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Abstract: 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. [Copyright &y& Elsevier]

Abstract: The heterodimeric hypoxia inducible factor-1 (HIF-1) complex is composed of the hypoxia inducible factor-1 alpha (HIF-1α) and the aryl hydrocarbon receptor nuclear translocator (ARNT). Activation of the HIF-1 function is essential for tumor growth and metastasis. We previously showed that transfection of a plasmid containing an ARNT-interacting peptide (Ainp1) cDNA suppresses the HIF-1 signaling in Hep3B cells. Here we generated TAT fusion of the Ainp1 peptide (6His-TAT-Ainp1) to determine whether and how the Ainp1 peptide suppresses the HIF-1 function. The bacterially expressed 6His-TAT-Ainp1 was purified under denatured condition and then refolded by limited dialysis. The refolded 6His-TAT-Ainp1 interacts with the helix–loop–helix (HLH) domain of ARNT in a similar fashion as the native 6His-Ainp1. 6His-TAT-Ainp1 colocalizes with ARNT in the nucleus of HeLa and Hep3B cells after protein transduction. The transduced protein reaches the maximum intracellular levels within 2h while remains detectable up to 96h in HeLa cells. At 2μM concentration, 6His-TAT-Ainp1 is not cytotoxic in HeLa cells but suppresses the cobalt chloride-activated, hypoxia responsive enhancer-driven luciferase expression in a dose-dependent manner. In addition, it decreases the cobalt chloride-dependent induction of the HIF-1 target genes at both the message (vascular endothelial growth factor and aldolase C) and protein (carbonic anhydrase IX and glucose transporter 1) levels. The protein levels of HIF-1α and ARNT are not altered in the presence of 6His-TAT-Ainp1. In summary, we provided evidence to support that the Ainp1 peptide directly suppresses the HIF-1 function by interacting with the ARNT HLH domain, and in turn interfering with the heterodimerization of HIF-1α and ARNT. [Copyright &y& Elsevier]

Abstract: In an effort to better understand the Ah receptor nuclear translocator (Arnt)-dependent signaling mechanisms, we employed a phage display system to identify Arnt-interacting peptides. Human liver cDNA library was utilized to screen for Arnt-interacting peptides using an Arnt construct fused to thioredoxin (TH-ArntCΔ418). Two clones, namely Ainp1 and Ainp2 (Arnt-interacting peptide), were identified and subsequently Ainp2 was further characterized. Ainp2 interacts with TH-ArntCΔ418 in the GST pull-down and mammalian two-hybrid assays. Northern blot results revealed that Ainp2 is predominantly expressed in human liver. The putative full-length Ainp2 cDNA sequence was subsequently cloned using RACE PCR. Endogenous expression of Ainp2 was found in Jurkat cells at the mRNA and protein levels. Results from the transient transfection studies using a DRE-driven reporter plasmid and the real-time QPCR experiments examining the endogenous CYP1A1 expression showed that Ainp2 enhances the 3-methylchloranthrene-induced activity in HepG2 cells, suggesting that Ainp2 plays a role in the Arnt-dependent function. [Copyright &y& Elsevier]

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner aryl hydrocarbon receptor nuclear translocator (Arnt). The AhR–Arnt heterodimer binds to the dioxin response element (DRE) to regulate target gene expression. Using baculovirus expressed human AhR and Arnt, we showed that the formation of the ligand-dependent AhR–Arnt–DRE complex requires protein factors in vitro. Recently, we provided evidence that p23, an Hsp90-associated protein, is involved in the complex formation. The aim of this study was to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR–Arnt–DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CyP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR–Arnt–DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex. [Copyright &y& Elsevier]

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that requires heterodimerization with its partner, the Ah receptor nuclear translocator (Arnt), for activation of transcription. The heterodimer specifically recognizes the dioxin response element (DRE), which contains a core sequence (5′-TNGCGTG-3′). This AhR/Arnt/DRE complex has been well characterized and can be observed readily by the gel shift assay. Human AhR and Arnt with a C-terminal histidine tag have been expressed functionally using a baculovirus expression system. However, after purification of these proteins using the metal resin, they are not able to bind the response element in a ligand-dependent manner unless crude extracts, such as the rabbit reticulocyte lysate (RRL), are reconstituted with these proteins. Proteins in the RRL are responsible for this restoration of the gel shift complex because the activity is sensitive to both heat and proteolytic treatments. We have examined whether hsp90 and p23 are among the protein factors in the RRL that are responsible for this activity. By performing fractionation studies using filtration devices and immunodepletion studies, we have selectively fractionated these proteins. Among all the fractions, the centricon-10 retentate, which contains 100% of p23 but no hsp90, possessed the most enriched activity. Purified bacterial-expressed p23 restored the gel shift complex; the mechanism was mediated at the heterodimerization step and was hsp90-dependent. [Copyright &y& Elsevier]

The thioredoxin fusion protein expression system from invitrogen was modified so that 32P-labelled recombinant proteins can be easily obtained in large quantities for functional studies. Proteins that are prone to form the inclusion bodies can be functionally expressed as thioredoxin fusion proteins in Escherichia coli. After expression, the recombinant proteins can be easily phosphorylated with 32P-gamma ATP and the 32P-labelled protein can be obtained functionally via a mild proteolytic digestion to cleave off the thioredoxin moiety. A deletion construct of the Ah receptor nuclear translocator protein was used as an example to illustrate how this protein expression system works. [Copyright &y& Elsevier]

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which is involved in diverse biological functions ranging from cancer metastasis to immune regulation. This receptor forms a cytoplasmic complex with Hsp90, p23, and XAP2. We have previously reported that down-regulation of p23 triggers degradation of the AHR protein, uncovering a potentially dynamic event which controls the cellular AHR levels without ligand treatment. Here we investigate the underlying mechanisms for this p23 effect using wild-type HeLa and the p23 knockdown HeLa cells. Reduction of the Hsp90 and XAP2 contents, however, did not affect the AHR protein levels, implying that this p23 effect on AHR is more than just alteration of the cytoplasmic complex dynamics. Association of p23 with Hsp90 is not important for the modulation of the AHR levels since exogenous expression of p23 mutants with modest Hsp90-binding affinity effectively restored the AHR message and protein levels. The protein folding property of p23 which resides at the terminal 50-amino acid region is not involved for this p23 effect. Results from our interaction study using the affinity purified thioredoxin fusion proteins and GST fusion proteins showed that p23 directly interacts with AHR and the interaction surface lies within AHR amino acid 1–216 and p23 amino acid 1–110. Down-regulation of the p23 protein content promotes the ubiquitination of AHR, indicating that p23 protects AHR from the ubiquitin-meditated protein degradation. [ABSTRACT FROM AUTHOR]

The human aryl hydrocarbon receptor is a cytosolic signaling molecule which affects immune response and aberrant cell growth. Canonical signaling of the receptor requires the recruitment of coactivators to the promoter region to remodel local chromatin structure. We predicted that interference of this recruitment would block the aryl hydrocarbon receptor function. To prove that, we employed phage display to identify nine peptides of twelve-amino-acid in length which target the C-terminal half of the human aryl hydrocarbon receptor, including the region where coactivators bind. Eight 12mer peptides, in the form of GFP fusion, suppressed the ligand-dependent transcription of six AHR target genes ( cyp1a1 , cyp1a2 , cyp1b1 , ugt1a1 , nqo1 , and ahrr ) in different patterns in Hep3B cells, whereas the AHR antagonist CH-223191 suppressed all these target genes similarly. Three of the 12mer peptides (namely 11-3, 1-7, and 7-3) suppressed the 3MC-induced, CYP1A1-dependent EROD activity and the ROS production caused by benzo[a]pyrene. These 12mer peptides suppressed the AHR function synergistically with CH-223191. In conclusion, we provide evidence that targeting the C-terminal half of the human aryl hydrocarbon receptor is a viable, new approach to selectively block the receptor function. [ABSTRACT FROM AUTHOR]

Abstract: The aryl hydrocarbon receptor (AhR) heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) for transcriptional regulation. We generated three N-terminal deletion constructs of the human AhR of 12–24kDa in size – namely D1, D2, and D3 – to suppress the Arnt function. We observed that all three deletions interact with the human Arnt with similar affinities. D2, which contains part of the AhR PAS-A domain and interacts with the PAS-A domain of Arnt, inhibits the formation of the AhR gel shift complex. D2 suppresses the 3-methylcholanthrene-induced, dioxin response element (DRE)-driven luciferase activity in Hep3B cells and exogenous Arnt reverses this D2 suppression. D2 suppresses the induction of CYP1A1 at both the message and protein levels in Hep3B cells; however, the CYP1B1 induction is not affected. D2 suppresses the recruitment of Arnt to the cyp1a1 promoter but not to the cyp1b1 promoter, partly because the AhR/Arnt heterodimer binds better to the cyp1b1 DRE than to the cyp1a1 DRE. Interestingly, D2 has no effect on the cobalt chloride-induced, hypoxia inducible factor-1 (HIF-1)-dependent expression of vegf, aldolase c, and ldh-a messages. Our data reveal that the flanking sequences of the DRE contribute to the binding affinity of the AhR/Arnt heterodimer to its endogenous enhancers and the function of AhR and HIF-1 can be differentially suppressed by the D2 inhibitory molecule. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 up-regulation 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. [ABSTRACT FROM AUTHOR]

Abstract: We have been studying the requirement for the aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent DNA complex formation, which precedes the activation of gene transcription. Using DEAE chromatography, we have obtained a Sf9 insect fraction F5 that is highly enriched with β-tubulin. F5 inhibits the formation of the AhR gel shift complex and this inhibition is sensitive to protease, suggesting that proteins that are present in this F5 fraction are responsible for the inhibition. Additional experiments have revealed that this inhibition is less pronounced in the presence of anti-β-tubulin IgG and β-tubulin enriched fraction from pig brain also inhibits the AhR gel shift formation. Sf9 β-tubulin interacts with Arnt and suppresses the binding of the AhR/Arnt heterodimer to its corresponding enhancer. Human β4-tubulin, which shares high sequence identity with Sf9 β-tubulin, suppresses the AhR-dependent luciferase expression by reducing the nuclear Arnt content and retaining Arnt in the cytoplasm. Fluorescence studies using the GFP fusion of human β4-tubulin have revealed that β4-tubulin prevents the localization of Arnt in Sf9 cells. Here we have provided evidence suggesting that β-tubulin may regulate the physiological content of Arnt. [Copyright &y& Elsevier]