Crosstalk of MAP3K1 and EGFR signaling mediates gene-environment interactions that block developmental tissue closure.

Aberrant regulation of signal transduction pathways can adversely derail biological processes for tissue development. One such process is the embryonic eyelid closure that is dependent on the Mitogen-Activated Protein Kinase Kinase Kinase 1 (MAP3K1). Map3k1 knockout in mice results in defective eyelid closure and an autosomal recessive eye-open at birth phenotype. We have shown that in utero exposure to dioxin, a persistent environmental toxicant, induces the same eye defect in Map3k1+/- heterozygous but not wild type pups. Here we explore the mechanisms of the Map3k1 (gene) and dioxin (environment) interactions (GxE) underlying defective eyelid closure. We show that, acting through the Aryl Hydrocarbon Receptor (AHR), dioxin activates Epidermal Growth Factor Receptor (EGFR) signaling, which in turn depresses MAP3K1-dependent Jun N-terminal Kinase (JNK) activity. The dioxin mediated JNK repression is moderate but is exacerbated by Map3k1 heterozygosity. Therefore, dioxin exposed Map3k1+/- embryonic eyelids have a marked reduction of JNK activity, accelerated differentiation and impeded polarization in the epithelial cells. Knocking out Ahr or Egfr in eyelid epithelium attenuates the open-eye defects in dioxin-treated Map3k1+/- pups, whereas knockout of Jnk1 and S1pr that encodes the Sphigosin-1-phosphate (S1P) receptors upstream of the MAP3K1-JNK pathway potentiates the dioxin toxicity. Our novel findings show that the crosstalk of AHR, EGFR and S1P-MAP3K1-JNK pathways determines the outcome of dioxin exposure. Thus, gene mutations targeting these pathways are potential risk factors for the toxicity of environmental chemicals.

MAP3K1 regulates female reproductive tract development.

Mitogen-activated protein 3 kinase 1 (MAP3K1) has a plethora of cell type-specific functions not yet fully understood. Herein, we describe a role for MAP3K1 in female reproductive tract (FRT) development. MAP3K1 kinase domain-deficient female mice exhibited an imperforate vagina, labor failure and infertility. These defects corresponded with shunted Müllerian ducts (MDs), the embryonic precursors of FRT, that manifested as a contorted caudal vagina and abrogated vaginal-urogenital sinus fusion in neonates. The MAP3K1 kinase domain is required for optimal activation of the Jun-N-terminal kinase (JNK) and cell polarity in the MD epithelium, and for upregulation of WNT signaling in the mesenchyme surrounding the caudal MD. The MAP3K1-deficient epithelial cells and MD epithelium had reduced expression of WNT7B ligands. Correspondingly, conditioned media derived from MAP3K1-competent, but not -deficient, epithelial cells activated a TCF/Lef-luciferase reporter in fibroblasts. These observations indicate that MAP3K1 regulates MD caudal elongation and FRT development, in part through the induction of paracrine factors in the epithelium that trans-activate WNT signaling in the mesenchyme.

The Role of MAP3K1 in the Development of the Female Reproductive Tract.

Mitogen-Activated Protein 3 Kinase 1 (MAP3K1) is a dynamic signaling molecule with a plethora of cell-type specific functions, most of which are yet to be understood. Here we describe a role for MAP3K1 in the development of female reproductive tract (FRT). MAP3K1 kinase domain-deficient ( Map3k1 ΔKD ) females exhibit imperforate vagina, labor failure, and infertility. These defects correspond to a shunted Müllerian duct (MD), the principle precursor of the FRT, in embryos, while they manifest as a contorted caudal vagina with abrogated vaginal-urogenital sinus fusion in neonates. In epithelial cells, MAP3K1 acts through JNK and ERK to activate WNT, yet in vivo MAP3K1 is crucial for WNT activity in mesenchyme associated with the caudal MD. Expression of Wnt7b is high in wild type, but low in Map3k1 knockout MD epithelium and MAP3K1-deficient keratinocytes. Correspondingly, conditioned media derived from MAP3K1-competent epithelial cells activate TCF/Lef-luciferase reporter in fibroblasts, suggesting that MAP3K1-induced factors released from epithelial cells trans-activate WNT signaling in fibroblasts. Our results reveal a temporal-spatial and paracrine MAP3K1-WNT crosstalk contributing to MD caudal elongation and FRT development. Highlights: MAP3K1 deficient female mice exhibit imperforate vagina and infertilityLoss of MAP3K1 kinase activity impedes Müllerian duct (MD) caudal elongation and fusion with urogenital sinus (UGS) in embryogenesisThe MAP3K1-MAPK pathway up-regulates WNT signaling in epithelial cellsMAP3K1 deficiency down-regulates Wnt7b expression in the MD epithelium and prevents WNT activity in mesenchyme of the caudal MD.

Mechanisms of chromate carcinogenesis by chromatin alterations.

In a dynamic environment, organisms must constantly mount an adaptive response to new environmental conditions in order to survive. Novel patterns of gene expression, driven by attendant changes in chromatin architecture, aid in adaptation and survival. Critical mechanisms in the control of gene transcription govern new spatiotemporal chromatin-chromatin interactions that make regulatory DNA elements accessible to the transcription factors that control the response. Consequently, agents that disrupt chromatin structure are likely to have a direct impact on the transcriptional programs of cells and organisms and to drive alterations in fundamental physiological processes. In this regard, hexavalent chromium (Cr(VI)) is of special interest because it interacts directly with cellular proteins, DNA, and other macromolecules, and is likely to upset cell functions that may cause generalized damage to the organism. Here, we will highlight chromium-mediated mechanisms that disrupt chromatin architecture and discuss how these mechanisms are integral to its carcinogenic properties. Emerging evidence indicates that Cr(VI) targets euchromatin, particularly in genomic locations flanking the binding sites of the essential transcription factors CTCF and AP1, and, in so doing, they disrupt nucleosomal architecture. Ultimately, the ensuing changes, if occurring in critical regulatory domains, may establish a new chromatin state, either toxic or adaptive, that will be governed by the corresponding gene transcription changes in key biological processes associated with that state.

The aryl hydrocarbon receptor directs the differentiation of murine progenitor blastomeres.

Key regulatory decisions during cleavage divisions in mammalian embryogenesis determine the fate of preimplantation embryonic cells. Single-cell RNA sequencing of early-stage-2-cell, 4-cell, and 8-cell-blastomeres show that the aryl hydrocarbon receptor (AHR), traditionally considered as an environmental sensor, directs blastomere differentiation. Disruption of AHR functions in Ahr knockout embryos or in embryos from dams exposed to dioxin, the prototypic xenobiotic AHR agonist, significantly impairs blastocyst formation, causing repression and loss of transcriptional heterogeneity of OCT4 and CDX2 and incidence of nonspecific downregulation of pluripotency. Trajectory-the path of differentiation-and gene variability analyses further confirm that deregulation of OCT4 functions and changes of transcriptional heterogeneity resulting from disruption of AHR functions restrict the emergence of differentiating blastomeres in 4-cell embryos. It appears that AHR directs the differentiation of progenitor blastomeres and that disruption of preimplantation AHR functions may significantly perturb embryogenesis leading to long-lasting conditions at the heart of disease in offspring's adulthood.

Hexavalent chromium disrupts chromatin architecture.

Accessibility of DNA elements and the orchestration of spatiotemporal chromatin-chromatin interactions are critical mechanisms in the regulation of gene transcription. Thus, in an ever-changing milieu, cells mount an adaptive response to environmental stimuli by modulating gene expression that is orchestrated by coordinated changes in chromatin architecture. Correspondingly, agents that alter chromatin structure directly impact transcriptional programs in cells. Heavy metals, including hexavalent chromium (Cr(VI)), are of special interest because of their ability to interact directly with cellular protein, DNA and other macromolecules, resulting in general damage or altered function. In this review we highlight the chromium-mediated mechanisms that promote disruption of chromatin architecture and how these processes are integral to its carcinogenic properties. Emerging evidence shows that Cr(VI) targets nucleosomal architecture in euchromatin, particularly in genomic locations flanking binding sites of the essential transcription factors CTCF and AP1. Ultimately, these changes contribute to an altered chromatin state in critical gene regulatory regions, which disrupts gene transcription in functionally relevant biological processes.

Converging Roles of the Aryl Hydrocarbon Receptor in Early Embryonic Development, Maintenance of Stemness, and Tissue Repair.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor well-known for its adaptive role as a sensor of environmental toxicants and mediator of the metabolic detoxification of xenobiotic ligands. In addition, a growing body of experimental data has provided indisputable evidence that the AHR regulates critical functions of cell physiology and embryonic development. Recent studies have shown that the naïve AHR-that is, unliganded to xenobiotics but activated endogenously-has a crucial role in maintenance of embryonic stem cell pluripotency, tissue repair, and regulation of cancer stem cell stemness. Depending on the cellular context, AHR silences the expression of pluripotency genes Oct4 and Nanog and potentiates differentiation, whereas curtailing cellular plasticity and stemness. In these processes, AHR-mediated contextual responses and outcomes are dictated by changes of interacting partners in signaling pathways, gene networks, and cell-type-specific genomic structures. In this review, we focus on AHR-mediated changes of genomic architecture as an emerging mechanism for the AHR to regulate gene expression at the transcriptional level. Collective evidence places this receptor as a physiological hub connecting multiple biological processes whose disruption impacts on embryonic development, tissue repair, and maintenance or loss of stemness.

Developmental and lifelong dioxin exposure induces measurable changes in cardiac structure and function in adulthood.

Congenital heart disease (CHD) is the most common congenital abnormality. A precise etiology for CHD remains elusive, but likely results from interactions between genetic and environmental factors during development, when the heart adapts to physiological and pathophysiological conditions. Further, it has become clearer that early exposure to toxins that do not result in overt CHD may be associated with adverse cardiac outcomes that are not manifested until later life. Previously, interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, was shown to cause structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. Here, we show that continuous exposure to TCDD from fertilization throughout adulthood caused male mice to underperform at exercise tolerance tests compared to their control and female counterparts, confirming previous observations of a sexually dimorphic phenotype. Renin-angiotensin stimulation by angiotensin II (Ang II) caused measurable increases in blood pressure and left ventricle mass, along with decreased end diastolic volume and preserved ejection fraction. Interestingly, TCDD exposure caused measurable reductions in the myocardial hypertrophic effects of Ang II, suggesting that endogenous AHR signaling present in adulthood may play a role in the pathogenesis of hypertrophy. Overall, the findings reported in this pilot study highlight the complex systems underlying TCDD exposure in the development of cardiac dysfunction in later life.

Hexavalent chromium promotes differential binding of CTCF to its cognate sites in Euchromatin.

Hexavalent chromium compounds are well-established respiratory carcinogens to which humans are commonly exposed in industrial and occupational settings. In addition, natural and anthropogenic sources of these compounds contribute to the exposure of global populations through multiple routes, including dermal, ingestion and inhalation that elevate the risk of cancer by largely unresolved mechanisms. Cr(VI) has genotoxic properties that include ternary adduct formation with DNA, increases in DNA damage, mostly by double-strand break formation, and altered transcriptional responses. Our previous work using ATAC-seq showed that CTCF motifs were enriched in Cr(VI)-dependent differentially accessible chromatin, suggesting that CTCF, a key transcription factor responsible for the regulation of the transcriptome, might be a chromium target. To test this hypothesis, we investigated the effect of Cr(VI) treatment on the binding of CTCF to its cognate sites and ensuing changes in transcription-related histone modifications. Differentially bound CTCF sites were enriched by Cr(VI) treatment within transcription-related regions, specifically transcription start sites and upstream genic regions. Functional annotation of the affected genes highlighted biological processes previously associated with Cr(VI) exposure. Notably, we found that differentially bound CTCF sites proximal to the promoters of this subset of genes were frequently associated with the active histone marks H3K27ac, H3K4me3, and H3K36me3, in agreement with the concept that Cr(VI) targets CTCF in euchromatic regions of the genome. Our results support the conclusion that Cr(VI) treatment promotes the differential binding of CTCF to its cognate sites in genes near transcription-active boundaries, targeting these genes for dysregulation.

Dioxin Disrupts Dynamic DNA Methylation Patterns in Genes That Govern Cardiomyocyte Maturation.

Congenital heart disease (CHD), the leading birth defect worldwide, has a largely unknown etiology, likely to result from complex interactions between genetic and environmental factors during heart development, at a time when the heart adapts to diverse physiological and pathophysiological conditions. Crucial among these is the regulation of cardiomyocyte development and postnatal maturation, governed by dynamic changes in DNA methylation. Previous work from our laboratory has shown that exposure to the environmental toxicant tetrachlorodibenzo-p-dioxin (TCDD) disrupts several molecular networks responsible for heart development and function. To test the hypothesis that the disruption caused by TCDD in the heart results from changes in DNA methylation and gene expression patterns of cardiomyocytes, we established a stable mouse embryonic stem cell line expressing a puromycin resistance selectable marker under control of the cardiomyocyte-specific Nkx2-5 promoter. Differentiation of these cells in the presence of puromycin induces the expression of a large suite of cardiomyocyte-specific markers. To assess the consequences of TCDD treatment on gene expression and DNA methylation in these cardiomyocytes, we subjected them to transcriptome and methylome analyses in the presence of TCDD. Unlike control cardiomyocytes maintained in vehicle, the TCDD-treated cardiomyocytes showed extensive gene expression changes, with a significant correlation between differential RNA expression and DNA methylation in 111 genes, many of which are key elements of pathways that regulate cardiovascular development and function. Our findings provide an important clue toward the elucidation of the complex interactions between genetic and epigenetic mechanisms after developmental TCDD exposure that may contribute to CHD.

Regulation of a long noncoding RNA MALAT1 by aryl hydrocarbon receptor in pancreatic cancer cells and tissues.

Environmental toxicants such as dioxins and polycyclic aromatic carbons are risk factors for pancreatitis and pancreatic cancer. These toxicants activate aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, of which activation regulates many downstream biological events, including xenobiotic metabolism, inflammation, and cancer cell growth and transformation. Here, we identified that environmental toxicant-activated AHR increased expression of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in pancreatic cancer cells and pancreatic tissues. The MALAT1 is a long noncoding (lnc) RNA which interacts with Enhancer of Zeste 2 (EZH2), a histone methyltransferase with epigenetic silencer activity, and the MALAT1-EZH2 interaction increased its epigenetic silencing activity. In contrast, AHR antagonist, CH223191 or resveratrol, counteracted the AHR-mediated MALAT1 induction and MALAT1-enahnced EZH2 activity. Collectively, these results revealed a novel pathway of how environmental exposure leads to epigenetic alteration via activation of AHR-MALAT1-EZH2 signaling axis under pancreatic tissue- and cancer cell-context.

Chromium exposure disrupts chromatin architecture upsetting the mechanisms that regulate transcription.

IMPACT STATEMENT: This mini-review highlights current evidence on the mechanisms through which hexavalent chromium (Cr(VI)) disrupts transcriptional regulation, an emerging area of interest and one of the central processes by which chromium induces carcinogenesis. Several studies have shown that Cr(VI) causes widespread DNA damage and disrupts epigenetic signatures, suggesting that chromatin may be a direct Cr(VI) target. The findings discussed here suggest that Cr(VI) disrupts transcriptional regulation by causing genomic architecture changes.

Prenatal exposure to PCBs in Cyp1a2 knock-out mice interferes with F1 fertility, impairs long-term potentiation, reduces acoustic startle and impairs conditioned freezing contextual memory with minimal transgenerational effects.

Polychlorinated biphenyls (PCBs) are toxic environmental pollutants. Humans are exposed to PCB mixtures via contaminated food or water. PCB exposure causes adverse effects in adults and after exposure in utero. PCB toxicity depends on the congener mixture and CYP1A2 gene activity. For coplanar PCBs, toxicity depends on ligand affinity for the aryl hydrocarbon receptor (AHR). Previously, we found that perinatal exposure of mice to a three-coplanar/five-noncoplanar PCB mixture induced deficits in novel object recognition and trial failures in the Morris water maze in Cyp1a2-/- ::Ahrb1 C57BL6/J mice compared with wild-type mice (Ahrb1  = high AHR affinity). Here we exposed gravid Cyp1a2-/- ::Ahrb1 mice to a PCB mixture on embryonic day 10.5 by gavage and examined the F1 and F3 offspring (not F2 ). PCB-exposed F1 mice exhibited increased open-field central time, reduced acoustic startle, greater conditioned contextual freezing and reduced CA1 hippocampal long-term potentiation with no change in spatial learning or memory. F1 mice also had inhibited growth, decreased heart rate and cardiac output, and impaired fertility. F3 mice showed few effects. Gene expression changes were primarily in F1 PCB males compared with wild-type males. There were minimal RNA and DNA methylation changes in the hippocampus from F1 to F3 with no clear relevance to the functional effects. F0 PCB exposure during a period of rapid DNA de-/remethylation in a susceptible genotype produced clear F1 effects with little evidence of transgenerational effects in the F3 generation. While PCBs show clear developmental neurotoxicity, their effects do not persist across generations for effects assessed herein.

Old Receptor, New Tricks-The Ever-Expanding Universe of Aryl Hydrocarbon Receptor Functions. Report from the 4th AHR Meeting, 29⁻31 August 2018 in Paris, France.

In a time where "translational" science has become a mantra in the biomedical field, it is reassuring when years of research into a biological phenomenon suddenly points towards novel prevention or therapeutic approaches to disease, thereby demonstrating once again that basic science and translational science are intimately linked. The studies on the aryl hydrocarbon receptor (AHR) discussed here provide a perfect example of how years of basic toxicological research on a molecule, whose normal physiological function remained a mystery for so long, has now yielded a treasure trove of actionable information on the development of targeted therapeutics. Examples are autoimmunity, metabolic imbalance, inflammatory skin and gastro-intestinal diseases, cancer, development and perhaps ageing. Indeed, the AHR field no longer asks, "What does this receptor do in the absence of xenobiotics?" It now asks, "What doesn't this receptor do?".

Chromium disrupts chromatin organization and CTCF access to its cognate sites in promoters of differentially expressed genes.

Hexavalent chromium compounds are well-established respiratory carcinogens used in industrial processes. While inhalation exposure constitutes an occupational risk affecting mostly chromium workers, environmental exposure from drinking water is a widespread gastrointestinal cancer risk, affecting millions of people throughout the world. Cr(VI) is genotoxic, forming protein-Cr-DNA adducts and silencing tumor suppressor genes, but its mechanism of action at the molecular level is poorly understood. Our prior work using FAIRE showed that Cr(VI) disrupted the binding of transcription factors CTCF and AP-1 to their cognate chromatin sites. Here, we used two complementary approaches to test the hypothesis that chromium perturbs chromatin organization and dynamics. DANPOS2 analyses of MNase-seq data identified several chromatin alterations induced by Cr(VI) affecting nucleosome architecture, including occupancy changes at specific genome locations; position shifts of 10 nucleotides or more; and changes in position amplitude or fuzziness. ATAC-seq analysis revealed that Cr(VI) disrupted the accessibility of chromatin regions enriched for CTCF and AP-1 binding motifs, with a significant co-occurrence of binding sites for both factors in the same region. Cr(VI)-enriched CTCF sites were confirmed by ChIP-seq and found to correlate with evolutionarily conserved sites occupied by CTCF in vivo, as determined by comparison with ENCODE-validated CTCF datasets from mouse liver. In addition, more than 30% of the Cr(VI)-enriched CTCF sites were located in promoters of genes differentially expressed from chromium treatment. Our results support the conclusion that Cr(VI) exposure promotes broad changes in chromatin accessibility and suggest that the subsequent effects on transcription regulation may result from disruption of CTCF binding and nucleosome spacing, implicating transcription regulatory mechanisms as primary Cr(VI) targets.

Aryl Hydrocarbon Receptor Ablation in Cardiomyocytes Protects Male Mice From Heart Dysfunction Induced by NKX2.5 Haploinsufficiency.

Epidemiological studies in humans and research in vertebrates indicates that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous and biopersistent environmental toxicant, is associated with incidence of early congenital heart disease in the embryo and later in the adult. TCDD-mediated toxicity depends on the aryl hydrocarbon receptor (AHR) but the role of the TCDD-activated AHR in cardiac function is not well-defined. To characterize the mechanisms responsible for AHR-mediated disruption of heart function, we generated several mouse strains with cardiomyocyte-specific Ahr gene knockout. Here, we report results on one of these strains in which the Ahr gene was deleted by cre recombinase regulated by the promoter of the cardiomyocyte-specific Nkx2.5 gene. We crossed mice with loxP-targeted Ahrfx/fx alleles with Nkx2.5+/cre mice bearing a "knock-in" cre recombinase gene integrated into one of the Nkx2.5 alleles. In these mice, loss of one Nkx2.5 allele is associated with disrupted cardiac development. In males, Nkx2.5 hemizygosity resulted in cardiac haploinsufficiency characterized by hypertrophy, dilated cardiomyopathy, and impaired ejection fraction. Ahr ablation protected Nkx2.5+/cre haploinsufficient males from cardiac dysfunction while inducing a significant increase in body weight. These effects were absent or largely blunted in females. Starting at 3 months of age, mice were exposed by oral gavage to 1 µg/kg/week of TCDD or control vehicle for an additional 2 months. TCDD exposure restored cardiac physiology in aging males, appearing to compensate for the heart dysfunction caused by Nkx2.5 hemizygosity. Our findings underscore the conclusion that deletion of the Ahr gene in cardiomyocytes protects males from heart dysfunction due to NKX2.5 haploinsufficiency.

Loss of NR2E3 represses AHR by LSD1 reprogramming, is associated with poor prognosis in liver cancer.

The aryl hydrocarbon receptor (AHR) plays crucial roles in inflammation, metabolic disorder, and cancer. However, the molecular mechanisms regulating AHR expression remain unknown. Here, we found that an orphan nuclear NR2E3 maintains AHR expression, and forms an active transcriptional complex with transcription factor Sp1 and coactivator GRIP1 in MCF-7 human breast and HepG2 liver cancer cell lines. NR2E3 loss promotes the recruitment of LSD1, a histone demethylase of histone 3 lysine 4 di-methylation (H3K4me2), to the AHR gene promoter region, resulting in repression of AHR expression. AHR expression and responsiveness along with H3K4me2 were significantly reduced in the livers of Nr2e3rd7 (Rd7) mice that express low NR2E3 relative to the livers of wild-type mice. SP2509, an LSD1 inhibitor, fully restored AHR expression and H3K4me2 levels in Rd7 mice. Lastly, we demonstrated that both AHR and NR2E3 are significantly associated with good clinical outcomes in liver cancer. Together, our results reveal a novel link between NR2E3, AHR, and liver cancer via LSD1-mediated H3K4me2 histone modification in liver cancer development.

Does the Aryl Hydrocarbon Receptor Regulate Pluripotency?

Recent evidence from embryonic stem cells suggests that the aryl hydrocarbon receptor (AHR) plays a central role in the regulation of pluripotency, a short-lived property of cells in the early blastula inner cell mass (ICM). Four key observations support this conclusion. The first is the temporal association between upregulation of AHR expression and the onset of cell differentiation, which argues for the AHR as a determinant of cell fate decisions. The second is the repression of the pluripotency factors OCT4 and NANOG by the AHR, which depresses their function and contributes to the cell's exit from pluripotency. The third is the temporal association between changes in global DNA methylation and stage-dependent AHR expression, which parallel each other during embryonic development, suggesting that AHR helps configure a repressive chromatin structure that controls differentiation. The fourth is the incidence of early developmental aberrations that take place in Ahr-null mice and cause the disruption of their embryonic program, which is likely to be a consequence of the loss of pluripotency of the Ahr-/- ICM cells. In this short review, we will focus on the modulation of pluripotency as a novel function of the AHR, and on the potentially detrimental developmental outcomes that may result from exposure to environmental toxicants. This line of enquiry brings us to the tantalizing conclusion that by activating mechanisms that modulate pluripotency, AHR regulates embryonic development. The likelihood that exposure to environmental AHR ligands might disrupt developmental processes is a reasonable corollary to this conclusion.

Quinone-mediated induction of cytochrome P450 1A1 in HepG2 cells through increased interaction of aryl hydrocarbon receptor with aryl hydrocarbon receptor nuclear translocator.

While it has long been believed that benzenes and naphthalenes are unable to activate the aryl hydrocarbon receptor (AhR) because they are poor ligands, we recently reported that these quinoid metabolites upregulated cytochrome P450 1A1 (CYP1A1) in Hepa1c1c7 cells (Abiko et al., 2015). In the current study, AhR activation, measured with a bioluminescence-based cell free assay, was induced by 1,2-naphthoquinone (1,2-NQ), a metabolite of naphthalene. Consistent with this, 1,4-benzoquinone (1,4-BQ), tert-butyl-1,4-BQ, and 1,4-NQ, as well as 1,2-NQ, all electrophilic mono- and bi-cyclic quinones, upregulated CYP1A1 mRNA and protein in HepG2 cells, whereas their parent aromatic hydrocarbons had little effect. Furthermore, immunofluorescence analysis confirmed that these quinones enhanced translocation of AhR to the nucleus.

Repression of the Aryl Hydrocarbon Receptor Is Required to Maintain Mitotic Progression and Prevent Loss of Pluripotency of Embryonic Stem Cells.

Lack of cell cycle checkpoints and uninterrupted passage through S-phase continuously renew the embryonic stem (ES) cell population and maintain pluripotency. Here, we show that to regulate mitotic progression and pluripotency ES cells must keep the aryl hydrocarbon receptor (AHR), an environmental sensor and transcriptional regulator, in a persistent state of repression. This repression, however, is not always absolute, causing the AHR to fluctuate between reversible states of expression and repression, with a fraction of the cells escaping repression at any one time. Cells that escape AHR repression exhibit reduced levels of the pluripotency factors OCT4 and SOX2 and show an extended mitotic traverse time due to AHR-dependent MID1 repression and the subsequent disruption of the MID1-PP2A-CDC25B-CDK1 signaling pathway that regulates mitosis. Unlike the bulk of the cell population that differentiates into cardiomyocytes upon stimulation, AHR-expressing ES cells restrict cardiogenesis and commit to a neuroglia cell fate. It appears that the untimely expression of the Ahr gene needs to be repressed to maintain ES cell mitotic progression and prevent premature loss of pluripotency. Stem Cells 2016;34:2825-2839.