Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor.

A single human cell contains more than 5.0 × 10(5) copies of long interspersed element-1 (L1), 80-100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein ß. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis.

Disruption of aryl hydrocarbon receptor (AhR) induces regression of the seminal vesicle in aged male mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates diverse dioxin toxicities. Despite mediating the adverse effects, the AhR gene is conserved among animal species, suggesting important physiological functions for AhR. In fact, a recent study revealed that AhR has an intrinsic function in female reproduction, though its role in male reproduction is largely unknown. In this study, we show age-dependent regression of the seminal vesicles, probably together with the coagulating gland, in AhR(-/-) male mice. Knockout mice had abnormal vaginal plugs, low sperm counts in the epididymis, and low fertility. Moreover, serum testosterone concentrations and expression of steroidogenic 3betahydroxysteroiddehydrogenase (3betaHsd) and steroidogenic acute regulatory protein (StAR) in testicular Leydig cells were decreased in AhR(-/-) males. Taken together, our results suggest that impaired testosterone synthesis in aged mice induces regression of seminal vesicles and the coagulating glands. Such tissue disappearance likely resulted in abnormal vaginal plug formation, and eventually in low fertility. Together with previous findings demonstrating AhR function in female reproduction, AhR has essential functions in animal reproduction in both sexes.

Molecular mechanisms of AhR functions in the regulation of cytochrome P450 genes.

AhR, a ligand-activated transcription factor, mediates xenobiotic signaling to enhance the expression of target genes, including drug-metabolizing cytochrome P450s. The recent development of several new techniques, including chromatin immunoprecipitation and RNA interference, has expanded and deepened our knowledge of AhR function in the xenobiotic signal transduction. In this review, we briefly summarize our current understanding of the activation and inactivation of AhR activities and discuss the future directions of AhR research.

Parathyroid hormone (PTH) down-regulates PTH/PTH-related protein receptor gene expression in UMR-106 osteoblast-like cells via a 3',5'-cyclic adenosine monophosphate-dependent, protein kinase A-independent pathway.

Parathyroid hormone (PTH) regulates osteoblast function via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. We have studied the mechanisms of PTH/PTHrP receptor gene repression by PTH in UMR-106 osteoblast-like cells. Inhibition of PTH/PTHrP receptor mRNA expression by rat (r) PTH(1-34) and Insulin-like growth factor-I (IGF-I) at 10(-7)M was significant at 1 h and 3 h, and maximal at 2 h and 6 h. A maximal decrease in receptor mRNA abundance by rPTH(1-34) and IGF-I was maintained for 24 h. Inhibition of receptor gene expression by rPTH(1-34) was mimicked in UMR-106 cells by the addition of forskolin (an adenylyl cyclase activator), or 8-(4-chlorophenylthio)-adenine 3',5'-cyclic monophosphate (8-pCPTcAMP; a cAMP analogue). Although H89, a selective protein kinase A (PKA) inhibitor, completely inhibited PKA activity stimulated by rPTH(1-34), forskolin or 8-pCPTcAMP, suppression of PTH/PTHrP receptor mRNA synthesis induced by these substances in UMR-106 cells was not affected by H89. In primary osteoblast cultures, rPTH(1-34) inhibited synthesis of PTH/PTHrP receptor mRNA irrespective of H89. The down-regulation effect of rPTH(1-34) was also unaltered by PD98059 (an extracellularly regulated kinase 1/2 mitogen-activated protein kinase pathway inhibitor). Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter the inhibition of PTH/PTHrP receptor mRNA expression by rPTH(1-34), indicating that receptor mRNA suppression does not require new protein synthesis. Transcriptional activation of PTH/PTHrP receptor gene promoter (U3P or U4P)-luciferase constructs was decreased by rPTH(1-34), forskolin and 8-pCPTcAMP irrespective of H89. Thus, PTH transcriptionally down-regulates PTH/PTHrP receptor gene expression in osteoblast-like cells via a cAMP-dependent, PKA-independent pathway.

Expression of human and mouse genes encoding polkappa: testis-specific developmental regulation and AhR-dependent inducible transcription.

BACKGROUND: Human polkappa is a newly identified low-fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene-adduct in an error-prone manner, it bypasses benzo[a]pyrene-N2-dG lesions in a mostly error-free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand-activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin. RESULTS: We examined promoter structures of the human POLK and mouse Polk genes to study how their expressions are regulated. The mouse Polk gene is developmentally regulated in testis and utilizes two transcription start sites during spermatogenesis, while it utilizes only one site in tissues other than testis. Both of the mouse Polk and the human POLK genes have two AhR-binding sites in the promoter regions and the expression of the mouse Polk gene is indeed enhanced upon AhR-activation. CONCLUSIONS: The AhR activation increases expression of the mouse Polk gene and probably the human POLK gene, the product of which bypasses benzo[a]pyrene-N2-dG lesions in a mostly accurate manner. Thus, polkappa seems to function to reduce mutagenesis at benzo[a]pyrene-adducts, although it may also have a role related to spermatogenesis.

Dioxin induces a novel nuclear factor, DIF-3, that is implicated in spermatogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), a member of a class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans, is one of the most toxic artificial compounds ever developed. In this study, we identified a novel TCDD target gene, DIF-3 (dioxin inducible factor-3), by cDNA representational difference analysis. DIF-3 protein is a nuclear factor and possesses a zinc-finger motif at its N-terminus. High DIF-3 mRNA expression in the testes was demonstrated by Northern blot analysis and abundant DIF-3 protein was detected during spermatogenesis. Thus, these results suggest that DIF-3 may be a target gene mediating the reproductive toxicity induced by TCDD.

Parathyroid hormone (PTH) suppresses rat PTH/PTH-related protein receptor gene promoter.

Parathyroid hormone (PTH) regulates osteoblasts via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. PTH effects on PTH/PTHrP receptor gene expression were studied in UMR 106 osteoblast-like cells. In heterogeneous nuclear RNA and Northern analysis, PTH suppressed PTH/PTHrP receptor transcription. We cloned the 7-kb promoter region of the rat PTH/PTHrP receptor gene and transiently transfected chimeric deletion constructs containing the 5'-flanking region and the luciferase gene into UMR 106 cells. In transfected cells the minimal region for basal promoter activity was between positions -128 and +103. The 5'-flanking region of exon U1 contained several putative-binding sites for Sp1 and the myc-associated zinc finger protein (MAZ). The minimal PTH-suppressive region (PTHSR) was between +1 and +25 in exon U1, but the 5'-flanking region or Sp1 and MAZ-binding sites also were required for PTH-mediated repression. By gel mobility shift assay PTH markedly decreased binding of PTHSR-protein complex in UMR 106 cells. The mutation experiments showed that the most critical sequence for the repression of PTH was 5'-GGGGGAGGGGAG-3' (+1 to +12) of PTHSR. This represents the first characterization of a PTH-suppressive region of the PTH/PTHrP receptor gene in rat.

Dioxin suppresses the checkpoint protein, MAD2, by an aryl hydrocarbon receptor-independent pathway.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown recently to be carcinogenic, but little is currently known about the molecular mechanism of TCDD affecting cell proliferation and carcinogenesis. In this report, we demonstrate that TCDD suppresses the expression of the checkpoint protein, Mad2. Suppression of Mad2 was also observed in aryl hydrocarbon receptor-deficient mouse embryonic fibroblasts, suggesting that TCDD suppresses Mad2 by a novel TCDD receptor signaling mechanism. In addition, HeLa cells treated with TCDD failed to arrest in mitosis after nocodazole treatment. The Mad2 protein plays a significant role in accurate chromosome segregation in mitotic cells. Our data suggest that TCDD may increase chromosomal instability through the suppression of Mad2 expression.

Structure and expression of the Ah receptor repressor gene.

The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library. The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-kappaB-binding sites have been identified in the 5'-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence. We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.

Defective development of secretory neurones in the hypothalamus of Arnt2-knockout mice.

BACKGROUND: Within the basic region-helix-loop-helix (bHLH)-PAS family of transcription factors, Arnt and Arnt2 play unique roles; these two factors not only heterodimerize with themselves, but also with other members of this family and they act as transcription regulators which bind to specific DNA elements. Whereas Arnt is broadly expressed in various tissues, the expression of Arnt2 is known to be limited to the neural tissues. RESULTS: To elucidate the function of Arnt2 in detail, we cloned the mouse Arnt2 gene and its gene structure was determined. We subsequently generated germ line Arnt2 mutant mice by gene targeting technology. Heterozygous Arnt2 mice were viable, but homozygous Arnt2 gene knockout mice died shortly after birth. Histological and immunological analyses revealed that the supraoptic nuclei (SON) and the paraventricular nuclei (PVN) are hypocellular. Moreover, secretory neurones identified by the expression of neurosecretory hormone such as arginine vasopressin, oxytocin, corticotrophin-releasing hormone and somatostatin are completely absent in SON and PVN in the mutant Arnt2 mice. Consistent with these observations, prospective SON and PVN neurones which express Brn2 appeared around E13.5 in the mantle zone, but no neurones which expressed the neurosecretory hormones were found in the SON and PVN regions. CONCLUSIONS: These data show that the transcription factor Arnt2 controls the development of the secretory neurones at the later or final stages of differentiation rather than at the beginning stage. Strikingly similar observations have been reported with the Sim1 deficient mice. Taken together, our results demonstrate that Arnt2 is an indispensable transcription factor for the development of the hypothalamus, and suggest that Arnt2 is an obligatory partner molecule of Sim1 in the developmental process of the neuroendocrinological cell lineages.

Aryl hydrocarbon receptor (AhR)-mediated induction of xanthine oxidase/xanthine dehydrogenase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, induced xanthine oxidase and xanthine dehydrogenase (XO/XDH) activities, in addition to ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in liver of mice. When TCDD was given to mice as a single oral dose of 40 microg/kg, the activities of XO and XDH increased about threefold within 3 days and the increased levels were maintained for 4 weeks. The treatment of mice with 3-methylcholanthrene also induced XO/XDH activities, but phenobarbital and dexamethasone had no effect. The level of aldehyde oxidase, a molybdenum flavoenzyme related to XO/XDH, in mouse liver was also enhanced about 1.5-fold by TCDD treatment. The inducing effect of TCDD and 3-methylcholanthrene was not observed in null mice (AhR(-/-)), which lack the AhR gene. XO and XDH activities were induced by TCDD in heterozygous mice (AhR(+/-)). The lipid peroxidation in liver was stimulated by TCDD. The induction of XO and XDH, which produces reactive oxygen species, may contribute to the various toxicities of TCDD.

Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3.

BACKGROUND: The Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin. RESULTS: We investigated the transcriptional mechanism of mArnt3/mClock heterodimer. While mClock did not possess any distinct activation domain, mArnt3 contained a transcriptional activation domain at the most C-terminal end, the activity of which was not expressed, even in the one hybrid system, until it was bound by mClock. It has been suggested that mClock plays a regulatory or structural role in exerting a transcription enhancing effect of the mArnt3/mClock heterodimer. Deletion proceeding from amino acids 559-492 of mClock markedly reduced the transactivation activity of mArnt3/mClock heterodimer, in consistence with the results of the Clock-delta 19 mutant. Yeast and mammalian two-hybrid systems revealed that CBP and p300 interacted with mArnt3 via the CREB binding domain. The In vivo interaction between mArnt3 and CBP was confirmed by the GST pull down assay. CONCLUSION: Taken together, these results suggest that the mArnt3/mClock heterodimer exerted its transactivation activity via CBP or p300 interacting with mArnt3 in the heterodimer with mClock playing a structural or regulatory role in the transactivation process.

Repression of heme oxygenase-1 by hypoxia in vascular endothelial cells.

Heme oxygenase 1 (HO-1), a rate-limiting enzyme in heme catabolism, has been reported to be induced by hypoxia. Unexpectedly, here we show that expression of HO-1 mRNA is repressed by hypoxia in primary cultures of human umbilical vein endothelial cells (HUVECs), but is increased by cobalt chloride (CoCl(2)) that is known to mimic hypoxia. Under the culture conditions used, the DNA-binding and transactivation activities of hypoxia-inducible factor 1 were increased in HUVECs by hypoxia or CoCl(2). Therefore, hypoxia and cobalt showed opposing effects on HO-1 mRNA expression, despite activation of hypoxia-inducible factor 1. The half-life of HO-1 mRNA was not changed by hypoxia, but was significantly prolonged by CoCl(2). Hypoxia also represses HO-1 mRNA expression in human coronary artery endothelial cells and astrocytes. The repression of HO-1 expression may represent the adaptation to hypoxia in certain cell types.

Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor.

The contribution of the aryl hydrocarbon receptor (AhR) in induction of a battery of xenobiotic-metabolizing enzymes has been studied extensively. However, no direct proof has been obtained that it plays a role in modulating carcinogenesis. To address the question of whether AhR is required for tumor induction, we have investigated the response of AhR-deficient mice to benzo[a]pyrene (B[a]P), a widely distributed environmental carcinogen. B[a]P treatment induced expression of the cytochrome P450 gene Cyp1a1 in the skin and liver of AhR-positive mice bearing +/+ and +/- genotypes and did not induce expression of the cytochrome P450 gene Cyp1a1 in AhR-null mice in either skin or liver. In contrast, Cyp1a2 gene expression was positive in liver irrespective of the presence or absence of the AhR gene, or B[a]P treatment, although its inducibility was lost in the AhR(-/-) mouse. All AhR-positive male mice of both +/+ and +/- genotypes that received subcutaneous injection of B[a]P (2 mg) on the first and the eighth days had developed subcutaneous tumors at the site of injection at the end of the 18-week experiment. In contrast, no tumors were apparent in any of the AhR-deficient mice. Likewise, topical application of B[a]P (200 microg) at weekly intervals to the skin of female mice for 25 weeks produced skin tumors only in the AhR-positive mice. Thus the carcinogenic action of B[a]P may be determined primarily by AhR, a transcriptional regulator of the gene for CYP1A1. The results of the present study provide direct evidence that AhR is involved in carcinogenesis.

Basic transcription element-binding protein (BTEB) is a thyroid hormone-regulated gene in the developing central nervous system. Evidence for a role in neurite outgrowth.

Thyroid hormone (3,5,3'-triiodothyronine; T(3)) is essential for normal development of the vertebrate brain, influencing diverse processes such as neuronal migration, myelin formation, axonal maturation, and dendritic outgrowth. We have identified basic transcription element-binding protein (BTEB), a small GC box-binding protein, as a T(3)-regulated gene in developing rat brain. BTEB mRNA levels in cerebral cortex exhibit developmental regulation and thyroid hormone dependence. T(3) regulation of BTEB mRNA is neural cell-specific, being up-regulated in primary cultures of embryonic neurons (E16) and in neonatal astrocytes (P2), but not in neonatal oligodendrocytes (P2). T(3) rapidly up-regulated BTEB mRNA in neuro-2a cells engineered to express thyroid hormone receptor (TR) beta1 but not in cells expressing TRalpha1, suggesting that the regulation of this gene is specific to the TRbeta1 isoform. Several lines of evidence support a transcriptional action of T(3) on BTEB gene expression. Overexpression of BTEB in Neuro-2a cells dramatically increased the number and length of neurites in a dose-dependent manner suggesting a role for this transcription factor in neuronal process formation. However, other T(3)-dependent changes were not altered; i.e. overexpression of BTEB had no effect on the rate of cell proliferation nor on the expression of acetylcholinesterase activity.

Mild impairment of learning and memory in mice overexpressing the mSim2 gene located on chromosome 16: an animal model of Down's syndrome.

Human Sim2 is a product of one of the genes located on human chromosome 21q22 and is a homolog of Drosophila single-minded ( sim ) which is a critical player in midline development of the central nervous system of the fly. Since Sim2 mRNA is expressed in facial, skull, palate and vertebra primordia in human and rodent embryos, features that are associated with phenotypes of Down's syndrome (DS), its trisomic state is suspected to contribute to the symptoms of DS. Here we describe that mSim2 mRNA is expressed in hippocampus and amygdala of adult mice, and that while mice overexpressing mSim2 under the control of the beta-actin promoter are viable and fertile and have superficially normal skeletal, brain and heart structures, they exhibit a moderate defect in context-dependent fear conditioning and a mild defect in the Morris water maze test. Taken together, our data show that overdosage of Sim2 may be important for the pathogenesis of Down's syndrome, especially mental retardation.

Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300.

Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.

Identification of a novel mechanism of regulation of Ah (dioxin) receptor function.

Ah receptor (AhR) is a ligand-activated transcription factor that mediates pleiotropic effects of environmental pollutants such as 2,3, 7,8-tetrachlorodibenzo-p-dioxin on host animals. In addition to induction of drug-metabolizing enzymes, the liganded AhR complex was found to activate gene expression of a factor designated AhR repressor (AhRR), which inhibits AhR function by competing with AhR for dimerizing with Arnt and binding to the XRE sequence. Thus, AhR and AhRR form a regulatory circuit in the xenobiotic signal transduction pathway and provide a novel mechanism of regulation of AhR function that may determine tissue-specific sensitivity to environmental pollutants.