Identification of a novel C-terminal domain involved in the negative function of the rainbow trout Ah receptor nuclear translocator protein isoform a (rtARNTa) in Ah receptor-mediated signaling.

Rainbow trout aryl hydrocarbon receptor (AHR) nuclear translocator isoform a (rtARNTa) has a negative function in AHR-mediated signal transduction. Previous analyses suggest that the negative function is at the level of DNA binding and may be due to the presence of 57 C-terminal amino acids that are strongly hydrophobic. To assess the negative activity of rtARNTa at the molecular level, hydrophobic-rich domains corresponding to amino acids 601-637, 601-631, and 616-631 were analyzed for the ability to affect the function of truncated rtARNT proteins in complementation and gel shift assays. Addition of the hydrophobic-rich domains to these proteins reduced their ability to complement AHR-mediated signal transduction in mouse hepatoma cells by 65-95%. The decrease in function was related to a reduced ability of the AHR. rtARNT complex to bind DNA and not due to a lack of dimerization with AHR. Expression of the hydrophobic-rich domains on Gal4 proteins showed that the C-terminal domain of rtARNTa was unlikely to contain transactivation function; however, the hydrophobic domains reduced the ability of the Gal4 proteins to bind DNA. Immunoprecipitation and mutational experiments indicate that the hydrophobic-rich domains do not interact with the bHLH motif of AHR. Interestingly, immunoprecipitation experiments also revealed that the C-terminal hydrophobic-rich region of rtARNTa could oligomerize in vitro in a chimera with the Gal4 DNA binding domain. These findings indicate that the C-terminal hydrophobic amino acids are critical for the negative function of rtARNTa in AHR-mediated signaling and suggest that multiple mechanisms may be involved in the repression of DNA binding.

An aryl hydrocarbon receptor independent mechanism of JP-8 jet fuel immunotoxicity in Ah-responsive and Ah-nonresponsive mice.

JP-8 jet fuel is handled extensively by personnel in the military and commercial airlines, despite the paucity of information regarding its potential human health effects. JP-8 is a complex mixture primarily consisting of kerosene plus aliphatic and aromatic hydrocarbons. Recent reports indicate that acute JP-8 exposure via inhalation or dermal routes can overtly and persistently impair immune function in mice. Data from preliminary studies in this laboratory assessing the immunotoxicity of JP-8 indicated that oral JP-8 exposure caused an increase in liver weight, a decrease in thymus weight, and a decrease in the PFC response. As these results were similar to classic effects elicited by TCDD, a strong AhR ligand, it was hypothesized that JP-8 may exert immunosuppression via a similar mechanism. To test this hypothesis, an Ah-responsive mouse strain (B6C3F1) and a classically non-responsive mouse strain (DBA/2) bearing a lower affinity AhR were gavaged with JP-8 for 7 days. The results suggest that both mouse strains were equally sensitive to JP-8's toxicity at several endpoints including thymus weight and cellularity, liver weight, and specific IgM antibody responses. Furthermore, JP-8 did not induce CYP1A1 or promote down regulation of the AhR when evaluated by Western blot in either B6C3F1 or DBA/2 mice. In vitro studies corroborated these findings as JP-8 did not induce CYP1A1, promote down regulation of the AhR, or activate an XRE-driven reporter gene in murine Hepa-1 cells. These results suggest that JP-8 may exert its toxicity via an AhR-independent mechanism.

Expression of aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms in juvenile and adult rainbow trout tissues.

Whether and where rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNT) isoforms are expressed in juvenile and adult tissues of rainbow trout is unknown. Using reverse transcriptase polymerase chain reaction (RT-PCR), expression of the rtARNT(b) isoform messenger RNA was identified in day 19 and 23 embryos, in day 27, 35, 39, and 42 sac fry, and in all adult tissues investigated. The rtARNT(a) isoform mRNA was expressed in all juvenile trout except day 42 sac fry and in all adult tissues except skeletal muscle. Western blot analysis and immunohistochemistry demonstrated that the rtARNT(b) protein was present in all juvenile trout and adult tissues investigated, except skeletal muscle, and was primarily localized to the nucleus. In contrast, rtARNT(a) protein was not detected at any developmental stage but was expressed in the adult gill. These results imply that rtARNT(b) is involved in signaling events at many developmental stages, while the functionality of the dominant negative rtARNT(a) is restricted.

Identification and characterization of a cDNA clone encoding the heat shock protein (Hsp60) from the biting midge, Culicoides variipennis sonorensis Wirth and Jones.

A cDNA expression library constructed from Culicoides variipennis sonorensis was screened using an antibody specific for Hsp60 of Heliothis virescens. A single clone encoding the complete heat shock protein (Hsp60) of C. variipennis was identified and its 2400-bp insert was sequenced. The encoded 62-kDa protein contains 581 amino acids and includes a 26-amino acid putative mitochondrial targeting sequence at its N terminus and a GGM motif at its carboxyl terminus. Deduced amino acid sequences are highly similar (67-78%) to Hsp60 of other species, including the fruit fly, the house mouse, the Norwegian rat, the Chinese hamster, the human, a nematode, and the tobacco budworm moth. This is the initial isolation of a coding sequence for a stress-induced protein in C. variipennis.

Expression and subcellular localization of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein in mouse and chicken over developmental time.

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.

Analysis of the complex relationship between nuclear export and aryl hydrocarbon receptor-mediated gene regulation.

The aryl hydrocarbon receptor (AHR) contains signals for both nuclear import and nuclear export (NES). The purpose of the studies in this report was to determine the relationship between the nuclear export of the AHR and AHR-mediated gene regulation. Blockage of nuclear export in HepG2 cells with leptomycin B (LMB) resulted in increased levels of AHR-AHR nuclear translocator (ARNT) complex in the nucleus and correlative reductions in agonist-stimulated AHR degradation. However, LMB exposure inhibited agonist-mediated induction of numerous AHR-responsive reporter genes by 75 to 89% and also inhibited induction of endogenous CYP1A1. LMB did not transform the AHR to a ligand binding species or affect activation by TCDD (2, 3,7,8-tetrachlorodibenzo-p-dioxin). Mutagenesis of leucines 66 and 71 of the putative AHR NES resulted in a protein with reduced function in dimerization to ARNT and binding to DNA, while alanine substitution at leucine 69 (AHR(A69)) resulted in an AHR that bound with ARNT and associated with DNA. AHR(A69) protein injected directly into the nuclei of E36 cells remained nuclear following 6 h of agonist stimulation. In transient-transfection assays, AHR(A69) accumulated within the nucleus was not degraded efficiently following agonist exposure. Finally, AHR(A69) supported induction of AHR-responsive reporter genes in an agonist-dependent manner. These findings show that it is possible to generate an AHR protein defective in nuclear export that is functional in agonist-mediated gene induction. This implies that the negative effect of LMB on agonist-mediated gene induction is independent of the nuclear export of the AHR.

Role of heat shock protein 90 dissociation in mediating agonist-induced activation of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a cytosolic basic helix-loop-helix protein that associates with a chaperone complex that includes two molecules of heat shock protein 90 (HSP90). It has been hypothesized that after ligand binding, the AhR dissociates from its chaperone complex and translocates into the nucleus, where it heterodimerizes with its DNA binding partner, the AhR nuclear translocator (ARNT), and activates specific genes. However, it remains unclear whether nuclear translocation of the AhR occurs before or after dissociation of the HSP90/chaperone complex. Because sodium molybdate stabilizes the AhR-HSP90 interaction and inhibits the gene activation of a number of steroid receptors, we reasoned that molybdate would be a useful tool in delineating the role of HSP90 dissociation in AhR nuclear translocation. In this study, we demonstrate that molybdate inhibits AhR gene activation in both HepG2 and Hepa-1 cells in a concentration-dependent manner and protects the AhR against agonist-induced proteolysis. In addition, we demonstrate that AhR/ARNT dimerization, but not nuclear translocation of the AhR, is inhibited by molybdate. This indicates that 1) HSP90 dissociation is not required for nuclear translocation of the AhR, 2) HSP90 dissociation is essential for formation of the AhR/ARNT heterodimer, and 3) an additional undefined regulatory step is required for AhR/ARNT dimerization in the nucleus.

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Analysis of aryl hydrocarbon receptor-mediated signaling during physiological hypoxia reveals lack of competition for the aryl hydrocarbon nuclear translocator transcription factor.

The aryl hydrocarbon nuclear translocator (ARNT) protein functions as a transcription factor after dimerization with other basic helix-loop-helix proteins. Thus, dimerization of ARNT within one pathway may limit the availability of this protein to others. To investigate this issue, aryl hydrocarbon receptor (AHR)-mediated signaling was investigated in mouse (Hepa-1), rat (H4IIE), and human (HepG2) hepatoma cell lines undergoing physiologically induced hypoxia (<1% O(2)). Basal levels of ARNT protein were not affected by hypoxia in any cell line, and ARNT remained exclusively nuclear. Furthermore, quantitative Western blotting revealed that the concentration of ARNT sequestered during hypoxia represented a small fraction of the total ARNT protein pool (12 and 15% in Hepa-1 and H4 cells, respectively). When the AHR-mediated signaling pathway was activated during hypoxia by 2,3,7,8-tetrachlorodibenzo-p-dioxin, the induction of P4501A1 protein was reduced by 55% without changes in the level of mRNA in Hepa-1 cells, whereas the levels of induction of both P4501A1 protein and CYP1A1 mRNA were reduced by >80% in the H4 cell line. Importantly, gel mobility shift analysis and Western blotting showed that the same level of AHR/ARNT complexes could be detected in cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin during hypoxia and normoxia. These data suggest that the effects of hypoxia on AHR-mediated gene regulation occur distal to the formation of AHR/ARNT complexes and imply that functional interference between hypoxia and AHR-mediated signaling does not occur through competition for ARNT protein.

Aryl hydrocarbon receptor imported into the nucleus following ligand binding is rapidly degraded via the cytosplasmic proteasome following nuclear export.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that dimerizes with the AHR nuclear translocator protein to mediate gene regulation. However, the AHR protein is rapidly depleted in vitro and in vivo following exposure to ligands. The purpose of the studies in this report was to characterize the mechanism of AHR degradation and determine the consequence of blocking the degradation process. Western blot and immunological analysis of rat smooth muscle (A7), murine Hepa-1, and human HepG2 cells show that ligand-induced degradation of AHR is blocked when the proteasome is inhibited by MG-132. AHR degradation is also blocked in Hepa-1 and HepG2 cells when nuclear export is inhibited with leptomycin B. Mutation of a putative nuclear export signal present in the AHR results in the accumulation of AHR in the nucleus and reduced levels of degradation following ligand exposure. In addition, inhibition of AHR degradation results in an increase in the concentration of AHR.AHR nuclear translocator complexes associated with DNA and extends the duration that the complex resides in the nucleus. These findings show that nuclear export and degradation of the AHR protein are two additional steps in the AHR-mediated signal transduction pathway and suggest novel areas for regulatory control.

Flavone antagonists bind competitively with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation.

Previous analyses suggested that potent aryl hydrocarbon receptor (AhR) antagonists were planar, with a lateral electron-rich center. To further define structural requirements and mechanism for antagonism, ten additional flavone derivatives were synthesized. Based on their ability to 1) compete with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the AhR; 2) inhibit TCDD-elicited binding of AhR to dioxin-responsive elements (DRE) in vitro; and 3) inhibit TCDD-induced transcription of DRE-dependent luciferase in stably transfected hepatoma cells, the most potent flavones contained a 3'-methoxy group and a 4'-substituent having one or more terminal atoms of high electron density (-N3, -NO2, or -NCS). Furthermore, these had low agonist activity as assessed by their inability to elicit AhR. DRE binding or to induce luciferase. Compounds containing bulkier 3' or 4'-substituents, or a 3'-OH group were less potent antagonists, and some were partial agonists. In rat liver cytosol, 3'-methoxy-4'-azido- and 3'-methoxy-4'-nitroflavones bound competitively (with TCDD) to the AhR, indicating that they bind to the TCDD-binding site. When hepatoma cells were exposed to these flavones, AhR complexes were primarily immunoprecipitable from the cytosol and contained 90 kDa heat shock protein. In contrast, AhR in TCDD-treated cells was primarily immunoprecipitated from nuclear extracts and was associated with Arnt but not 90 kDa heat shock protein. Immunocytofluorescence analysis in intact cells further indicated that the potent antagonist inhibited nuclear uptake of AhR and blocked TCDD-dependent down-regulation of AhR. Together, these data indicate that the most potent antagonists bind the AhR with high affinity but cannot initiate receptor transformation and nuclear localization.

Ah receptor and ARNT protein and mRNA concentrations in rat prostate: effects of stage of development and 2,3,7, 8-tetrachlorodibenzo-p-dioxin treatment.

Effects of stage of development and 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) protein concentrations in reproductive organs of male rats were determined. AhR protein levels in developing rat ventral and dorsolateral prostate decreased with age, declining approximately 70% between Postnatal Days (PND) 1 and 21. ARNT protein levels also decreased with age in dorsolateral, but not ventral prostate. The developmental decreases in prostatic AhR and ARNT protein were associated with decreases in AhR and ARNT mRNA. AhR and ARNT protein concentrations in fetal urogenital sinus on Gestation Days (GD) 16, 18, and 20 were similar to levels in ventral prostate on PND 7. TCDD exposure of adult male rats (0.2, 1, 5, or 25 micrograms/kg po, 24 h) decreased AhR but not ARNT protein in ventral and dorsolateral prostate, vas deferens, and epididymis. In utero and lactational TCDD exposure (1.0 micrograms/kg dam po, GD 15) did not alter ARNT levels but reduced prostatic AhR protein levels on PND 7 and delayed the developmental decrease in AhR protein in ventral and dorsolateral prostate. Finally, pretreatment of rat pups for 24 h with TCDD (5 micrograms/kg ip) down-regulated prostatic AhR protein on PND 7, but not on PND 1. Thus, prostatic AhR and ARNT protein and mRNA levels are regulated with age, whereas only AhR protein concentration is altered by TCDD exposure. Because in utero and lactational TCDD exposure only decreased prostatic AhR on PND 7, it is unlikely that down-regulation of AhR is the mechanism by which perinatal TCDD exposure impairs prostate development.

Functional analysis of activation and repression domains of the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNT) protein isoforms.

The aryl hydrocarbon receptor nuclear translocator (ARNT) protein is involved in many signaling pathways. Rainbow trout express isoforms of ARNT protein that are divergent in their C-terminal domains due to alternative RNA splicing. Rainbow trout ARNT(b) (rtARNT(b)) contains a C-terminal domain rich in glutamine and asparagine (QN), whereas the C-terminal domain of rtARNT(a) is rich in proline, serine, and threonine (PST). rtARNT(b) functions positively in AH receptor-mediated signaling, whereas rtARNT(a) functions negatively. Studies were performed to understand how changes in the C-terminal domains of the two rtARNT isoforms affect function. Deletion of the QN-rich C-terminal domain of rtARNT(b) did not affect function in aryl hydrocarbon receptor (AHR)-mediated signaling, whereas deletion of the PST-rich domain of rtARNT(a) restored function. Expression of the PST-rich domain on truncated rtARNT(b) or mouse ARNT (mARNT) reduced function of this protein by 50-80%. Gel shift assays revealed that the PST-rich domain affected AHR-mediated signaling by inhibiting DNA binding of the AHR*ARNT heterodimer. Gal4 transactivation assays revealed a potent transactivation domain in the QN-rich domain of rtARNT(b). In contrast, Gal4 proteins containing the PST-rich domain of rtARNT(a) did not transactivate because the proteins did not bind to DNA. Secondary structure analysis of the PST-rich domain revealed hydrophilic and hydrophobic regions. Truncation of the hydrophobic domain that spanned the final 20-40 amino acids of the rtARNT(a) restored function to the protein, suggesting that repressor function was related to protein misfolding or masking of the basic DNA binding domain. Functional diversity within the C-terminal domain is consistent with other negatively acting transcription factors and illustrates a common biological theme.

Responsiveness of the adult male rat reproductive tract to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: Ah receptor and ARNT expression, CYP1A1 induction, and Ah receptor down-regulation.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) either in adulthood or during late fetal and early postnatal development causes a variety of adverse effects on the male rat reproductive system. It was therefore of interest to identify male rat reproductive organs and cell types within these organs that might be direct targets of TCDD exposure. Because TCDD toxicity could possibly be the result of alterations in gene transcription mediated by the TCDD/aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex, the presence of the AhR and ARNT in the various organs of the adult male reproductive tract was examined using Western blotting. Both proteins were detectable in all organs examined (testis, epididymis, vas deferens, ventral prostate, dorsolateral [combined dorsal and lateral] prostate, and seminal vesicle). Although technical difficulties precluded the immunohistochemical evaluation of AhR distribution in these organs, ARNT was localized in all organs in a variety of cell types, including germ cells, epithelial cells, fibroblasts, smooth muscle cells, and endothelial cells. Subcellular localization varied across organs and across cell types within these organs. In order to determine whether TCDD exposure could alter gene expression in these organs, animals were dosed with TCDD (25 micrograms/kg po) or vehicle and euthanized at 24 h, and cytochrome P4501A1 (CYP1A1) expression was evaluated. By Western blotting, only the ventral and dorsolateral prostates exhibited significant induction of CYP1A1. Immunohistochemistry confirmed this induction and localized CYP1A1 expression to epithelial cells of the ventral and lateral lobes of the prostate. Immunohistochemistry also revealed CYP1A1 induction in select epithelial cells in the epididymis and seminal vesicle, as well as endothelial cells in the vas deferens and seminal vesicle. No induction was observed in the testis. Finally, AhR and ARNT expression in TCDD-exposed and control animals was evaluated by Western blotting. Results revealed no effect of TCDD exposure on ARNT protein expression, while AhR expression was decreased to 5-51% of control in all organs examined. In summary, both AhR and ARNT were expressed in all organs of the adult male rat reproductive tract examined, and epithelial and/or endothelial cells within each of these organs (with the exception of the testis) were responsive to TCDD exposure in terms of CYP1A1 induction. In addition, all tissues exhibited marked reductions in AhR protein content after TCDD exposure that did not correlate with the magnitude of the CYP1A1 response.

Female Sprague-Dawley rats exposed to a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibit sustained depletion of aryl hydrocarbon receptor protein in liver, spleen, thymus, and lung.

There is currently little information concerning the time-dependent relationship between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) protein concentration in vivo. Therefore, female Sprague-Dawley rats were given a single oral dose of TCDD (10 micrograms/kg), and the AHR and ARNT protein concentrations in liver, spleen, thymus, and lung determined by Western blotting. In liver, the concentration of AHR protein was significantly reduced 8 and 24 h postdosing as compared to time-matched controls. In spleen and lung, the concentration of AHR protein was reduced 3, 8, 24, and 168 h posttreatment compared to time-matched controls but returned to control levels by 336 h. In thymus, reductions in AHR protein concentration were observed 8, 24, 168, and 336 h postdosing as compared to time-matched controls. Significant reductions in the concentration of ARNT protein were not observed in any of the TCDD-exposed tissues. Functional studies in cell culture showed that exposure of a mouse hepatoma cell line (Hepa-1c1c7) and a rat smooth muscle cell line (A-7) to TCDD (1 nM) for 12 days resulted in a 50% reduction in TCDD-inducible reporter gene expression following subsequent challenge by an additional dose of TCDD (1 nM). Collectively, these results show that (i) TCDD-mediated depletion of AHR occurs in vivo, (ii) AHR protein does not rapidly recover to pretreatment levels even though the tissue concentration of TCDD has fallen, and (iii) reduction in AHR protein concentration correlates with reduction in TCDD-mediated reporter gene expression in mammalian culture cells.

Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates.

Western blot analysis was used to determine the concentration of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and aryl hydrocarbon receptor (AHR) in 11 mammalian cell culture lines derived from hepatic and nonhepatic tissues. The strategy was to first use Western blot analysis to determine the expression of ARNT or AHR in each cell line relative to its concentration in murine wild-type Hepa-1c1c7 (Hepa-1) cells. Actual ARNT and AHR concentrations in known amounts of total cell lysates were then determined by generating a standard curve with defined amounts of a highly purified ARNT or AHR protein and performing regression analysis. The results show that the level of ARNT expression in each of the cell lines is similar and represents approximately 0.001-0.002% of total cellular protein. The range of expression was only approximately 3-fold with wild-type Hepa-1 cells expressing the highest level of ARNT (33,000/cell) and canine kidney cells (MDCK line) expressing 14,000 ARNT molecules/cell. In contrast, the concentration of AHR varied by 65-fold over the different cell lines with the wild-type Hepa-1 expressing 323,000 AHR/cell and rat hepatoma cells (H4IIE) expressing 4700. The ratio of AHR to ARNT ranged from 0.3 in H4IIE cells to 10 in the Hepa-1 line with the majority of cells expressing 1-5 times more AHR than ARNT protein. Immunocytochemical staining of each cell line showed that ARNT was exclusively localized to the nuclear compartment and that a conserved nuclear localization signal mapped to the NH-terminal portion of the protein.

Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced heart defects.

We examined cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the chick embryo and the cardiac expression of transcription factors, the aryl hydrocarbon receptor (AhR) which binds TCDD, and its dimer partner, the AhR nuclear translocator (Arnt). Chicken eggs were injected with control (triolein) or 1.0 pmol TCDD/g egg prior to incubation and collected on Day 10 when cardiomorphogenesis is complete. Relative to controls, TCDD increased heart wet weight (27.2 +/- 0.5 versus 36.6 +/- 1.3 mg, p < 0.001) and dry weight (2.7 +/- 0.1 versus 3.1 +/- 0.1 mg, p < 0.01), and tended to increase heart myosin content (3.5 +/- 0.6 versus 6.3 +/- 2.5 microg, p < 0.07), suggesting an increase in cardiac muscle mass and edema. Histologic and morphometric analyses revealed that 10/13 TCDD-exposed hearts exhibited enlarged right and left ventricles, thickened ventricular septum, and a thinner left ventricular wall with increased trabeculation, and 4/13 exhibited ventricular septal defects compared to controls (0/23). To evaluate AhR and Arnt expression, untreated chick embryos were collected on Days 2.2, 3, 4, 5, and 8 of incubation, preserved in Bouin's fixative, sectioned, and stained with AhR and Arnt antibodies. The AhR was expressed ubiquitously in cardiac myocytes, while Arnt expression was restricted to myocytes overlying developing septa: atrioventricular canal, outflow tract, and atrial and ventricular septa. Both proteins were absent from endocardium and endocardial-derived mesenchyme. In addition, cardiac expression of an AhR/Arnt target, cytochrome P4501A1, was restricted to myocardium coexpressing AhR and Arnt. Thus, the spatial and temporal expression of AhR and Arnt suggests that the developing myocardium and cardiac septa are potential targets of TCDD-induced teratogenicity, and such targets are also consistent with cardiac hypertrophy and septal defects observed following TCDD exposure.

Isolation and expression of cDNAs from rainbow trout (Oncorhynchus mykiss) that encode two novel basic helix-loop-Helix/PER-ARNT-SIM (bHLH/PAS) proteins with distinct functions in the presence of the aryl hydrocarbon receptor. Evidence for alternative mRNA splicing and dominant negative activity in the bHLH/PAS family.

cDNAs encoding two distinct basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) proteins with similarity to the mammalian aryl hydrocarbon nuclear translocator (ARNT) protein were isolated from RTG-2 rainbow trout gonad cells. The deduced proteins, termed rtARNTa and rtARNTb, are identical over the first 533 amino acids and contain a basic helix-loop-helix domain that is 100% identical to human ARNT. rtARNTa and rtARNTb differ in their COOH-terminal domains due to the presence of an additional 373 base pairs of sequence that have the characteristics of an alternatively spliced exon. The presence of the 373-base pair region causes a shift in the reading frame. rtARNTa lacks the sequence and has a COOH-terminal domain of 104 residues rich in proline, serine, and threonine. rtARNTb contains the sequence and has a COOH-terminal domain of 190 residues rich in glutamine and asparagine. mRNAs for both rtARNT splice variants were detected in RTG-2 gonad cells, trout liver, and gonad tissue. rtARNTa and rtARNb protein were identified in cell lysates from RTG-2 cells. Transfection of rtARNT expression vectors into murine Hepa-1 cells that are defective in ARNT function (type II) result in rtARNT protein expression localized to the nucleus. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a 20-fold greater induction of endogenous P4501A1 protein in cells expressing rtARNTb when compared with rtARNTa, even though both proteins effectively dimerize with the aryl hydrocarbon receptor. The decreased function of rtARNTa appears to be due to inefficient binding of rtARNTa.AHR complexes to DNA. In addition, the presence of rtARNTa can reduce the aryl hydrocarbon receptor-dependent function of rtARNTb in vivo and in vitro.

The aryl-hydrocarbon receptor, but not the aryl-hydrocarbon receptor nuclear translocator protein, is rapidly depleted in hepatic and nonhepatic culture cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Western blot analysis and indirect immunofluorescence microscopy were used to evaluate the fate of the aryl-hydrocarbon receptor (AhR) and aryl-hydrocarbon receptor nuclear translocator (Arnt) protein in culture cell models exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In wild-type (WT) murine Hepa-1c1c7 cells, AhR protein was depleted by 85% after 4 hr of TCDD treatment as measured in total cell lysates. In contrast, the concentration of Arnt protein was unaffected by TCDD treatment in WT cells. Analysis of the AhR with immunofluorescence microscopy revealed that nuclear translocation of the liganded AhR preceded its depletion from cells. AhR protein was depleted from Hepa-1 type I variants (that contain a concentration of AhR that is 10% of WT) with a similar time course and to the same maximal level observed in WT cells (85%). The EC50 for AhR depletion in Hepa-1 cells was 39 pm TCDD and correspond to the EC50 for induction of P4501A1 protein. Murine embryonic fibroblasts (NIH-3T3), rat aortic smooth muscle cells (A7), and murine skeletal muscle cells (C2C12) all exhibited >90% depletion of the AhR after 2-4 hr of TCDD treatment. Arnt concentration was not affected by TCDD in these cell lines. These results indicate that the liganded AhR is rapidly depleted within the nuclear compartment of hepatic and nonhepatic cells in a manner independent of the Arnt protein.

The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy.

The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) protein were evaluated in the Hepa 1c1c7 (Hepa-1) cell line by indirect immunofluorescence microscopy and Western blot analysis. Wild-type (WT) Hepa-1 cells stained for AhR show intense cytoplasmic fluorescence with minimal nuclear reactivity. WT cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show a time-dependent decrease in cytoplasmic AhR staining and a concomitant increase in nuclear fluorescence. WT cells stained for Arnt show nuclear fluorescence with minimal cytoplasmic reactivity, a pattern unchanged after TCDD treatment. Hepa-1 type II variants express normal levels of AhR but are defective in TCDD-mediated induction of cytochrome P4501A1. Type II variants stained for Arnt show reduced nuclear fluorescence, compared with WT cells, and express minimal levels of Arnt protein, as determined by Western blot analysis. Type II variants stained for the AhR show intense cytoplasmic fluorescence that becomes nuclear after TCDD treatment. Detailed evaluation by immunoelectron microscopy of the AhR and Arnt present in the nuclear compartment of WT cells shows that both proteins are uniformly distributed and do not appear to be associated with nuclear pores, membranes, or nucleoli. Western blot analysis of nuclei isolated from WT Hepa-1 cells fractionated with Nonidet P-40 shows that minimal levels of AhR or Arnt are retained in the nuclear fraction after TCDD treatment. Collectively, these results indicate that the unliganded AhR resides in the cytoplasm, Arnt is localized to the nucleus, and Hepa-1 cells defective in Arnt expression exhibit TCDD-mediated nuclear accumulation of the AhR.