Brett D. Hollingshead

Aryl‐hydrocarbon receptor activation regulates constitutive androstane receptor levels in murine and human liver

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 β‐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. (HEPATOLOGY 2007;46:209–218.)

Inflammatory Signaling and Aryl Hydrocarbon Receptor Mediate Synergistic Induction of Interleukin 6 in MCF-7 Cells

The pleiotropic cytokine interleukin 6 (IL-6) is involved in immune cell homeostasis. Additionally, IL-6 expression and signaling in tumor cells have been shown to elicit both protumor and antitumor properties. There is a plethora of mechanistic knowledge regarding how IL-6 signal transduction translates to biological responses. However, there is little understanding as to what factors control IL-6 expression within a tumor cell environment. The studies presented herein show that, in MCF-7 breast and ECC-1 endocervical cancer cells, the stimulation of aryl hydrocarbon receptor (AHR) activity, in combination with IL-1beta or phorbol 12-myristate 13-acetate (PMA) treatment, results in a marked synergistic induction of IL-6 levels over what is seen without AHR activation. Chromatin immunoprecipitation experiments suggest that the regulation of IL-6 mRNA expression occurs at the chromatin level, as AHR presence on the IL-6 promoter was observed in response to treatment with AHR ligand. Synergistic induction of IL-6 expression was sustained for 72 hours, with accumulation of IL-6 protein reaching levels 4.8-fold above IL-1beta treatment alone. In addition, transcriptional regulation of the prototypic AHR responsive gene Cyp1a1 was negatively regulated by PMA and IL-1beta treatment. Silencing of RELA expression alleviated IL-1beta-mediated repression of AHR transcriptional activity, whereas PMA-mediated repression was maintained. Additionally, small interfering RNA studies reveal that AHR and RELA are necessary for synergistic induction of IL-6. The findings presented here reveal the AHR as a potential therapeutic target for selective modulation of IL-6 expression in some tumor cell types. The data also suggest a possible previously unrecognized mechanism of AHR-mediated tumor promotion.

Distinct roles for aryl hydrocarbon receptor nuclear translocator and ah receptor in estrogen-mediated signaling in human cancer cell lines.

The activated AHR/ARNT complex (AHRC) regulates the expression of target genes upon exposure to environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Importantly, evidence has shown that TCDD represses estrogen receptor (ER) target gene activation through the AHRC. Our data indicates that AHR and ARNT act independently from each other at non-dioxin response element sites. Therefore, we sought to determine the specific functions of AHR and ARNT in estrogen-dependent signaling in human MCF7 breast cancer and human ECC-1 endometrial carcinoma cells. Knockdown of AHR with siRNA abrogates dioxin-inducible repression of estrogen-dependent gene transcription. Intriguingly, knockdown of ARNT does not effect TCDD-mediated repression of estrogen-regulated transcription, suggesting that AHR represses ER function independently of ARNT. This theory is supported by the ability of the selective AHR modulator 3′,4′-dimethoxy-α-naphthoflavone (DiMNF) to repress estrogen-inducible transcription. Furthermore, basal and estrogen-activated transcription of the genes encoding cathepsin-D and pS2 are down-regulated in MCF7 cells but up-regulated in ECC-1 cells in response to loss of ARNT. These responses are mirrored at the protein level with cathepsin-D. Furthermore, knock-down of ARNT led to opposite but corresponding changes in estrogen-stimulated proliferation in both MCF7 and ECC-1 cells. We have obtained experimental evidence demonstrating a dioxin-dependent repressor function for AHR and a dioxin-independent co-activator/co-repressor function for ARNT in estrogen signalling. These results provide us with further insight into the mechanisms of transcription factor crosstalk and putative therapeutic targets in estrogen-positive cancers.

Endogenous Hepatic Expression of the Hepatitis B Virus X-Associated Protein 2 Is Adequate for Maximal Association with Aryl Hydrocarbon Receptor-90-kDa Heat Shock Protein Complexes

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 β-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.

Estrogen Receptor Expression Is Required for Low-Dose Resveratrol-Mediated Repression of Aryl Hydrocarbon Receptor Activity

The putative cardioprotective and chemopreventive properties of the red wine phenolic resveratrol (RES) have made it the subject of a growing body of clinical and basic research. We have begun investigations focusing on the effects of RES on the activity of the aryl hydrocarbon receptor (AHR) complex. Our evidence suggests that RES is a potent repressor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible gene transcription in estrogen receptor (ER)-positive human breast, lung, and colon cancer cell lines. RES activates the transcription of the ER target genes to the same degree as estradiol (E2) in human MCF-7 breast cancer cells. Unlike E2, which can only diminish TCDD-inducible CYP1A1 gene transcription by approximately 50%, RES can completely abrogate this response. Furthermore, 50% repression of TCDD-inducible transcription can be achieved with 100 nM RES, approximately 2.5 orders of magnitude lower than concentrations required for maximal inhibition, suggesting that multiple mechanisms are responsible for this effect. RES (100 nM) does not prevent ligand binding of a TCDD analog, nor does it prevent AHR from binding to its response element in the 5′-regulatory region of the CYP1A1 gene. Small inhibitory RNAs directed to ERα have demonstrated that RES-mediated repression of CYP1A1 depends on ERα. Whereas CYP1A1 protein levels in MCF-7 cells are refractory to the low-dose transcriptional effects of RES, a concomitant decrease in CYP1A1 protein levels is observed in Caco-2 cells. These results highlight a low-dose RES effect that could occur at nutritionally relevant exposures and are distinct from the high-dose effects often characterized.