify AHR's ability to function in a DNA-independent manner.
[Show abstract][Hide abstract] ABSTRACT: The aryl hydrocarbon receptor (AHR) is a ligand-regulated transcription factor that can be activated by structurally diverse chemicals, ranging from environmental carcinogens to dietary metabolites. Evidence supporting a necessary role for the AHR in normal biology has been established; however, identification of key endogenous ligand/activator remains to be established. Here, we report the ability of 12(R)-hydroxy-5(Z),8(Z),10(E), 14(Z)-eicosatetraenoic acid [12(R)-HETE], an arachidonic acid metabolite produced by either a lipoxygenase or cytochrome P-450 pathway, to act as a potent indirect modulator of the AHR pathway. In contrast, structurally similar HETE isomers failed to demonstrate significant activation of the AHR. Electrophoretic mobility shift assays, together with ligand competition binding experiments, have demonstrated the inability of 12(R)-HETE to directly bind or directly activate the AHR to a DNA binding species in vitro. However, cell-based xenobiotic-responsive element-driven luciferase reporter assays indicate the ability of 12(R)-HETE to modulate AHR activity, and quantitation of induction of an AHR target gene confirmed 12(R)-HETE's ability to activate AHR-mediated transcription, even at high nanomolar concentrations in human hepatoma (HepG2)- and keratinocyte (HaCaT)-derived cell lines. One explanation for these results is that a metabolite of 12(R)-HETE is acting as a direct ligand for the AHR. However, several known metabolites failed to exhibit AHR activity. The ability of 12(R)-HETE to activate AHR target genes required receptor expression. These results indicate that 12(R)-HETE can serve as a potent activator of AHR activity and suggest that in normal and inflammatory disease conditions in skin, 12(R)-HETE is produced, perhaps leading to AHR activation.
[Show abstract][Hide abstract] ABSTRACT: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsible for mediating the cellular response to the toxic compound 2,3,7,8,-tetrachlorodibenzo-p-dioxin. An essential role for the AhR in cellular biology has been established previously, but no high-affinity endogenous ligand has yet been identified. We have confirmed the presence of a putative endogenous ligand(s) in CV-1 cells through transient transfection with various cytochrome P450 isoforms. Expression of cytochromes P450 1A1, 1A2, or 1B1 reduced AhR-mediated luciferase reporter activity, whereas cytochrome P450 2E1 exhibited no significant effect. Studies with 2,4,3',5'-tetramethoxystilbene, a potent and specific inhibitor of cytochrome P450 1B1, was able to partially block cytochrome P450 1B1-mediated reduction in reporter gene activity. These results provide evidence of the existence of a possible feedback mechanism in which AhR-regulated cytochromes P450 from the CYP1A and CYP1B families are able to metabolically alter putative endogenous ligand(s). Several experiments were performed to provide initial characterization of these putative endogenous ligands, including electrophoretic mobility shift assay analyses, which demonstrated that these ligands directly activate the AhR. Soluble extracts from various C57BL/6J and Ahr-null mouse tissues were also analyzed for the presence of AhR activators. Studies revealed that Ahr-null mouse lung tissue had a 4-fold increase in AhR-mediated reporter activity in cells. Quantitative polymerase chain reaction analysis revealed that lung tissue exhibits relatively high constitutive CYP1A1 mRNA levels. These results suggest that there is an autoregulatory feedback loop between the AhR and cytochrome P450 1A1 in mouse lung.
[Show abstract][Hide abstract] ABSTRACT: The aryl-hydrocarbon receptor (AhR) is a basic helix-loop-helix/Per-Arnt-Sim transcription factor that can be activated by exogenous as well as endogenous ligands. AhR is traditionally associated with xenobiotic metabolism. In an attempt to identify novel target genes, C57BL/6J mice were treated with beta-naphthoflavone (BNF), a known AhR ligand, and genome-wide expression analysis studies were performed using high-density microarrays. Constitutive androstane receptor (CAR) was found to be one of the differentially regulated genes. Real-time quantitative polymerase chain reaction (qPCR) verified the increase in CAR messenger RNA (mRNA) level. BNF treatment did not increase CAR mRNA in AhR-null mice. Time-course studies in mice revealed that the regulation of CAR mRNA mimicked that of Cyp1A1, a known AhR target gene. To demonstrate that the increase in CAR mRNA translates to an increase in functional CAR protein, mice were sequentially treated with BNF (6 hours) followed by the selective CAR agonist, TCPOBOP (3 hours). qPCR revealed an increase in the mRNA level of Cyp2b10, previously known to be regulated by CAR. This also suggests that CAR protein is present in limiting amounts with respect to its transactivation ability. Finally, CAR was also up-regulated in primary human hepatocytes in response to AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene. CONCLUSION: This study identifies a mode of up-regulating CAR and potentially expands the role of AhR in drug metabolism. This study also demonstrates in vivo up-regulation of CAR through chemical exposure.
[Show abstract][Hide abstract] ABSTRACT: The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that acts as an environmental sensor by binding to a variety of xenobiotics. AHR activation serves to combat xenotoxic stress by inducing metabolic enzyme expression in the liver. The hepatitis B virus X-associated protein (XAP2) is a component of the cytosolic AHR complex and modulates AHR transcriptional properties in vitro and in cell culture and yeast systems. Expression of XAP2 is low in liver compared with other nonhepatic tissues and the AHR exhibits high ligand-induced transcriptional activity. Because XAP2 has been demonstrated to repress AHR activity, we hypothesized that XAP2 may be limiting in liver and that increasing XAP2 levels would attenuate AHR transcriptional activity. To this end, transgenic mice were generated that exhibit hepatocyte-specific elevation in XAP2 expression. Transgenic XAP2 expression was restricted to liver, and its ability to complex with the AHR was verified. Gene expression experiments were performed by inducing AHR transcriptional activity with beta-naphthoflavone via intraperitoneal injection, and mRNA quantification was done by real-time polymerase chain reaction. Wild-type and transgenic animals showed little difference in constitutive or ligand-induced CYP1A1; CYP1A2; UDP glucuronosyltransferase 1A2; NAD(P)H dehydrogenase, quinone 1; constitutive androstane receptor; or nuclear factor erythroid 2-related factor 2 mRNA expression. Sucrose density fractionation and AHR immunoprecipitation experiments found little or no stoichiometric increase in bound XAP2 to the AHR between genotypes. Gene array studies were performed to identify novel XAP2-regulated targets. Taken together, this work shows that despite the relatively low level of XAP2 in liver, it is not a limiting component in AHR regulation.
[Show abstract][Hide abstract] ABSTRACT: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause a large number of adverse effects, mediated largely by its binding to the aryl-hydrocarbon receptor (AhR) and subsequent modulation of gene expression. It is thought that AhR mediates these effects through the untimely and disproportionate expression of specific genes. However, the exact mechanism, or the genes involved, through which TCDD leads to these effects is still unknown. This study reports the discovery of a novel target gene, epiregulin, which is regulated by TCDD-activated AhR. Epiregulin is a growth regulator which belongs to the epidermal growth factor (EGF) family. Using real time quantitative PCR (qPCR), it was established that TCDD upregulates epiregulin gene expression. The promoter region of epiregulin has a dioxin responsive element (DRE) 56 nucleotides upstream of the transcription start site, along with three potential Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR antibody showed promoter occupancy upon TCDD treatment. Luciferase reporter assays using a vector harboring the first 125 base pairs of the epiregulin rat promoter revealed an increase in signal on TCDD treatment, which was lost upon mutation of the DRE. Epiregulin and TCDD treatment mediated a dose-dependent increase in primary mouse keratinocyte growth. These results demonstrate that AhR directly increases epiregulin expression, which could play an important role in TCDD mediated tumor promotion observed in rodent models.