Alvaro Puga

Role of the Ah Receptor and the Dioxin‐Inducible [Ah] Gene Battery in Toxicity, Cancer, and Signal Transductiona

1. On the basis of our current knowledge about the evolution of drug-metabolizing enzymes, it appears to be extremely likely that these enzymes play a critical role in maintaining steady-state levels of the ligands involved in ligand-modulated transcription of genes effecting growth, differentiation, homeostasis, and neuroendocrine functions. 2. The original observations about genetic differences in CYP1A1 (cytochrome P1-450) induction by TCDD or benzo[a]pyrene in the mouse have led to an appreciation for a similar polymorphism in the human and the recent cloning of the murine Ah receptor (Ahr) and human Ah receptor nuclear translocator (ARNT) genes. It is most likely that the correlation between genetic differences in human or murine CYP1A1 inducibility by polycyclic hydrocarbons or TCDD and increased risk of cancer will be explained by differences in the AHR gene, leading to enhanced tumor promotion (rather than in the CYP1A1 structural gene). Perhaps the same will be found for birth defects, immunotoxicity, and other forms of toxic damage caused by these environmental chemicals. 3. In a manner similar to that of the phorbol ester tumor promoter, TCDD induces intracellular Ca2+ changes, accumulation of FOS and JUN mRNAs, and large increases in AP-1 transcription factor activity. Interestingly, these early effects of TCDD, and also of benzo[a]pyrene, appear not to require the Ah receptor. 4. Many genes are induced by TCDD, and many others are induced by electrophilic metabolites such as quinones and H2O2; using several mouse experimental systems, we have defined a subset of six of these genes as constituting the [Ah] battery by the sole criterion that a functional CYP1A1 or CYP1A2 enzyme is able to repress the expression of genes that are members of this gene battery.

The aryl hydrocarbon receptor cross-talks with multiple signal transduction pathways

Exposure to toxic polycyclic aromatic hydrocarbons raises a number of toxic and carcinogenic responses in experimental animals and humans mediated for the most part by the aryl hydrocarbon -- or dioxin -- receptor (AHR). The AHR is a ligand-activated transcription factor whose central role in the induction of drug-metabolizing enzymes has long been recognized. For quite some time now, it has become clear that the AHR also functions in pathways outside of its role in detoxification and that perturbation of these pathways by xenobiotic ligands may be an important part of the toxicity of these compounds. AHR activation by some of its ligands participates among others in pathways critical to cell cycle regulation, mitogen-activated protein kinase cascades, immediate-early gene induction, cross-talk within the RB/E2F axis and mobilization of crucial calcium stores. Ultimately, the effect of a particular AHR ligand may depend as much on the adaptive interactions that it established with pathways and proteins expressed in a specific cell or tissue as on the toxic responses that it raises.

Aromatic Hydrocarbon Receptor Interaction with the Retinoblastoma Protein Potentiates Repression of E2F-dependent Transcription and Cell Cycle Arrest

Polyhalogenated aromatic hydrocarbons, of which 2,3,7,8-tetrachloro-p-dioxin (TCDD) is the prototype compound, elicit a variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. In cultured cells, TCDD shows marked effects on the regulation of cell cycle progression, including thymocyte apoptosis, induction of keratinocyte proliferation and terminal differentiation, and inhibition of estrogen-dependent proliferation in breast cancer cells. The presence of an LXCXE domain in the dioxin aromatic hydrocarbon receptor (AHR), suggested that the effects of TCDD on cell cycle regulation might be mediated by protein-protein interactions between AHR and the retinoblastoma protein (RB). Using the yeast two-hybrid system, AHR and RB were in fact shown to bind to each other. In vitro pull-down experiments with truncated AHR peptides indicated that at least two separate AHR domains form independent complexes with hypophosphorylated RB. Coimmunoprecipitation of whole cell lysates from human breast carcinoma MCF-7 cells, which express both proteins endogenously, revealed that AHR associates with RB in vivo only after receptor transformation and nuclear translocation. However, the AHR nuclear translocator and transcriptional heterodimerization partner, is not required for (nor is it a part of) the AHR·RB complexes detected in vitro. Ectopic expression of AHR and RB in human osteosarcoma SAOS-2 cells, which lack endogenous expression of both proteins, showed that AHR synergizes with RB to repress E2F-dependent transcription and to induce cell cycle arrest. Furthermore, AHR partly blocked T-antigen-mediated reversal of RB-dependent transcriptional repression. These results uncover a potential function for the AHR in cell cycle regulation and suggest that this function may be that of serving as an environmental sensor that signals cell cycle arrest when cells are exposed to certain environmental toxicants.

Aryl hydrocarbon receptor, cell cycle regulation, toxicity, and tumorigenesis.

Most effects of exposure to halogenated and polycyclic aromatic hydrocarbons are mediated by the aryl hydrocarbon receptor (AHR). It has long been recognized that the AHR is a ligand‐activated transcription factor that plays a central role in the induction of drug‐metabolizing enzymes and hence in xenobiotic detoxification. Of late, it has become evident that outside this well‐characterized role, the AHR also functions as a modulator of cellular signaling pathways. In this Prospect, we discuss the involvement of the AHR in pathways critical to cell cycle regulation, mitogen‐activated protein kinase cascades, immediate‐early gene induction, and the functions of the RB protein. Ultimately, the toxicity of AHR xenobiotic ligands may be intrinsically connected with the perturbation of these pathways and depend on the many critical signaling pathways and effectors with which the AHR itself interacts. J. Cell. Biochem.

Constitutive activation of the aromatic hydrocarbon receptor.

ABSTRACT The ligand-activated aromatic hydrocarbon receptor (AHR) dimerizes with the AHR nuclear translocator (ARNT) to form a functional complex that transactivates expression of the cytochrome P-450CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. Previous work from this laboratory has shown that the activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation we used CYP1A1-deficient mouse hepatomac37 cells and CYP1A1- and AHR-deficient African green monkey kidney CV-1 cells. Using gel mobility shift and luciferase reporter gene expression assays, we found that c37 cells that had not been exposed to exogenous Ah receptor ligands already contained transcriptionally active AHR-ARNT complexes, a finding that we also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AHR and ARNT leads to high levels of AHR–ARNT-dependent luciferase gene expression even in the absence of an agonist. Using a green fluorescent protein-tagged AHR, we showed that elevated reporter gene expression correlates with constitutive nuclear localization of the AHR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (i) expression of a functional CYP1A1 enzyme, (ii) competition with chimeric or truncated AHR proteins containing the AHR ligand-binding domain, and (iii) treatment with the AHR antagonist α-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AHR ligand responsible for endogenous activation of the Ah receptor.

The transcriptional signature of dioxin in human hepatoma HepG2 cells

We have used a high density microarray hybridization approach to characterize the transcriptional response of human hepatoma HepG2 cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We find that exposure to 10 nM TCDD for 8 hr alters by at least a factor of 2.1 the expression of 310 known genes and of an equivalent number of expressed sequence tags. Treatment with TCDD in the presence of 20 microg/mL of cycloheximide blocked the effect on 202 of these genes, allowing us to distinguish between primary effects of TCDD exposure, which take place whether cycloheximide is present or not, and secondary effects, which are blocked by inhibition of protein synthesis. Of the 310 known genes affected by TCDD, 30 are up-regulated and 78 are down-regulated regardless of cycloheximide treatment, and 84 are up-regulated and 118 are down-regulated only when protein synthesis is not inhibited. Functional clustering of genes regulated by TCDD reveals many potential physiological interactions that might shed light on the multiple biological effects of this compound. Our results, however, suggest that arriving at a sound understanding of the molecular mechanisms governing the biological outcome of TCDD exposure promises to be orders of magnitude more complicated than might have been previously imagined.

Heme oxygenase-1 induction by NRF2 requires inactivation of the transcriptional repressor BACH1

Oxidative stress activates the transcription factor NRF2, which in turn binds cis-acting antioxidant response element (ARE) enhancers and induces expression of protective antioxidant genes. In contrast, the transcriptional repressor BACH1 binds ARE-like enhancers in cells naïve to oxidative stress and antagonizes NRF2 binding until it becomes inactivated by pro-oxidants. Here, we describe the dynamic roles of BACH1 and NRF2 in the transcription of the heme oxygenase-1 (HMOX1) gene. HMOX1 induction, elicited by arsenite-mediated oxidative stress, follows inactivation of BACH1 and precedes activation of NRF2. BACH1 repression is dominant over NRF2-mediated HMOX1 transcription and inactivation of BACH1 is a prerequisite for HMOX1 induction. In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1. By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction. Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

Effects of arsenic exposure on DNA methylation and epigenetic gene regulation

Arsenic is a nonmutagenic human carcinogen that induces tumors through unknown mechanisms. A growing body of evidence suggests that its carcinogenicity results from epigenetic changes, particularly in DNA methylation. Changes in gene methylation status, mediated by arsenic, have been proposed to activate oncogene expression or silence tumor suppressor genes, leading to long-term changes in the activity of genes controlling cell transformation. Mostly descriptive, and often contradictory, studies have demonstrated that arsenic exposure is associated with both hypo- and hyper-methylation at various genetic loci in vivo or in vitro. This ambiguity has made it difficult to assess whether the changes induced by arsenic are causally involved in the transformation process or are simply a reflection of the altered physiology of rapidly dividing cancer cells. Here, we discuss the evidence supporting changes in DNA methylation as a cause of arsenic carcinogenesis and highlight the strengths and limitations of these studies, as well as areas where consistencies and inconsistencies exist.

Role of the aryl hydrocarbon receptor in cell cycle regulation

Traditionally, the aryl hydrocarbon receptor (AHR) is considered to be a ligand-activated receptor and transcription factor responsible for the induction of drug-metabolizing enzymes. Its role in the combinatorial matrix of cell functions was neatly established long before the first report of an AHR cDNA sequence was published. Only recently, other functions of this protein have begun to be recognized. This review addresses novel findings relating to AHR functions that have resulted from experimental approaches markedly outside traditional receptor analyses. Here we examine the aspects of AHR biology relevant to its role in cell cycle regulation, from the activation of mitogen-activated protein kinases to the cross-talk between AHR and the RAS pathway and the functional significance of the interaction between AHR and the retinoblastoma protein. We have attempted to provide the reader with a balanced interpretation of the evidence, highlighting areas of consensus as well as areas still being contested.

Activation of mitogen-activated protein kinases (MAPKs) by aromatic hydrocarbons: role in the regulation of aryl hydrocarbon receptor (AHR) function.

The aromatic hydrocarbon (Ah) receptor (AHR) is the only known cellular receptor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of many other widespread environmental contaminants that cause diverse toxic effects in animals and humans. Most, if not all, the biological effects of TCDD are mediated by the activation of AHR, which is a ligand-activated transcription factor required for ligand-induced expression of several detoxification genes, including those encoding for cytochrome P450 enzymes CYP1A1, CYP1A2, and CYP1B1. Environmental agents also activate several mitogen-activated protein kinase (MAPK) pathways, believed to modulate transcription factor function and to regulate gene expression. However, the contribution to TCDD toxicity resulting from cross-talk between AHR and MAPK pathways has yet to be determined. In this study, we show that TCDD and other AHR ligands induced the immediate activation of the extracellular signal-regulated kinases and the Jun N-terminal kinases, but not the p38 MAPKs. MAPK activation by TCDD did not require the AHR, since it occurred equally well in AHR-negative CV-1 cells and in Ahr (-/-) mouse embryonic fibroblasts as in AHR-positive cells. Distinct from serum factors and the tumor promoter TPA-induced MAPKs, which resulted in transcriptional activation of ELK or c-JUN, TCDD-stimulated MAPKs were critical for the induction of AHR-dependent gene transcription and CYP1A1 expression. These data indicate that AHR ligands elicit AHR-independent non-genomic events that are essential for AHR activation and function.