Aryl hydrocarbon receptor is required for p300-mediated induction of DNA synthesis by adenovirus E1A.
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The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the biological responses to environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin. Embryonic fibroblast (EF) isolated from AHR-null mice exhibited slow cell growth compared with wild-type EF. Reintroduction of AHR into AHR-null EF increased cell growth, suggesting that AHR is involved in cell cycle control. The role of the AHR in cell cycle control was examined using the adenovirus oncoprotein E1A. EF, derived from wild-type and AHR-null mice, were transfected with two mutant E1A expression plasmids that inactivate either p300/CBP or retinoblastoma protein (pRb). Although DNA synthesis of wild-type EF was induced by both E1A mutants, DNA synthesis in the AHR-null EF was induced only by the mutant that binds pRb, not by the mutant to p300/CBP. These data show that both pRb and p300/CBP were the target of E1A-induced DNA synthesis in wild-type EF. In AHR-null mice, however, only pRb was the target of E1A-induced DNA synthesis and p300/CBP cannot be inactivated by E1A in the absence of AHR. Immunoprecipitation revealed that AHR directly bound to p300, thus suggesting the intriguing possibility that AHR is involved in control of the cell cycle via interaction with p300. (+info)
Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse uterus: critical role of the aryl hydrocarbon receptor in stromal tissue.
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The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the role of aryl hydrocarbon receptor (AhR) in estradiol (E(2))-induced uterine epithelial mitogenic activity and secretory protein mRNA expression were determined. Ovariectomized wild-type (wt) and AhR-knockout (AhRKO) mice received oil, E(2), or 5 microg/kg TCDD+E(2). E(2) stimulated similar large increases in the uterine epithelial labeling index (LI) and mRNA abundance for the E(2)-dependent epithelial secretory protein, lactoferrin (LF), in both wt and AhRKO mice. However, uterine epithelial LI and LF mRNA were significantly reduced by TCDD+E(2) in wt but not AhRKO mice. To determine the roles of stromal and epithelial AhR in the TCDD effect, uterine stroma and epithelium from AhRKO and wt mice were enzymatically separated and recombined into four types of tissue recombinants that either contained or lacked AhR in one or more tissue compartments. Tissue recombinants were grafted into nude mice, which were later ovariectomized and given oil, E(2), or TCDD+E(2). Epithelial LI was significantly reduced by TCDD in grafts containing stromal AhR, regardless of epithelial AhR status. However, LI was unaffected by TCDD in grafts lacking stromal AhR, even when epithelial AhR was present. Thus, TCDD inhibits E(2)-induced uterine epithelial mitogenic and secretory activity, and this requires AhR. Anti-proliferative effects of TCDD on uterine epithelia appear to be mediated indirectly through stromal AhR, suggesting that liganded AhR alters epithelial function by disrupting normal E(2)-induced stromal activity. This is the first demonstration that TCDD impairs uterine epithelial function by altering normal stromal-epithelial interactions in vivo. (+info)
Isolation and characterization of AINT: a novel ARNT interacting protein expressed during murine embryonic development.
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Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. Herein, we describe a novel, embryonically expressed, ARNT interacting protein (AINT) that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINT localizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development. (+info)
Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions.
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In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties. (+info)
Expression and subcellular localization of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein in mouse and chicken over developmental time.
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The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic-helix-loop-helix/Per- ARNT-Sim (bHLH/PAS) transcription factor that is involved in multiple signaling pathways. This study focuses on the tissue distribution and subcellular localization of ARNT during embryological development of the mouse and chicken. Two different species were chosen to determine the consistency of the ARNT staining pattern. Immunohistochemical techniques were used to stain sections of embryos over three developmental time points for each species. Mouse tissues evaluated from embryonic day 10.5, 12.5, and 15, exhibited predominant nuclear staining with little change in expression patterns over time. Chicken tissues evaluated from embryonic day 2, 4, and 10 also showed predominant nuclear staining within all cells and little change in expression over developmental time, as well as, low levels of cytoplasmic ARNT staining in some cells. Importantly, in all tissues, the level of ARNT staining within the nuclear compartment was greater than staining observed in the cytoplasm. Thus, the overall conclusions from these studies are that i) the predominant subcellular localization of ARNT protein is nuclear, and ii) that mouse and chicken appear to maintain ARNT protein expression in many cell types over developmental time. These data support vertebrate ARNT as a nuclear transcription factor and a model in which dimerization partners require nuclear localization for interaction. (+info)
Placental defects in ARNT-knockout conceptus correlate with localized decreases in VEGF-R2, Ang-1, and Tie-2.
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The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcriptional regulator that heterodimerizes with Per-ARNT-Sim (PAS) proteins. ARNT also dimerizes with hypoxia inducible factor1alpha (HIF1alpha), inducing expression of vascular endothelial cell growth factor (VEGF) to promote angiogenesis. The angiogenesis/vasculogenesis pathway is required for embryonic survival and includes several receptors (VEGFR1, VEGFR2, Tie2) and ligands (VEGF, Ang1, Ang2, neuropillin). Transgenic knockout of ARNT in mice is lethal due to abnormal placentation. This study examines the VEGF pathway in GD9.5 embryos of wild-type (+/+), heterozygous (+/-), or knockout (-/-) ARNT genotype. All genotypes expressed abundant VEGF in trophoblastic giant cells. However, -/- conceptuses had less VEGFR2 in placental labyrinth and trophoblastic giant cells. Ang1 and Tie2 decreased in trophoblastic giant cells and Ang2 was decreased in placental endothelial cells. Abnormal development of the labyrinth correlated with decreased binding of VEGF and decreased expression of VEGFR2. In addition, VEGFR2 seemed to be the primary VEGF binding receptor in the labyrinth and blood lacunae of the placenta, as binding could be eliminated by masking the VEGFR2 receptor with inactive antibody complex. VEGFR1 may be primarily responsible for binding of VEGF to yolk sac and embryonic tissues, as masking VEGFR2 did not reduce VEGF binding in those areas, and it is interesting that major structural defects were also not found in those regions. In summary, in the ARNT knockout conceptus, the impact of ARNT deficiency on placental expression of VEGFR2 seems to provide an explanation for the failure of the placental labyrinth to progress, whereas the vascularization of the yolk sac and embryo appear relatively unaffected on GD9.5. Published 2000 Wiley-Liss, Inc. (+info)
Aromatic hydrocarbon receptor (AhR).AhR nuclear translocator- and p53-mediated induction of the murine multidrug resistance mdr1 gene by 3-methylcholanthrene and benzo(a)pyrene in hepatoma cells.
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The mouse multidrug resistance gene family consists of three genes (mdr1, mdr2, and mdr3) encoding P-glycoprotein. We show that the expression of mdr1 is increased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7 with the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC). This increase is not observed in the aromatic hydrocarbon receptor (AhR)-defective TAOc1BP(r)c1 and the AhR nuclear translocator (Arnt)-defective BP(r)c1 variants, demonstrating that the induction of mdr1 by 3-MC requires AhR.Arnt. We show that the mdr1 promoter (-1165 to +84) is able to activate the expression of a reporter gene in response to 3-MC in Hepa-1c1c7 but not in BP(r)c1 cells. Deletion analysis indicated that the region from -245 to -141 contains cis-acting sequences mediating the induction, including a potential p53 binding sequence. 3-MC treatment of the cells increased the levels of p53 and induced p53 binding to the mdr1 promoter in an AhR.Arnt-dependent manner. Mutations in the p53 binding site abrogated induction of mdr1 by 3-MC, indicating that p53 binding to the mdr1 promoter is essential for the induction. Benzo(a)pyrene, a polycyclic aromatic hydrocarbon and AhR ligand, which, like 3-MC, is oxidized by metabolizing enzymes regulated by AhR.Arnt, also activated p53 and induced mdr1 transcription. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an AhR ligand resistant to metabolic breakdown, had no effect. These results indicate that the transcriptional induction of mdr1 by 3-MC and benzo(a)pyrene is directly mediated by p53 but that the metabolic activation of these compounds into reactive species is necessary to trigger p53 activation. The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1 independently of AhR.Arnt further supports the proposition that mdr1 is transcriptionally up-regulated by p53 in response to DNA damage. (+info)
Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses.
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Placental development is profoundly influenced by oxygen (O(2)) tension. Human cytotrophoblasts proliferate in vitro under low O(2) conditions but differentiate at higher O(2) levels, mimicking the developmental transition they undergo as they invade the placental bed to establish the maternal-fetal circulation in vivo. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1alpha and ARNT subunits, activates many genes involved in the cellular and organismal response to O(2) deprivation. Analysis of Arnt(-/-) placentas reveals an aberrant cellular architecture due to altered cell fate determination of Arnt(-/-) trophoblasts. Specifically, Arnt(-/-) placentas show greatly reduced labyrinthine and spongiotrophoblast layers, and increased numbers of giant cells. We further show that hypoxia promotes the in vitro differentiation of trophoblast stem cells into spongiotrophoblasts as opposed to giant cells. Our results clearly establish that O(2) levels regulate cell fate determination in vivo and that HIF is essential for mammalian placentation. The unique placental phenotype of Arnt(-/-) animals also provides an important tool for studying the disease of preeclampsia. Interestingly, aggregation of Arnt(-/-) embryonic stem (ES) cells with tetraploid wild-type embryos rescues their placental defects; however, these embryos still die from yolk sac vascular and cardiac defects. (+info)