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3,**
Department of Biochemistry,
*
Fels Institute for Cancer Research and Molecular Biology
and Department of Microbiology and Immunology,
**
Temple University School of Medicine, Philadelphia, PA 19140
3To whom correspondence should be addressed. E-mail: dsoprano{at}nimbus.temple.edu.
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ABSTRACT |
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 TOP ABSTRACT Retinoid receptor pathwayAryl hydrocarbon receptor...CYP1A1 transcriptional...Aryl hydrocarbon receptor...ConclusionLITERATURE CITED |
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KEY WORDS: • aryl hydrocarbon receptor • AhR nuclear translocator • retinoic acid receptor • retinoids • retinoid X receptor
Vitamin A [retinol, retinal and retinoic acid (RA)4]is a potent regulator of a diverse group of biological processes including growth, differentiation, cell proliferation and morphogenesis (for review, see 1
). The most potent naturally occurring vitamin A metabolites are all-trans-RA and 9-cis-RA. In addition to these biologically important forms of vitamin A, many thousand synthetic vitamin A-like compounds (termed retinoids) have been synthesized by chemists principally for pharmacological applications in the areas of cancer chemotherapy, cancer chemoprevention and dermatology (2
–4
). In this review, we describe studies that demonstrate that some synthetic retinoids that had been designed to bind to retinoic acid receptors (RARs) and to modulate the retinoid receptor pathway also bind to the aryl hydrocarbon receptor (AhR) and activate a completely unrelated pathway to RARs, the AhR pathway (Fig. 1
).
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Retinoid receptor pathway |
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TOPABSTRACTRetinoid receptor pathwayAryl hydrocarbon receptor...CYP1A1 transcriptional...Aryl hydrocarbon receptor...ConclusionLITERATURE CITED |
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). Three subtypes, called 
, ß and 
, of both RARs and RXRs have been described along with several isoforms of each subtype. These isoforms arise due to alternative promoter use and differential splicing. Analysis of the deduced amino acid sequences has demonstrated that RARs and RXRs are quite homologous to the steroid/thyroid hormone receptor superfamily. Each contains a highly conserved DNA binding domain (domain C), a well conserved ligand binding domain (LBD) (domain E) and three or four additional domains that are not as well conserved (domains A, B, D and F). In addition, high resolution crystal structures of the LBD of several RAR and RXR subtypes have demonstrated that the LBDs of these receptors share a novel protein fold termed an antiparallel -helical sandwich (for review, see 6
). Binding studies in vitro have demonstrated that the natural metabolites all-trans-RA and 9-cis-RA are high affinity ligands for RARs, whereas only 9-cis-RA has been shown to bind RXRs. As heterodimers (RAR/RXR) or homodimers (RXR/RXR), these proteins function as RA-inducible transcriptional regulatory proteins by binding to DNA sequences called RA response elements (RARE) or retinoid X response elements (RXRE) located within the promoter of target genes. In addition, RA in conjunction with RARs and RXRs has been demonstrated to inhibit activator protein-1 (AP-1) activity and to induce apoptosis in certain cell types.
Although each of the RAR and RXR subtypes binds RA (all-trans and/or 9-cis) with similar affinity, chemists have successfully designed many retinoids that are targeted to a specific retinoid receptor subtype or function of RA, including RAR subtype-selective agonists, RXR-selective agonists, RAR-selective antagonists, anti-AP-1-specific retinoids and retinoids that induce apoptosis (e.g., 7
–11
). The bulk of these third-generation retinoids are conformationally restricted, nonisoprenoid in structure and are modeled after compounds like TTNPB, AM80, Ch55 and TTNN. It is hoped that these new retinoids will be more specific in function and cause fewer side effects than RA, including reduced toxicity.
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Aryl hydrocarbon receptor pathway |
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TOPABSTRACTRetinoid receptor pathwayAryl hydrocarbon receptor...CYP1A1 transcriptional...Aryl hydrocarbon receptor...ConclusionLITERATURE CITED |
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and 13
).
Although a natural ligand for AhR has yet to be demonstrated, recent studies using AhR knockout mice suggest that AhR has important functions in the liver and in immune cells beyond mediating the response to environmental contaminants (14
,15
). Furthermore, livers from AhR knockout mice display a threefold increase in retinoids and a 65% decrease in RA metabolism, suggesting a role for AhR in the regulation of retinoid metabolism (16
). Interestingly these AhR null mice appear to be resistant to the adverse effects of TCDD, including its potent teratogenic effects on developing embryos (17
,18
). Based on these data and the findings of many other groups, it is currently hypothesized that most, if not all, of the adverse/toxic effects of PAHs and HAHs are mediated by AhR/Arnt via their binding to specific DNA sequences, called dioxin response elements (DREs), located in the promoter region of genes that are involved in the toxic responses (19
).
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CYP1A1 transcriptional regulation and retinoids |
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TOPABSTRACTRetinoid receptor pathwayAryl hydrocarbon receptor...CYP1A1 transcriptional...Aryl hydrocarbon receptor...ConclusionLITERATURE CITED |
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). Analysis of CYP1A1 expression in Hepa-1c1c7 cells also demonstrated that CYP1A1 mRNA concentrations, protein concentrations and enzymatic activity were all elevated by 10-5 mol of AGN 193109/L (20
). The treatment of either murine embryos or Hepa-1c1c7 cells with similar concentrations of RA did not cause an increase in the expression of CYP1A1, suggesting that the induction of CYP1A1 expression by AGN 193109 may not be mediated by RARs or RXRs. Furthermore, an examination of the DNA sequence of the promoter of the mouse CYP1A1 gene did not reveal any RAREs or RXREs. Finally, an elevation in CYP1A1 expression was not observed in mutant Hepa-1c1c7 cells defective in either AhR (taoBpRc1) or Arnt (BpRc1), suggesting that the AhR/Arnt signaling pathway, rather than the RAR/RXR signaling pathway, may be mediating the increase in CYP1A1 expression by AGN 193109 (20
).
As a method to rapidly identify other synthetic retinoids that increase CYP1A1 expression and activate the AhR pathway, we used a luciferase bioassay system (H1L1.1c2 cells). H1L1.1c2 cells contain a stably integrated luciferase reporter construct under the control of a portion of the CYP1A1 promoter that contains 4 DREs (19
). Twenty-three retinoids were tested for their ability to increase CYP1A1 transcriptional activity using this luciferase bioassay system. Three retinoids (AGN 190730, AGN 193109 and AGN 192837) were found to greatly increase CYP1A1 transcriptional activity (25-, 17- and 8-fold, respectively) when H1L1.1c2 cells were treated with 10-5 mol of each retinoid/L (Table 1
). Four retinoids (AGN 190121, AGN 191650, AGN 191312 and AGN 190205) increased CYP1A1 transcriptional activity to a lesser extent (approximately threefold), and 16 retinoids caused a minimal elevation in luciferase activity (AGN 193762, AGN191526, AGN 193313, AGN 190186, AGN 190246, AGN 191440, AGN 192240,RA,AM-80,AM-580,SR11254,SR11253,LE540,LE135,LE555s and AZ-80) when H1L1.1c2 cells were treated with 10-5 mol of each retinoid/L (21
).
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10,000-fold less potent than TCDD in the CYP1A1 transcriptional assay, their potency is similar to that of many other previously reported AhR ligands, including 3-methylcholanthrene, indole[3,2-b]carbazole, YH439, omeprazole, and benz[a]anthracene.
Like the prototypical AhR ligands, AGN 190730, AGN 193109 and AGN 192837 are polycyclic, aromatic, relatively planar and hydrophobic. It is interesting to note that retinoids with a highly rigid structure have higher AhR activity than those with a less rigid structure. As should be expected with distinct receptors, there is no apparent relationship between the activity of these three compounds on the RARs and their AhR activity (Table 1)
. All three AGN compounds were inactive on the RXRs. However, two compounds, AGN 190730 and AGN 192837, have relatively weak RAR-dependent transactivation activity and affinity for RARs, whereas AGN 193109 has a high affinity for all three RARs but no RAR-dependent transactivation activity (antagonist). Molecular modeling of one of these retinoids, AGN 193109, has demonstrated that it fits reasonably well into the hypothetical planar rectangle proposed by Gillner et al. (22
) to accommodate compounds that interact tightly with AhR and to occupy a similar position in this rectangle as known AhR ligands such as benzo[a]pyrene and indole derivative compound IV (20
).
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Aryl hydrocarbon receptor pathway and retinoids |
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TOPABSTRACTRetinoid receptor pathwayAryl hydrocarbon receptor...CYP1A1 transcriptional...Aryl hydrocarbon receptor...ConclusionLITERATURE CITED |
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presents a schematic representation of the AhR/ARNT signaling pathway indicating the five steps in this pathway that have been demonstrated to be modulated by specific synthetic retinoids. The first step in the AhR signaling pathway is the interaction of a ligand with AhR in the cytosol of the cell (Step 1 in Fig. 2
). Competitive binding studies have demonstrated that AGN 190730 can effectively inhibit the binding of TCDD to AhR in cytosolic extracts prepared from either Hepa-1c1c7 cells or mouse liver (21
).
|
). Ultimately AhR is translocated from the cytosol into the nucleus, with both heat shock protein 90 (HSP90) and ligand bound to the receptor. This conformational change can be examined in vitro by comparing the pattern of trypsin digestion of the unliganded protein with that of the liganded protein using in vitro transcribed/translated AhR. Kronenberg et al. (23
) demonstrated the formation of a 35-kDa fragment of the AhR protein that is resistant to trypsin digestion after treatment with TCDD. Like TCDD, AGN 193109 is also capable of causing the formation of a 35-kDa trypsin-resistant fragment that is indistinguishable from that observed in TCDD-treated samples. This result suggests that the binding of AGN 193109, like TCDD, to AhR results in a similar conformational change in AhR (22
).
Once in the nucleus, AhR dimerizes with ARNT to form an active transcription factor that recognizes the DRE motif in the promoter of target genes (24
) (Step 3 in Fig. 2
). Neither AhR nor ARNT is capable of recognizing DREs as homodimers (25
). To address whether AGN 190730 is capable of causing the formation of the active AhR/ARNT complex that binds to DREs, electrophoretic mobility shift assays were performed using in vitro transcribed/translated AhR and ARNT. The treatment of AhR and ARNT protein with AGN 190730 caused a shift in the migration of a [32P]-labeled DRE probe, similar to that observed after treatment with TCDD. In addition, to ensure that this AhR/ARNT complex is specific for the consensus DRE sequence, a DRE with two mutations within the consensus sequence was used as a probe. The migration of this mutant probe was not retarded after treatment of AhR and ARNT with either TCDD or AGN 190730, confirming that the formation of the AhR/ARNT complex after treatment with AGN 190730 is specific for the DRE sequence. These results provide strong evidence that AGN 190730 is capable of interacting with AhR to form an AhR/ARNT complex that recognizes a DRE.
The interaction of the AhR/ARNT complex with DREs in the promoter of target genes has been shown to increase the rate of transcription of these genes (24
) (Step 4 in Fig. 2
). Using a bioassay system composed of a DRE-driven luciferase reporter that is responsive to AhR ligands stably integrated into Hepa-1c1c7 cells, we demonstrated that AGN 190730, along with two other synthetic retinoids (AGN 193109 and AGN 192837), can elevate luciferase activity. Because an increase in transcriptional activation of the luciferase gene is directly attributable to recognition and binding of activated AhR/ARNT complexes to DREs, it can be concluded that the these three retinoids are capable of increasing the transcription rate of genes containing a DRE.
A similar conclusion can be reached by measuring the CYP1A1 mRNA and protein concentrations on treatment with synthetic retinoids. It is well known that AhR regulates the expression of CYP1A1 mRNA in response to ligands, such as TCDD, through formation of the AhR/ARNT complex and recognition of DREs in the promoter region of the CYP1A1 gene. Thus an increase in CYP1A1 mRNA (Step 4 in Fig. 2
) and a subsequent increase in CYP1A1 protein (Step 5) are most often used as a marker of AhR activation (26
,27
). AGN 193109 was observed to elevate CYP1A1 mRNA concentrations (20
) and AGN 193109, AGN 190730 and AGN 192837 each elevated CYP1A1 protein concentrations (20
,21
).
In conclusion, the synthetic retinoid AGN 190730 has been demonstrated to activate each of the important steps in the AhR/ARNT signaling pathway, and two additional synthetic retinoids (AGN 193109 and AGN 192837) have been shown to increase AhR-dependent transcriptional activity and CYP1A1 protein concentrations. Furthermore, because steps 1–3 were assayed in vitro, it is most likely that AGN 190730, and not a metabolite, directly mediates each of these events.
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Conclusion |
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TOPABSTRACTRetinoid receptor pathwayAryl hydrocarbon receptor...CYP1A1 transcriptional...Aryl hydrocarbon receptor...ConclusionLITERATURE CITED |
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. Although classic AhR ligands, including TCDD and benzo[a]pyrene, have been linked to a variety of toxic and carcinogenic effects in animals and humans, other AhR ligands such as YH439 and indole-3-carbinol do not appear to be harmful. Further studies are necessary to ascertain the biochemical and physiological consequences, if any, of the activation of the AhR signaling pathway by pharmacological doses of specific retinoids.
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FOOTNOTES |
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2 Supported by National Institutes of Health grants DE11954 and CA82770.
4 Abbreviations used: AhR, arylhydrocarbon receptor; AP-1, activator protein-1; Arnt, AhR nuclear translocator; CYP, cytochrome P450; DRE, dioxin response element; HAH, halogenated aromatic hydrocarbon; HSP90, heat shock protein 90; LBD, ligand binding domain; NLS, nuclear localization signal; PAH, polycyclic aromatic hydrocarbon; PAS, Per-Arnt-Sim; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; RXR, retinoid X receptor; RXRE, retinoid X response element; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
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LITERATURE CITED |
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TOPABSTRACTRetinoid receptor pathwayAryl hydrocarbon receptor...CYP1A1 transcriptional...Aryl hydrocarbon receptor...ConclusionLITERATURE CITED |
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