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Xenia2 is a DV cluster actinobacteriophage that infects Gordonia rubripertincta NRRL B-16540. The genome is 68,135bp, has a GC content of 57.9% and 98 predicted protein-coding genes, 33 of which have a predicted function. Xenia2 has a lysis cassette with an endolysin (lysin A) and four different holin-like transmembrane proteins., Competing Interests: The authors declare no conflict of interest.

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes (~28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic no known functions proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Secretariat and Hydrus are phages grouped into the DJ cluster that were isolated on Gordonia rubripertincta NRRL B-16540. The phages have 75% nucleotide identity and share 73% gene content. Secretariat has a genome with 84 predicted genes, while Hydrus has 91 predicted genes and can also infect Gordonia terrae 3612., Competing Interests: The authors declare no conflict of interest.

Azira is a CT cluster actinobacteriophage that infects Gordonia rubripertincta NRRL B-16540. The genome contains 67 predicted protein coding genes, of which 31 have a putative function. Azira has a lysis cassette encoding two endolysins and three transmembrane proteins. Azira contains four genes predicted to encode enzymes involved in thymine synthesis., Competing Interests: The authors declare no conflict of interest.

Holins are bacteriophage-encoded transmembrane proteins that function to control the timing of bacterial lysis event, assist with the destabilization of the membrane proton motive force and in some models, generate large "pores" in the cell membrane to allow the exit of the phage-encoded endolysin so they can access the peptidoglycan components of the cell wall. The lysis mechanism has been rigorously evaluated through biochemical and genetic studies in very few phages, and the results indicate that phages utilize endolysins, holins and accessory proteins to the outer membrane to achieve cell lysis through several distinct operational models. This observation suggests the possibility that phages may evolve novel variations of how the lysis proteins functionally interact in an effort to improve fitness or evade host defenses. To begin to address this hypothesis, the current study utilized a comprehensive bioinformatic approach to systematically identify the proteins encoded by the genes within the lysis cassettes in 16 genetically diverse phages that infect the Gram-positive Gordonia rubripertincta NRLL B-16540 strain. The results show that there is a high level of diversity of the various lysis genes and 16 different genome organizations of the putative lysis cassette, many which have never been described. Thirty-four different genes encoding holin-like proteins were identified as well as a potential holin-major capsid fusion protein. The holin-like proteins contained between 1-4 transmembrane helices, were not shared to a high degree amongst the different phages and are present in the lysis cassette in a wide range of combinations of up to 4 genes in which none are duplicated. Detailed evaluation of the transmembrane domains and predicted membrane topologies of the holin-like proteins show that many have novel structures that have not been previously characterized. These results provide compelling support that there are novel operational lysis models yet to be discovered., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Pollenz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

CaiB is a DR cluster actinobacteriophage that was isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540 as the host. The genome is 61,620 bp, has a GC content of 68.6%, and contains 85 predicted protein coding genes. CaiB has several putative operons and has repeated intergenic regions that may be involved in gene regulation.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

VanLee is a singleton phage that was isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540 as the host. The genome is 84,560 bp and has a GC content of 67.8%. VanLee has 164 predicted protein-coding genes and one tRNA. VanLee can infect Gordonia terrae with the same efficiency as G. rubripertincta.

Cluster EK2 Akoni, Ashton, and Truong are lytic Podoviridae actinobacteriophages that were isolated from soil in Florida using Microbacterium foliorum NRRL B-24224 as the host. The genomes are 54,307 bp, 54,560 bp, and 54,309 bp, respectively, and are 60% GC rich. Each genome contains a novel 13,464-bp gene that encompasses 25% of the genome.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics., Competing Interests: The authors have declared that no competing interests exist.

In a previous report, we validated that a cohort of first-year undergraduates who participated in a weeklong pre-college engagement STEM Academy (SA) program were retained in science, technology, engineering, and mathematics (STEM) at a higher rate than a matched comparison group (MCG). In addition, SA students yielded increases in science identity and sense of belonging to STEM and to the university. Here, we report the ability to scale the size of the SA program to accommodate more students and replicate the previous findings with two additional cohorts. Longitudinal analysis of the 2015 and 2016 program cohorts demonstrate that both groups were retained to STEM disciplines and the university at higher rates than a MCG. To assess what underlying psychological mechanisms lead to increases in science identity and university belonging, we tested three exploratory models. These models indicate that positive changes in university and STEM belonging indirectly predict an increase in science identity. Further, positive changes in perceived family support indirectly predict increases in university belonging. Thus, through the evaluation of three different cohorts, we found robust evidence that the SA program increases sense of belonging and science identity, and these attitudinal changes promote undergraduate persistence in STEM.

The persistence of undergraduate students in science, technology, engineering, and mathematics (STEM) disciplines is a national issue based on STEM workforce projections. We implemented a weeklong pre-college engagement STEM Academy (SA) program aimed at addressing several areas related to STEM retention. We validated an instrument that was developed based on existing, validated measures and examined several psychosocial constructs related to STEM (science identity, self-efficacy, sense of belonging to the university and to STEM, career expectancies, and intention to leave STEM majors) before and after the program. We also compared students in the SA program with a matched comparison group of first-year students. Results show that SA students significantly increased in science identity and sense of belonging to STEM and to the university, all predictive of increased STEM retention and a primary aim of the program. Relative to the matched comparison group, SA students began their first semester with higher STEM self-efficacy, sense of belonging, and science identity, positive career expectancies, and lower intention to leave STEM. The SA cohort showed 98% first-year retention and 92% STEM major retention. The SA program serves as a model of a scalable, first-level, cocurricular engagement experience to enhance psychosocial factors that impact undergraduate persistence in STEM., (© 2017 D. Findley-Van Nostrand and R. S. Pollenz. CBE—Life Sciences Education © 2017 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of beta-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent beta-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc(Min/+) mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

ARNT and ARNT2 proteins are expressed in mammalian and aquatic species and exhibit a high level of amino acid identity in the basic-helix loop-helix PER/ARNT/SIM domains involved in protein interactions and DNA binding. Since the analysis of ARNT2 function at the protein level has been limited, ARNT2 function in aryl hydrocarbon receptor (AHR)-mediated signaling was evaluated and compared to ARNT. In vitro, ARNT and ARNT2 dimerized equally with the AHR in the presence of 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) and ARNT2 outcompeted ARNT for binding to the AHR when expressed in excess. In contrast, activation of the AHR with 3-methylcholanthrene or benzo[a]pyrene resulted in predominant formation of AHR*ARNT complexes. ARNT2 expressed in Hepa-1 cell culture lines with reduced ARNT protein resulted in minimal induction of endogenous CYP1A1 protein compared to cells expressing ARNT, and mutation of the putative proline residue at amino acid 352 to histidine failed to produce an ARNT2 that could function in AHR-mediated signaling. However, the expression of ARNT2 in wild-type Hepa-1 cells reduced TCDD-mediated induction of endogenous CYP1A1 protein by 30%, even though AHR*ARNT2 complexes could not be detected in nuclear extracts. Western blot analysis of numerous mouse tissues and various cell culture lines showed that both endogenous ARNT and ARNT2 could be detected in cells derived from kidney, central nervous system, and retinal epithelium. Thus, ARNT2 has the ability to dimerize with the liganded AHR in vitro and is influenced by the activating ligand yet appears to be limited in its ability to influence AHR-mediated signaling in cell culture.

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin causes altered gene expression and toxicity. The AHR repressor (AHRR) inhibits AHR signaling through a proposed mechanism involving competition with AHR for dimerization with AHR nuclear translocator (ARNT) and binding to AHR-responsive enhancer elements (AHREs). We sought to delineate the relative roles of competition for ARNT and AHREs in the mechanism of repression. In transient transfections in which AHR2-dependent transactivation was repressed by AHRR1 or AHRR2, increasing ARNT expression failed to reverse the repression, suggesting that AHRR inhibition of AHR signaling does not occur through sequestration of ARNT. An AHRR1 point mutant (AHRR1-Y9F) that could not bind to AHREs but that retained its nuclear localization was only slightly reduced in its ability to repress AHR2, demonstrating that AHRR repression does not occur solely through competition for AHREs. When both proposed mechanisms were blocked (AHRR1-Y9F plus excess ARNT), AHRR remained functional. AHRR1 neither blocked AHR nuclear translocation nor reduced the levels of AHR2 protein. Experiments using AHRR1 C-terminal deletion mutants showed that amino acids 270 to 550 are dispensable for repression. These results demonstrate that repression of AHR transactivation by AHRR involves the N-terminal portion of AHRR; does not involve competition for ARNT; and does not require binding to AHREs, although AHRE binding can contribute to the repression. We propose a mechanism of AHRR action involving "transrepression" of AHR signaling through protein-protein interactions rather than by inhibition of the formation or DNA binding of the AHR-ARNT complex.

In vitro mutagenesis was utilized to render the various xenobiotic response elements (XREs) within the zebrafish CYP1A promoter/enhancer region non-functional either independently or in combination. Reporter gene assays revealed that only XRE4, XRE7, and XRE8 contributed to maximal TCDD-mediated induction of luciferase and that the contribution of each XRE to maximal induction was not equal. XRE4 and XRE7 were capable of functioning independently, while XRE8 alone could not support TCDD-mediated induction but was required for the ability of XRE4 and XRE7 to support maximal induction. These results were observed in cell lines derived from human, mouse and zebrafish. Mutagenesis of 3' nucleotides flanking the non-functional XRE5, and functional XRE4 did not alter the function of these XREs in cell culture. In silico analyses revealed the presence of putative Sp1, AP2, CREB, and two HNF-3 transcription factor binding sites that were localized to common positions within the enhancer region of both the mouse and zebrafish CYP1A genes. In vitro mutagenesis of the binding sites showed that loss of the Sp1 or AP2 sites had minimal impact on TCDD-mediated gene induction while loss of the putative CREB site resulted in a modest decrease in basal and inducible activity and mutation of the HNF-3 reduced inducible activity by >90% of controls. Collectively, these findings suggest that the presence of XREs is not the sole determinant for regulation of aryl hydrocarbon receptor (AHR)-mediated gene and do not function in an additive manner.

To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75 microM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1-5 microM), proteasome inhibitor I (5-10 microM) or lactacystin (5-15 microM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.

Studies have shown that zebrafish and rodent aryl hydrocarbon receptors (AHRs) are degraded following ligand exposure and that reductions in AHR protein can impact growth and development in vivo. The current study was designed to evaluate the degradation of the AHR in seven human cell lines that were derived from various carcinomas or from normal tissue. Consistent with studies in other species, the results show that the human AHR (hAHR) is degraded in a ligand dependent manner following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin for up to 16h. However, the hAHRs expressed in the various cell lines show differences in the time course and magnitude of degradation. The ligand dependent degradation is completely blocked by treatment with the proteasome inhibitor, MG-132. Ligand-independent degradation of the hAHR following exposure to geldanamycin (GA) is also observed in the different cell lines, although the magnitude of hAHR degradation is also is variable. These findings are significant since they indicate that ligand-dependent and independent degradation of the AHR is a conserved aspect of this signal transduction cascade from fish to human. In addition, the study identifies several cell lines that may provide novel models to further assess the regulation of AHR-mediated signaling and degradation of the human AHR.

Studies using transient expression systems have implicated the hepatitis B virus X-associated protein (XAP2) in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Studies were performed in Hepa-1 cells to evaluate these functions of XAP2 on the mouse Ahb-1 receptor under endogenous stoichiometry. The Ahb-1 receptor is cytoplasmic, and it becomes predominantly nuclear after 30 to 60 min of ligand exposure with minimal degradation. During this time, XAP2 coprecipitates with the AHR, suggesting that it does not affect the nuclear localization of the liganded receptor. Overexpression of XAP2 in Hepa-1 cells does not result in increased association with the endogenous Ahb-1 complex or influence the receptors cytoplasmic localization. Knockdown of endogenous XAP2 by small interfering RNA results in >or=90% reduction in the amount of XAP2 associated with the endogenous Ahb-1 receptor complex. Despite the reduction in XAP2, the unliganded Ahb-1 receptor complex remains cytoplasmic, although inhibition of nuclear export results in accumulation of the receptor in the nucleus. Truncation of the C-terminal 305 amino acids of the Ahb-1 receptor (AHR500) results in proteins that exhibit a predominantly nuclear localization and remain associated with the same level of endogenous XAP2 as full-length AHRs. Together, these results support a model in which the majority of the unliganded Ahb-1 receptor complexes are associated with XAP2, and the association prevents dynamic nucleocytoplasmic shuttling in the unliganded state. After ligand binding, XAP2 remains associated with the Ahb-1 receptor complex, and it does not impair nuclear translocation but may function to limit receptor "transformation".

To assess the importance of transactivation domains (TAD), DNA binding and transcription on the degradation of the AH receptor (AHR), Hepa-1 cells were pre-treated with actinomycin D (AD) or cycloheximide (CHX) and exposed to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). AD or CHX did not affect nuclear localization or DNA binding of the AHR but inhibited ligand-induced degradation. In contrast, AD or CHX did not inhibit geldanamycin (GA) induced degradation of the AHR. To assess the role of the COOH-terminal TAD in AHR degradation, stop codons were placed at nucleotide 1501 and 1921 of the Ah(b-1) AHR coding region to generate AHR(500) and AHR(640). Stable cell lines were generated and exposed to TCDD. Cells expressing AHR(500) did not induce CYP1A1 protein, but exhibited significant degradation of AHR(500). Cells expressing AHR(640) induced CYP1A1 protein to 50% of the level of cells expressing wild type AHR and exhibited significant degradation of AHR(640). Importantly, AD and CHX did not inhibit the TCDD-induced degradation of either AHR(500) and AHR(640) and these receptors showed a more rapid profile of ligand-induced degradation compared to cells expressing wild type AHR. TCDD exposure to Hepa-1 cells with reduced aryl hydrocarbon receptor nuclear translocator (ARNT), showed ligand-induced degradation of the AHR that was not blocked by AD. However, AD inhibited TCDD-induced degradation when ARNT expression was restored. These results show that multiple mechanisms exist for the ligand and GA-induced degradation of the AHR and suggest that ligand-induced degradation can switch between two mechanisms depending on the presence of a functional TAD and the binding to DNA.

Studies using transient expression systems have implicated the XAP2 protein in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Thus, studies were performed in cell lines that expressed endogenous rat or mouse Ah(b-1) (C57BL/6) or Ah(b-2) (C3H) AHRs with similar levels of endogenous XAP2. Unliganded rat and mouse Ah(b-2) receptor complexes associated with reduced levels of XAP2 and exhibited dynamic nucleocytoplasmic shuttling in comparison with Ah(b-1) receptors. Rat and mouse Ah(b-2) receptors also exhibited a greater magnitude of ligand-induced degradation than Ah(b-1) receptors. Small interfering RNA reduction of endogenous XAP2 by >80% had minimal impact on the level of Ah(b-2) receptors but resulted in a 25-30% reduction of Ah(b-1) receptors. XAP2 reduction resulted in increased susceptibility of the Ah(b-1) receptor to ligand-induced degradation yet produced higher levels of endogenous CYP1A1 induction. Stable expression of the Ah(b-2) receptor in the C57BL/6 background resulted in a protein with reduced association with XAP2, dynamic nucleocytoplasmic shuttling, and increased levels of ligand-induced degradation. Small interfering RNA reduction of endogenous XAP2 in a C-terminal hsp70-interacting protein knockout mouse cell line, exhibited a 25-30% reduction in the level of endogenous Ah(b-1) AHR and showed high levels of ligand-induced degradation. Thus, endogenous XAP2 exerts a negative function on a small fraction of the endogenous Ah(b-1) receptor complex but appears to have a minimal impact on endogenous rat or Ah(b-2) receptors. This implies that the analysis of the AHR-mediated signaling via rat and mouse Ah(b-2) receptors may better represent the physiology of this signal transduction pathway.

To isolate the CYP1A1 promoter/enhancer from zebrafish, a PAC genomic library was screened with sequence derived from the 5'UTR of the zfCYP1A1 cDNA. Sequence was identified that contained CAAT and TATA boxes, had a large intron within the 5'UTR, and showed 100% sequence identity to zfCYP1A1 cDNAs in the 5'UTR and initial 300 bp of the open reading frame. Oligonucleotides complementary to the 5'UTR were used to detect zfCYP1A1 mRNA in zebrafish liver cells (ZFL) exposed to TCDD, thus identifying the gene as a TCDD-inducible CYP1A1. Sequence analysis revealed that the 5' flanking region contained eight putative xenobiotic response elements (XRE) arranged in two distinct clusters. One cluster was localized between -580 and -187 and contained three XREs and two XREs bound zfAHR2 . rtARNTb complexes in vitro. However, this region was incapable of conferring TCDD-responsiveness to an SV40 promoter. In contrast, the region between -2608 and -2100 contained five XREs and was capable of driving TCDD-inducible expression when placed in either a forward or reverse orientation upstream or downstream of an SV40 promoter. Thus, the zfCYP1A1 gene has similar structural features to mammalian CYP1A1s and will be ideal for the analysis of AHR-mediated gene regulation in an aquatic organism.

Cytochrome P4501B1 (CYP1B1), the major constitutively expressed CYP in the rat mammary gland, is induced by Ah-receptor (AhR) ligands, while CYP1A1 is predominantly expressed only after induction. These CYPs contribute to carcinogenic activation of polycyclic aromatic hydrocarbons (PAHs). AhR, ARNT, and CYP1B1 were only weakly expressed, even after 2,3,7,8-tetrachlorodibenzo-p-dioxin induction, when rat mammary epithelial cells (RMEC) were cultured on plastic. RMEC cultured on the extracellular matrix (ECM), Matrigel, or on a floating gel of collagen I demonstrated branching morphogenesis and substantially increased basal CYP1B1 and induced CYP1A1 expression, in parallel with large increases in AhR and ARNT expression. Branching was more pronounced in the Wistar Kyoto than in the Wistar Furth rat strain. Although EGF enhanced branching, neither strain nor growth factor treatment substantially impacted CYP expression. Increased AhR and ARNT expression is observed within 24 h of dispersal on Matrigel, substantially prior to branch formation. Culture on thin layers of collagen I, collagen IV, and laminin, respectively, failed to reproduce the branching morphogenesis or increases in AhR, ARNT, or CYP expression. However, adherent, gelled collagen I recapitulated the increased protein expression, without supporting branching. This increased protein expression was closely paralleled by enhanced expression of beta-catenin and E-cadherin, components of cell-cell adhesion complexes. A synthetic peptide that selectively antagonizes integrin-ECM interactions reduced branch formation, without diminishing AhR, ARNT, and CYP expression. These data demonstrate that early ECM surface adhesion interactions mediate AhR and ARNT expression, which enhances CYP expression, independent of branching morphogenesis.

The basic-helix-loop-helix/PAS (bHLH/PAS) family of proteins is a group of transcription factors that regulate key pathways during normal development and in the response to stress. The aryl hydrocarbon receptor (AHR) is a member of this family. Recently, Danio rerio (zebrafish) has become an important model system in the study of the signal transduction pathway and complements the results seen in mammalian models. However, studies of the AHR protein have been limited by the lack of antibody reagents and thus, little is known concerning the localization and degradation of the zebrafish AHR (zfAHR). In this report, we describe the production and characterization of specific polyclonal antibodies to the zfAHR2 protein and the analysis of AHR-mediated signal transduction in the zebrafish liver cell line (ZFL). The results show that the zfAHR2 is degraded via the 26S proteasome following exposure of cells to beta-naphthoflavone (BNF). Interestingly, the time course is slower and the magnitude of zfAHR2 degradation is not as great as seen for the mammalian AHR. Studies also show that the zfAHR2 is rapidly degraded in a ligand-independent manner by exposure of cells to geldanamycin (GA) to levels consistent with mammalian AHR. Finally, immunohistochemical staining of the ZFL cells suggest that the unliganded AHR resides in both the cytoplasm and nucleus and undergoes active nucleocytoplasmic shuttling in the absence of ligand. These results suggest that there is conservation of function between fish and mammals with respect to ligand-dependent and -independent degradation of the AHR and that the zfAHR2 is degraded via the 26S proteasome.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is also a substrate for the 26S proteasome. However, the subcellular location of the degradation events or the requirement for nuclear transport has not been resolved. To gain insight into both ligand-dependent and independent degradation of the AHR, studies were designed to evaluate the relationship between AHR localization, stability, and gene regulation in a defined cell culture model system. The strategy of these studies was to generate stable cell lines expressing murine AHR proteins that were defective in nuclear import and then to assess the location of the AHR, the time course of AHR degradation, and the level of induction of endogenous CYP1A1 protein after exposure to 2,3,7,8-tetrachlorodibezo-p-dioxin (TCDD), geldanamycin (GA), or the protease inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG-132). Mutation within the putative nuclear localization sequence (NLS) resulted in AHR mutants that were severely defective in nuclear import as evaluated by immunocytochemical staining after exposure to TCDD, GA, or MG-132. Importantly, the NLS mutants exhibited identical levels of degradation along a similar time course as wild-type AHR after exposure to TCDD or GA when stably expressed in either murine hepatoma cells (Hepa-1) or hamster lung cells (E36). In contrast, the NLS mutants were severely defective in ligand-mediated induction of CYP1A1 expression. These findings imply that the proteolytic machinery present in the cytoplasmic compartment is sufficient to degrade the AHR and that nuclear translocation, binding with ARNT, or DNA binding are not necessary for efficient degradation of the AHR.

Changes in the concentration or subcellular location of the key proteins involved in signal transduction pathways have been shown to impact gene regulation. Studies were designed to evaluate the relationship between aryl hydrocarbon receptor (AHR) localization, stability, and gene regulation in a defined system where the endogenous AHR protein could be evaluated. The findings indicate that treatment of cells with geldanamycin (GA) or MG-132 (an inhibitor of the 26S proteasome) results in nuclear translocation of the endogenous AHR in both human HepG2 and murine Hepa-1 cells without induction of endogenous CYP1A1 protein. Exposure to GA resulted in the degradation of AHR by >90% in the nucleus via the 26S proteasome. Importantly, the reduced level of AHR resulted in a 50% reduction in the maximal level of CYP1A1 induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In all treatments the concentration of the AHR nuclear translocator (ARNT) protein was unchanged and had no impact on the localization of the AHR. Thus, ligand-independent translocation of the AHR to the nucleus was not sufficient to induce CYP1A1 in the absence of ligand, but reductions in the level of the endogenous AHR protein pool shifted the dose-response curve for TCDD to the right.

The proteolytic degradation of transcription factors is an established mechanism of regulating signal transduction pathways. Recent reports have suggested that the aryl hydrocarbon receptor (AHR) protein is rapidly downregulated (degraded) following ligand binding. The downregulation of AHR has been observed in nine distinct cells culture lines derived from human and rodent tissues and has also been observed in rodent models following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The downregulation of AHR appears to be ubiquitin mediated and occurs via the 26S proteasome pathway following nuclear export of AHR. The consequence of blocking AHR degradation in cell culture appears to be an increase in both the magnitude and duration of gene regulation by the AHR.ARNT complex. Thus, the physiological role of AHR degradation may be to modulate AHR-mediated gene regulation. This review provides analysis of the studies that have focused on the degradation of AHR in vivo and in vitro and the hypothesis that the downregulation of AHR is critical in the attenuation of AHR-mediated gene regulation.

Efficient routes to three classes of 10-membered oxa-enediynes are presented. The electronic and stereoelectronic contributions to half-lives are supported by density functional theory calculations. One member of this class cyclizes to give an isochroman which binds to and degrades the aryl hydrocarbon receptor (AhR).

Oct2 transcription factors of the catfish (Ictalurus punctatus) are expressed as alternatively spliced alpha and beta isoforms. Functional analysis revealed an N-terminal glutamine (Q)-rich transactivation domain common to both isoforms of catfish Oct2. A C-terminal proline, serine, threonine (PST)-rich activation domain was identified exclusively in the beta isoform. Activation domains of fish and mammalian Oct2 showed cell type- and species-specific activity correlated with their biochemical composition (Q-rich vs PST-rich). In contrast the activation domains of the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator of fish and mammals showed no correlation of activity with biochemical composition or species of origin. Although isolated catfish Oct2 activation domains were unable to drive transcription from a site 1.9kb distal to the promoter, Oct2beta activated transcription from both an IgH enhancer and an array of octamer motifs at this distal position. The properties of catfish Oct2 activation domains differ depending on whether they are studied in isolation or as components of the intact transcription factor.

Two distinct aryl hydrocarbon receptor (AHR) cDNAs have been isolated from rainbow trout. The encoded receptor protein products termed rtAHR2alpha and rtAHR2ss are 97% identical at the amino acid level but are reported to have distinct functions with regard to AHR-mediated gene regulation. To test this hypothesis, the two proteins were evaluated functionally both in vitro and in a Chinese hamster lung cell line, E36. To facilitate analysis, both rtAHR2 isoforms were tagged with the FLAG peptide and could be expressed and quantified in a rabbit reticulocyte lysate. However, both proteins failed to form functional complexes with mammalian or rainbow trout AHR nuclear translocator protein (ARNT) that could associate with xenobiotic response elements (XREs) in a ligand-dependent manner in vitro. In contrast, both proteins exhibited positive function on AHR-mediated signaling when expressed in the E36 cell line. Both rtAHR2 isoforms showed a cytoplasmic distribution in the unliganded state and could drive the expression of a reporter gene under control of the trout CYP1A3 promoter. Although both proteins induced reporter gene activity to the same magnitude, the EC(50) values of the two isoforms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) differed by an order of magnitude, with the rtAHR2ss isoform less responsive to TCDD. When the functions of the rtAHR2 isoforms were tested in the context of the dominant negative rtARNT(a) protein, TCDD-mediated induction of reporter gene activity was reduced as the level of rtARNT(a) protein increased. In summary, both rtAHR2 isoforms appear to exhibit positive function in AHR-mediated signaling, suggesting conservation of function.

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.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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.

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., (Copyright 1999 Academic Press.)

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.

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.

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.

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.

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.

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.