Nuclear Aryl Hydrocarbon Receptor Research Articles

The Jun N-terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor.

Exposure of the immortalized human breast epithelial cell line MCF10A to the Jun N-terminal kinase (JNK) inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) suppressed, in a concentration-dependent manner (IC50 is approximately 2 microM), the induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with SP600125 also suppressed the accumulation of TCDD-induced nuclear aryl hydrocarbon receptor (AhR)-DNA complexes, as assessed by electrophoretic mobility shift assays. Concentrations of SP600125 < or = 50 microM did not transform the AhR into a DNA-binding species when added to rat liver cytosol. However, addition of SP600125 to cytosol just before TCDD addition completely suppressed AhR transformation and DNA binding (IC50 approximately 7 microM). Sucrose gradient analyses using rat liver and murine hepatoma 1c1c7 extracts demonstrated that SP600125 competed with TCDD for binding to the AhR. These results suggest that SP600125 is an AhR ligand and functions as an AhR antagonist at concentrations used to pharmacologically inhibit JNK.

Aint/Tacc3 is highly expressed in proliferating mouse tissues during development, spermatogenesis, and oogenesis.

Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.

Polychlorinated biphenyls affect gene expression in the rabbit preimplantation embryo.

Polychlorinated biphenyls (PCBs) have been shown to be embryotoxic. The mechanism(s) of action is not clearly understood. The toxic effects could be either direct or indirect. Furthermore, PCB congeners vary in their toxic potential. They can be classified in coplanar PCBs binding to the transcription factor aryl hydrocarbon receptor (AhR), which induce subsequent changes in gene expression, and noncoplanar PCBs exhibiting AhR-independent effects. In order to investigate possible mechanisms, 5 and 6 days old preimplantation rabbit embryos were exposed in vitro to low levels of coplanar (PCB 77, 126, and 169) or noncoplanar PCBs (PCB 28, 52, 101, 118, 138, 153, and 180). The PCB effects were studied by semiquantitative RT-PCR analysis of AhR target genes (cytochrome P450 (CYP) 1A1, 1A2, UDP-glucuronosyl transferase 1, glutathione S-transferase pi1 and aldehyde dehydrogenase) and dioxin-responsive genes (IL 1beta, PAI 2, Cox 2, TGFalpha, EGF, erbB 1-4, c-fos, c-jun, HSP 90, cyclophilin 40), and by differential display (DD) RT-PCR. CYP 1B1 mRNA and AhR protein were localized by in situ hybridization and immunohistochemistry, respectively. From the AhR target genes studied only CYP 1B1, and cyclooxygenase 2 showed an increase in mRNA levels after coplanar and noncoplanar PCB. Interleukin 1beta and plasminogen activator inhibitor 2 were downregulated. CYP 1B1 mRNA showed a stage specific inducibility at day 6, but not at day 5. By DD RT-PCR we identified six new genes previously not reported to be regulated by PCBs. The mRNAs encoding the subunits 1, 2, 4, and 5 of the NADH ubiquinone oxidoreductase and beta-globin showed a decrease, whereas trichohyalin mRNA was increased after PCB exposure. Coplanar and noncoplanar PCB congeners elicited similar responses on the mRNA levels of the studied genes. Exposure to coplanar PCBs did not result in the AhR being translocated to the nucleus. Our results show that (i). PCBs induce changes in gene expression in rabbit day 5 and 6 preimplantation embryos and imply (ii). that the transcriptional changes observed were not mediated by the nuclear AhR.

Identifying toxic mechanisms using DNA microarrays: evidence that an experimental inhibitor of cell adhesion molecule expression signals through the aryl hydrocarbon nuclear receptor

Microarray analysis of gene expression has become a powerful approach for exploring the biological effects of drugs and other chemicals. In toxicology research, gene expression profiling may help identify hazards by comparing results for an experimental compound with a database, and establish mechanistic hypotheses through examination of discrete gene changes. Here we examine the hepatic effects of a thienopyridine inhibitor of NF-κB-mediated expression of cellular adhesion proteins. In a 3-day toxicity study in Sprague–Dawley rats, A-277249 induced hypertrophy of the liver and elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). To investigate mechanism, microarray analysis was done on RNA from livers of A-277249-treated rats. Gene expression profiles from A-277249 were compared with a database of profiles from fifteen known hepatotoxins. Agglomerative hierarchical cluster analysis showed A-277249 to have a profile most similar to the aromatic hydrocarbons Aroclor 1254 and 3-methylcholanthrene (3MC), two known activators of the aryl hydrocarbon nuclear receptor (AhR). Several genes regulated by the AhR, including cytochrome P450 1A1, were upregulated by A-277249. In addition, several genes involved in apoptosis and cell cycle were differentially expressed consistent with cell turnover, hypertrophy and hyperplasia observed by histology. Results from this study indicate that A-277249 hepatic toxicity is mediated by the AhR either directly or through effects on NF-κB, and demonstrate the utility of microarray analysis for the rapid identification of toxic hazards for new chemical entities.

A cycloheximide-sensitive factor regulates TCDD-induced degradation of the aryl hydrocarbon receptor

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD), a prototype of environmental halogenated aromatic hydrocarbons, induces a rapid reduction in steady state aryl hydrocarbon receptor (AhR). Here, we analyzed the biochemical pathway and function of the downregulation. Our results reveal that TCDD downregulates the AhR protein by shortening the halflife of AhR. The TCDD-induced degradation of AhR is inhibited by MG132, a potent inhibitor of the 26S proteasome, indicating the ubiquitin-26S proteasome mediated proteolysis as a mechanism for the degradation of AhR. Furthermore, inhibition of protein synthesis by cycloheximide blocks the degradation of AhR by TCDD, suggesting a labile factor in controlling the stability of ligand-activated AhR (hence, designated as AhR degradation promoting factor, or ADPF). Analyses of nuclear AhR demonstrated that cycloheximide increases nuclear AhR protein and functional AhR/Arnt DNA-binding complex, resulting in superinduction of CYP1A1. Lastly, genetic analyses by using AhR- or Arnt-defective variant cells demonstrate that superinduction by cycloheximide requires the transcription activation (TA) domain of AhR, implicating the TA domain in the control of AhR turnover by ADPF. These findings provide new insights into the mechanism by which TCDD-activated AhR is regulated in nucleus through the 26S proteasome protein degradation pathway.

Involvement of a post-transcriptional mechanism in the inhibition of CYP1A1 expression by resveratrol in breast cancer cells

Resveratrol decreases basal and induced CYP1A1 mRNA/protein levels in both in vitro and in vivo models, and some studies suggest that resveratrol acts as an aryl hydrocarbon receptor (AhR) antagonist. Treatment of T47D or MCF-7 cells with 10 μM resveratrol inhibited induction of CYP1A1 mRNA and CYP1A1-dependent activity after treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), as previously reported. In contrast, resveratrol did not inhibit TCDD-induced reporter gene activity in cells transfected with an Ah-responsive construct containing a human CYP1A1 gene promoter insert, whereas 3′-methoxy-4′-nitroflavone, a “pure” AhR antagonist, inhibited this response. Resveratrol induced transformation of the rat cytosolic AhR and, after treatment of T47D and MCF-7 cells with resveratrol, a transformed nuclear AhR complex was observed. In contrast to 3′-methoxy-4′-nitroflavone, resveratrol did not block TCDD-induced AhR transformation in vitro or nuclear uptake of the AhR complex in breast cancer cells. Thus, the action of resveratrol on the AhR was consistent with that of an AhR agonist; however, resveratrol did not exhibit functional AhR agonist or antagonist activities in breast cancer cells. Actinomycin D chase experiments in T47D cells showed that resveratrol and dehydroepiandrosterone both increased the rate of CYP1A1 mRNA degradation, whereas resveratrol did not affect CYP1A1-dependent activity in cells pretreated with TCDD for 18 hr. These data suggest that resveratrol inhibits CYP1A1 via an AhR-independent post-transcriptional pathway.

Induction of CYP1A1. The AhR/DRE paradigm: transcription, receptor regulation, and expanding biological roles.

The CYP1A1 gene encodes microsomal cytochrome P4501A1 that catalyzes the metabolism of many xenobiotics, including the oxygenation of polycyclic aromatic hydrocarbons (PAH). Induction of CYP1A1 enhances the metabolism of PAHs, and therefore, represents an adaptive response to chemical exposure in mammalian cells. Mechanistic studies reveal an AhR/DRE paradigm for the induction, which involves activation of the aryl hydrocarbon receptor (AhR) by an agonist, dimerization of AhR with the Ah recceptor nuclear translocator (Arnt), followed by binding of the AhR/Arnt heterodimer to the dioxin-responsive enhancer (DRE) and transcription of the gene. The AhR mediated transcription is tightly regulated through, at least, two mechanisms: (a) the cytoplasmic AhR interacts with hsp90 and an immunophilin chaperone AIP for proper folding and receptivity, and (b) the agonist-activated, nuclear AhR is degraded through the ubiquitin-26S proteasome mediated protein turnover, such that the transcription by AhR is controlled at a physiologically adequate level. In addition to CYP1A1 induction, AhR mediates a broad range of biological responses to CYP1A1 inducers, typified by the environmental contaminant dioxin, via modulating gene expression. Thus, mechanistic studies of CYP1A1 induction have provided insights into P450 induction, PAH carcinogenesis, dioxin action, AhR function, and receptor-mediated mammalian gene expression.

Molecular biology of the Ah receptor and its role in carcinogenesis

The aryl hydrocarbon receptor (AhR) is a ligand-activated nuclear transcription factor that mediates responses to toxic halogenated aromatic toxins such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), polynuclear aromatic hydrocarbons, combustion products, and numerous phytochemicals such as flavonoids and indole-3-carbinol (I3C). The nuclear AhR complex is a heterodimer containing the AhR and AhR nuclear translocator (Arnt) proteins, and the molecular mechanism of AhR action is associated with binding of the heterodimer to dioxin responsive elements (DREs) in regulatory regions of Ah-responsive genes. TCDD, a ‘xenodioxin’, is a multi-site carcinogen in several species and possibly in humans, whereas natural AhR ligands including I3C and flavonoids tend to protect against cancer. Both TCDD and phytochemicals inhibit estrogen-induced breast and endometrial cancer, and the molecular mechanisms of this common response will be described.

Estrogen enhances induction of cytochrome P-4501A1 by 2,3,7, 8-tetrachlorodibenzo-p-dioxin in liver of female Long-Evans rats.

A human carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces liver tumors in female rats. In this study, we examined the effects of estrogen on an arylhydrocarbon receptor (AhR)-responsible protein, CYP1A1 expression induced by TCDD in female rat livers. The induction of CYP1A1 by a dose of 300 ng TCDD/kg and its resultant enzymatic activity were significantly enhanced by 5 microg 17beta-estradiol/kg body weight treatment to both intact and ovariectomized rats. Immunoblot analysis showed a increase in nuclear AhR due to estrogen, TCDD or both, suggesting that estrogen is involved in the activation of CYP1A1 gene after the formation of AhR-TCDD complex.

Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex.

The region of residues -145 to -119 (CD/L) of the cathepsin D gene promoter contains a GC-rich motif that binds Sp1 protein and an adjacent pentanucleotide (CACGC) that corresponds to the core sequence of a dioxin responsive element (DRE) and binds the aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (Arnt) complex. This Sp1(N)(4)DRE(core) motif has been identified in promoters of several genes in which Sp1 plays an important role in basal gene expression. In transient transfection assays with MCF-7 human breast cancer cells using wild-type pCD/L and constructs mutated in the core DRE (pCD/L(m1)) and Sp1 (pCD/L(m2)) sites, it was shown that both motifs were required for maximal basal activity. The requirements for AhR-Arnt interactions with Sp1 protein for maximal activity of pCD/L were confirmed in wild-type MCF-7 and Hepa 1c1c7 cells and Arnt-deficient Hepa 1c1c7 cells using antisense Arnt and Arnt expression plasmids. The functional interactions of Sp1 with AhR-Arnt were paralleled by physical interactions showing that AhR-Arnt and Sp1 proteins were co-immunoprecipitated and AhR-Arnt enhanced Sp1-[(32)P]CD/L binding in electrophoretic mobility shift assays. The physical and functional interactions of Sp1 with AhR-Arnt proteins bound to the Sp1(N)(4)DRE(core) motif were also dependent on the proximity of these sites, and both the activity and the extent of Sp1-DNA binding decreased as the number of intervening nucleotides increased from 4 to 20. These studies show that regulation of basal expression of some genes by Sp1 may also require interactions with AhR-Arnt.

Ah receptor agonists as endocrine disruptors: antiestrogenic activity and mechanisms

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds induce a broad spectrum of biochemical and toxic responses and disrupt multiple endocrine pathways. Research in this laboratory has focused on characterizing aryl hydrocarbon receptor (AhR)-mediated antiestrogenicity in the rodent uterus and mammary and in human breast cancer cells. TCDD inhibits multiple estrogen (E2)-induced responses in these tissues including development or growth of human mammary and endometrial cancer cells, carcinogen-induced mammary cancer in rats, and mammary cancer in mice bearing breast cancer cell xenografts. The mechanisms of AhR-mediated antiestrogenicity are complex; however, studies on the molecular biology of cross-talk between the AhR and estrogen-receptor (ER) signaling pathways have been initiated using several E2-regulated genes as models. The results indicate that the nuclear AhR complex targets specific genomic core inhibitory dioxin responsive elements (iDREs) in promoter regions of some E2-responsive target genes to inhibit hormone-induced transactivation. The pS2, cathepsin and c-fos genes have functional iDREs, whereas the iDRE in the progesterone receptor gene promoter was not functional. Research has also focused on development of AhR-based antiestrogens which inhibit mammary tumor development and growth but do not exhibit prototypical AhR-induced toxic responses.

Induction of Endothelial PAS Domain Protein-1 by Hypoxia: Characterization and Comparison With Hypoxia-Inducible Factor-1α

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1α and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1α is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1α, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1α responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1α in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1α nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1α transactivation) of the VEGF promoter than the LDH-A promoter.

Induction of Endothelial PAS Domain Protein-1 by Hypoxia: Characterization and Comparison With Hypoxia-Inducible Factor-1α

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1α and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1α is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1α, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1α responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1α in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1α nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1α transactivation) of the VEGF promoter than the LDH-A promoter.

Expression of the aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) in fetal, benign hyperplastic, and malignant prostate.

Androgen-dependent tissue has been reported to be affected by chemical ligands of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which heterodimerizes with the aryl hydrocarbon receptor nuclear translocator protein (ARNT). Fetal (n = 3), benign hyperplastic (BPH) (n = 10), and carcinomatous (CaP) (n = 19) prostate tissues were analyzed using immunohistochemistry. Western blot analysis was used to confirm the identity of the recognized proteins. Immunoblotting of enriched prostatic epithelial cells (EC) and stromal cells revealed constitutive expression of bands at around 110 kDa and 90 kDa, using anti-AhR and anti-ARNT, respectively. Immunohistology of the fetal specimens revealed heterogeneous cytoplasmic and nuclear AhR expression of immature EC and mesenchymal cells. Constitutive expression of AhR (primarily cytoplasmic) and ARNT (nuclear and cytoplasmic) by the majority of adult basal and secretory EC, CaP, and smooth muscle cells was confirmed in situ. The most intense anti-AhR/-ARNT reactivity was found on smooth muscle cells, followed by EC and fibrocytes. Secretory BPH-EC revealed significantly decreased AhR expression when compared to normal tissue segments. By contrast, anti-AhR reactivity was frequently increased in the more dedifferentiated tumor areas. These findings suggest that an undefined physiologic AhR ligand(s) as well as environmental factors may exert effects on EC and smooth muscle cells in the prostate through binding to these receptors.

Mechanisms of ligand-induced aryl hydrocarbon receptor-mediated biochemical and toxic responses.

The ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a member of a broad group of halogenated aromatic hydrocarbons (HAHs) that is known to induce a wide range of toxic and biochemical responses in laboratory animals and humans. The effects of HAH exposure are mediated by binding to the cytosolic aryl hydrocarbon receptor (AhR), which is expressed in a tissue- and cell type-specific manner. The AhR is a ligand-activated transcription factor belonging to the basic helix-loop-helix/Per-AhR-Arnt-Sim (bHLH/PAS) superfamily of proteins. The mechanism of induction of gene transcription by TCDD involves ligand recognition and binding by the AhR, nuclear translocation, and dimerization with the AhR cofactor, AhR nuclear translocator (Arnt). The nuclear heterodimer interacts with cognate xenobiotic responsive elements (XREs) in promoter/enhancer regions of multiple Ah-responsive genes. Subsequent changes in chromatin structure and/or interaction of the AhR complex with the basal transcriptional machinery play a significant role in AhR-mediated gene expression. Although Arnt is a necessary component of a functional nuclear AhR complex, this protein also forms transcriptionally active heterodimers with other bHLH/PAS factors, including those involved in the transcriptional response to hypoxia. Arnt is ubiquitously expressed in mammalian systems, and results from transgenic mouse studies suggest that this protein plays a vital role in early mammalian embryonic development. Similar experiments suggest that the AhR may be involved in development of various organ systems. Thus, molecular mechanistic studies of TCDD action have contributed significantly to an improved understanding of the role of at least 2 bHLH/PAS proteins, as well as organ- and tissue-specific biochemical and toxic responses to this class of environmental toxins.

Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane.

Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of cruciferous vegetables which exhibit antitumorigenic activity associated with altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with 10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and induction of CYP1A1 gene expression was observed at concentrations >50 microM. Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats. Results of this study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells, reporter gene activity in cells transiently transfected with an E2-responsive plasmid (containing a frog vitellogenin A2 gene promoter insert) and down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and this was not accompanied by induction of hepatic CYP1A1-dependent activity. Thus, DIM represents a new class of relatively non-toxic AhR-based antiestrogens that inhibit E2-dependent tumor growth in rodents and current studies are focused on development of analogs for clinical treatment of breast cancer.

Depletion of Arylhydrocarbon Receptor during Adipose Differentiation in 3T3-L1 Cells

Arylhydrocarbon receptor (AhR) is the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although a physiological ligand for AhR has yet to be identified, several lines of evidence suggest that AhR may play an important role not only in the regulation of xenobiotic metabolism but also in the maintenance of homeostatic functions. When TCDD is administrated in vivo, this compound is primarily deposited in adipose tissue. Therefore, it is critical to know the states of AhR in adipose cells for assessing the expression of toxicities of TCDD and related compounds in vivo. In this report, we examined the levels of AhR protein and its associated protein (Arnt) during the adipose differentiation in 3T3-L1 cells. The level of AhR protein was found to decrease with ongoing adipose differentiation in 3T3-L1 cells. The binding activity to the xenobiotic response element and the cellular response to TCDD were also lowered as a result of adipose differentiation. These results indicate that the depletion of AhR is a novel event associated with adipose differentiation in 3T3-L1 cells and that the magnitude of the depletion of AhR is sufficient for 3T3-L1 cells to lose the functional response to xenobiotics. We also found a population of 3T3-L1 cells which have an adipose differentiation capability in the presence of high doses of TCDD. These cells lack nuclear AhR but not cytoplasmic AhR, suggesting a possible negative role of liganded nuclear AhR in adipose differentiation. The level of the Arnt protein also decreased as a result of the differentiation. However, the pattern of the depletion of the Arnt protein was distinct from that of the AhR protein. The data presented in this study will provide opportunities to carry out studies to better understand the roles of AhR in adipose cells which are the primary targets of TCDD.

Functional and physical interactions between the estrogen receptor Sp1 and nuclear aryl hydrocarbon receptor complexes.

17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells and previous analyses of the proximal promoter region of this gene identified two functional enhancer sequences; namely an Sp1(N)23estrogen-responsive element (ERE) half-site (-199 to -165) and an imperfect palindromic ERE (-119 to -107). A third region of the cathepsin D gene promoter (CD/L, -145 to -119) was also E2 responsive in transient transfection assays. A GC-rich sequence which contains two overlapping Sp1 binding sites (-145 to -135) was responsible for ER-mediated transactivation and required formation of an ER/Sp1 complex in which only the Sp1 protein bound DNA. E2 responsiveness of the CD/L sequence was also dependent on an adjacent overlapping GCGTG motif corresponding to the dioxin-responsive element (DRE) core binding sequence, which is the cognate response element for the heterodimeric aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) transcription factor complex. The results show that ER-mediated transactivation of CD/L was associated with the Sp1(N)2-4DRE (core) motif and involved formation of a multiprotein ER/Sp1-AhR/ARNT complex. These results illustrate a unique example of an endogenous role for AhR/ARNT in the absence of added AhR agonist and indicate that the cathepsin D gene proximal promoter region contains at least three different functional motifs associated with ER-mediated transactivation.

Characterization of the Aryl Hydrocarbon Receptor Complex in Human B Lymphocytes: Evidence for a Distinct Nuclear DNA-Binding Form

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses B lymphocyte proliferation and immunoglobulin production. We previously reported that the aryl hydrocarbon receptor (AhR) complex, composed of the AhR ligand binding subunit and the Ah receptor nuclear translocator (ARNT), was constitutively present in nuclear extracts from two human B lymphocyte cell lines (Biochem. Biophys. Res. Commun.212, 27–34, 1995). The present study compared the AhR complex in the IM-9 and PJS-91 human B lymphocyte and HepG2 human hepatoma cell lines. AhR mRNA levels in the two lymphocyte cell lines were substantially lower than those in HepG2 cells, as was immunoreactive AhR protein. In contrast, ARNT mRNA and protein were expressed at a high level in all three cell lines. TCDD induction of cytochrome P450 1A1 mRNA and protein was detected in only the PJS-91 lymphocyte cell line, and at a markedly lower level than that in HepG2 cells. In gel shift assays, the cytosolic DNA-binding AhR complex in IM-9 and PJS-91 cells was indistinguishable from that in HepG2 cells. In contrast, the nuclear DNA-binding AhR complex in IM-9 and PJS-91 cells consisted of several closely migrating species, one being recognized by an AhR antibody, while an ARNT antibody reacted with all species. Protein:DNA cross-linking analysis revealed the presence of a novelMr100,000 DNA-binding protein in nuclear extracts from IM-9 and PJS-91, but not HepG2, cells that was not recognized by either AhR or ARNT antibodies. These results show that IM-9 and PJS-91 human B cells constitutively express a distinct nuclear DNA-binding form of the AhR complex that may result from the presence of an additional protein or a structural variant of the AhR.

Protein Kinase C Modulates Regulation of the CYP1A1 Gene by the Aryl Hydrocarbon Receptor

Transcriptional activation of the human CYP1A1 gene by halogenated and polycyclic aromatic hydrocarbons is mediated by the aryl hydrocarbon receptor (AhR) complex, a ligand-dependent transcription factor. A competent AhR comprises at least two components following nuclear translocation and DNA binding, the AhR and the AhR nuclear translocator (Arnt) protein, whose combined action on human CYP1A1 gene transcription is shown to be dependent upon functional protein kinase C (PKC). In the present study, we examined the effects of phorbol 12-myristate 13-acetate, a potent PKC activator, on the ligand-induced transcriptional activation of the CYP1A1 gene and cellular function of the AhR in human HepG2 101L cells. The 101L cells carry a stable transgene consisting of 1800 bases of 5'-flanking DNA and the promoter of the human CYP1A1 gene linked to the firefly luciferase structural gene (Postlind, H., Vu, T. P., Tukey, R. H. & Quattrochi, L. C. (1993) Toxicol. Appl. Pharmacol. 118, 255-262). Pretreatment of cells with 12-myristate 13-acetate enhanced ligand-induced CYP1A1 gene expression 2-3-fold. Inhibition of PKC activity blocked directly the transcriptional activation and the transactivation of the CYP1A1 gene, indicating a role for PKC in the AhR-mediated transcriptional activation process. However, the DNA binding activities of the in vitro activated and the induced nuclear AhR as measured by electrophoretic mobility shift analysis were not affected when CYP1A1 transcription was inhibited, indicating the actions of PKC to be a nuclear event that works in concert with or precedes AhR binding to the gene. These results illustrate that PKC is absolutely essential for the cellular and molecular events that control induction of CYP1A1 gene transcription.