Colin A. Flaveny

Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells

Stem Cell Expansion The ability to expand hematopoietic stem cells (HSCs) during ex-vivo culture has been an important goal for over 20 years. Using a high-throughput chemical screen, Boitano et al. (p. 1345, published online 5 August; see the Perspective by Sauvageau and Humphries) found that a purine derivative, StemRegenin1 (SR1), promoted the expansion of human HSCs. Treatment of HSCs with SR1 (which blocked the activity of the aryl hydrocarbon receptor) led to the expansion of CD34+ cells and a 12 to 17-fold increase in the number of HSCs that engraft immune deficient mice. The identification of a mechanism for ex vivo amplification may facilitate clinical application of hematopoietic stem cell therapies. Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.

Kynurenic Acid Is a Potent Endogenous Aryl Hydrocarbon Receptor Ligand that Synergistically Induces Interleukin-6 in the Presence of Inflammatory Signaling

Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations.

The uremic toxin 3-indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in the regulation of multiple cellular pathways, such as xenobiotic metabolism and Th17 cell differentiation. Identification of key physiologically relevant ligands that regulate AHR function remains to be accomplished. Screening of indole metabolites has identified indoxyl 3-sulfate (I3S) as a potent endogenous ligand that selectively activates the human AHR at nanomolar concentrations in primary human hepatocytes, regulating transcription of multiple genes, including CYP1A1, CYP1A2, CYP1B1, UGT1A1, UGT1A6, IL6, and SAA1. Furthermore, I3S exhibits an approximately 500-fold greater potency in terms of transcriptional activation of the human AHR relative to the mouse AHR in cell lines. Structure-function studies reveal that the sulfate group is an important determinant for efficient AHR activation. This is the first phase II enzymatic product identified that can significantly activate the AHR, and ligand competition binding assays indicate that I3S is a direct AHR ligand. I3S failed to activate either CAR or PXR. The physiological importance of I3S lies in the fact that it is a key uremic toxin that accumulates to high micromolar concentrations in kidney dialysis patients, but its mechanism of action is unknown. I3S represents the first identified relatively high potency endogenous AHR ligand that plays a key role in human disease progression. These studies provide evidence that the production of I3S can lead to AHR activation and altered drug metabolism. Our results also suggest that prolonged activation of the AHR by I3S may contribute to toxicity observed in kidney dialysis patients and thus represent a possible therapeutic target.

Ligand Selectivity and Gene Regulation by the Human Aryl Hydrocarbon Receptor in Transgenic Mice

The aryl hydrocarbon receptor (AHR) is a ligand-inducible transcription factor that displays interspecies differences with the human and mouse AHR C-terminal region sequences sharing only 58% amino acid sequence identity. Compared with the mouse AHR (mAHR), the human AHR (hAHR) displays ∼10-fold lower relative affinity for prototypical AHR ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, which has been attributed to the amino acid residue valine 381 (alanine 375 in the mAHR) in the ligand binding domain of the hAHR. We investigated whether the 10-fold difference in ligand-binding affinity between the mAHR and hAHR would be observed with a diverse range of AHR ligands. To test this hypothesis, ligand binding assays were performed using the photo-affinity ligand 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin and liver cytosol isolated from hepatocyte-specific transgenic hAHR mice and C57BL/6J mice. It is noteworthy that competitive ligand-binding assays revealed that, compared with the mAHR, the hAHR has a higher relative affinity for certain compounds, including indirubin [(2Z)-2,3-biindole-2,3 (1′H,1′H)-dione and quercetin (2-(3,4dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one]. Electrophoretic mobility shift assays revealed that indirubin was more efficient at transforming the hAHR compared with the mAHR. Indirubin was also a more potent inducer of Cyp1a1 expression in transgenic hAHR mouse hepatocytes compared with C57BL/6J mouse hepatocytes. These observations suggest that indirubin is a potent hAHR ligand that is able to selectively bind to and activate the hAHR. These discoveries imply that there may be a significant degree of structural divergence between mAHR and hAHR ligands and highlights the importance of the hAHR transgenic mouse as a model to study the hAHR in vivo.

Ah receptor represses acute-phase response gene expression without binding to its cognate response element.

Repression of the nuclear factor-κB (NF-κB) pathway has been extensively researched because of its pivotal role in inflammation. We investigated the potential of the aryl hydrocarbon receptor (AHR) to suppress NF-κB regulated-gene expression, especially acute-phase genes, such as serum amyloid A (Saa). Using AHR mutants, it was determined that nuclear translocation and heterodimerization with AHR-nuclear translocator are essential, but DNA binding is not involved in AHR-mediated Saa repression. A number of AHR ligands were capable of repressing Saa3 expression. AHR activation leads to a decrease in RELA and C/EBP/β recruitment to and histone acetylation at Saa3 gene promoter. A battery of acute-phase genes (eg C-reactive protein and haptoglobin) induced by cytokine exposure was repressed by AHR activation in mouse hepatocytes. Dietary exposure to an AHR ligand represses cytokine-induced acute-phase response in the liver. Use of a human liver-derived cell line revealed similar repression of Saa mRNA levels and secreted protein. Repression of AHR expression also enhanced Saa induction in response to cytokines, suggesting that AHR is capable of constitutively repressing Saa gene expression. These results establish a role for AHR in inflammatory signaling within the liver, presenting a new therapeutic opportunity, and signify AHRs ability to function in a DNA-independent manner.

Differential Gene Regulation by the Human and Mouse Aryl Hydrocarbon Receptor

The human aryl hydrocarbon receptor (hAHR) and mouse aryl hydrocarbon receptor (mAHR(b)) share limited (58%) transactivation domain (TAD) sequence identity. Compared to the mAHR(b) allele, the hAHR displays 10-fold lower relative affinity for prototypical ligands, such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). However, in previous studies, we have demonstrated that the hAHR can display a higher relative ligand-binding affinity than the mAHR(b) for specific AHR ligands, such as indirubin. Each receptor has also been shown to differentially recruit LXXLL coactivator motif proteins and to utilize different TAD subdomains in gene transactivation. Using hepatocytes isolated from C57BL/6J mice (Ahr(b/b)) and AHR(Ttr) transgenic mice, which express hAHR protein specifically in hepatocytes, we investigated whether the hAHR and mAHR(b) differentially regulate genes. DNA microarray and quantitative PCR analysis of Ahr(b/b) and AHR(Ttr) primary mouse hepatocytes treated with 10nM TCDD revealed that a number of established AHR target genes such as Cyp1a1 and Cyp1b1 are significantly induced by both receptors. Remarkably, of the 1752 genes induced by mAHR(b) and 1186 genes induced by hAHR, only 265 genes (approximately 18%) were significantly activated by both receptors in response to TCDD. Conversely, of the 1100 and 779 genes significantly repressed in mAHR(b) and hAHR hepatocytes, respectively, only 462 (approximately 49%) genes were significantly repressed by both receptors in response to TCDD treatment. Genes identified as differentially expressed are known to be involved in a number of biological pathways, including cell proliferation and inflammatory response, which suggest that compared to the mAHR(b), the hAHR may play contrasting roles in TCDD-induced toxicity and endogenous AHR-mediated gene regulation.

Evidence for Ligand-Mediated Selective Modulation of Aryl Hydrocarbon Receptor Activity

The concept of selective receptor modulators has been established for the nuclear steroid hormone receptors. Such selective modulators have been used therapeutically with great success in the treatment of cancer. However, this concept has not been examined with regard to the aryl hydrocarbon receptor (AHR) because of the latent toxicity commonly associated with AHR activation. AHR-mediated toxicity is primarily derived from AHR binding to its dioxin response element (DRE) and driving expression of CYP1 family members, which have the capacity to metabolize procarcinogens to genotoxic carcinogens. Recent evidence using a non-DRE binding AHR mutant has established the DRE-independent suppression of inflammatory markers by the AHR. We wished to determine whether such DRE-independent repression with wild-type AHR could be dissociated from canonical DRE-dependent transactivation in a ligand-dependent manner and, in doing so, prove the concept of a selective AHR modulator (SAhRM). Here, we identify the selective estrogen receptor (ER) modulator Way-169916 as a dually selective modulator, binding both ER and AHR. Inflammatory gene expression associated with the cytokine-inducible acute-phase response (e.g., SAA1 and CRP) are diminished by Way-169916 in an AHR-dependent manner. Furthermore, activation of AHR by Way-169916 fails to stimulate canonical DRE-driven AHR-mediated CYP1A1 expression, thus eliminating the potential for AHR-mediated genotoxic stress. Such anti-inflammatory activity in the absence of DRE-mediated expression fulfills the major criteria of an SAhRM, which suggests that selective modulation of AHR is possible and renders the AHR a therapeutically viable drug target for the amelioration of inflammatory disease.

Development of a Selective Modulator of Aryl Hydrocarbon (Ah) Receptor Activity that Exhibits Anti-Inflammatory Properties

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, the role of the AHR in normal physiology is still an area of intense investigation. For example, this receptor plays an important role in certain immune responses. We have previously determined that the AHR can mediate repression of acute-phase genes in the liver. For this observation to be therapeutically useful, selective activation of the AHR would likely be necessary. Recently, the selective estrogen receptor ligand WAY-169916 has also been shown to be a selective AHR ligand. WAY-169916 can efficiently repress cytokine-mediated acute-phase gene expression (e.g., SAA1) yet fail to mediate a dioxin response element-driven increase in transcriptional activity. The goals of this study were to structurally modify WAY-169916 to block binding to the estrogen receptor and increase its affinity for the AHR. A number of WAY-169916 derivatives were synthesized and subjected to characterization as AHR ligands. The substitution of a key hydroxy group for a methoxy group ablates binding to the estrogen receptor and increases its affinity for the AHR. The compound 1-allyl-7-trifluoromethyl-1H-indazol-3-yl]-4-methoxyphenol (SGA 360), in particular, exhibited essentially no AHR agonist activity yet was able to repress cytokine-mediated SAA1 gene expression in Huh7 cells. SGA 360 was tested in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated ear inflammatory edema model using C57BL6/J and Ahr(-/-) mice. Our findings indicate that SGA 360 significantly inhibits TPA-mediated ear swelling and induction of a number of inflammatory genes (e.g., Saa3, Cox2, and Il6) in C57BL6/J mice. In contrast, SGA 360 had no effect on TPA-mediated ear swelling or inflammatory gene expression in Ahr(-/-) mice. Collectively, these results indicate that SGA 360 is a selective Ah receptor modulator (SAhRM) that exhibits anti-inflammatory properties in vivo.

Antagonism of aryl hydrocarbon receptor signaling by 6, 2', 4'-trimethoxyflavone

The aryl hydrocarbon receptor (AHR) is regarded as an important homeostatic transcriptional regulator within physiological and pathophysiological processes, including xenobiotic metabolism, endocrine function, immunity, and cancer. Agonist activation of the AHR is considered deleterious based on toxicological evidence obtained with environmental pollutants, which mediate toxic effects through AHR. However, a multitude of plant-derived constituents, e.g., polyphenols that exhibit beneficial properties, have also been described as ligands for the AHR. It is conceivable that some of the positive aspects of such compounds can be attributed to suppression of AHR activity through antagonism. Therefore, we conducted a dioxin response element reporter-based screen to assess the AHR activity associated with a range of flavonoid compounds. Our screen identified two flavonoids (5-methoxyflavone and 7,4′-dimethoxyisoflavone) with previously unidentified AHR agonist potential. In addition, we have identified and characterized 6,2′,4′-trimethoxyflavone (TMF) as an AHR ligand that possesses the characteristics of an antagonist having the capacity to compete with agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene, thus effectively inhibiting AHR-mediated transactivation of a heterologous reporter and endogenous targets, e.g., CYP1A1, independent of cell lineage or species. Furthermore, TMF displays superior action by virtue of having no partial agonist activity, in contrast to other documented antagonists, e.g., α-napthoflavone, which are partial weak agonists. TMF also exhibits no species or promoter dependence with regard to AHR antagonism. TMF therefore represents an improved tool allowing for more precise dissection of AHR function in the absence of any conflicting agonist activity.

Suppression of Cytokine-Mediated Complement Factor Gene Expression through Selective Activation of the Ah Receptor with 3′,4′-Dimethoxy-α-naphthoflavone

We have characterized previously a class of aryl hydrocarbon receptor (AHR) ligand termed selective AHR modulators (SAhRMs). SAhRMs exhibit anti-inflammatory properties, including suppression of cytokine-mediated acute phase genes (e.g., Saa1), through dissociation of non–dioxin-response element (DRE) AHR activity from DRE-dependent xenobiotic gene expression. The partial AHR agonist α-naphthoflavone (αNF) mediates the suppressive, non-DRE dependent effects on SAA1 expression and partial DRE-mediated CYP1A1 induction. These observations suggest that αNF may be structurally modified to a derivative exhibiting only SAhRM activity. A screen of αNF derivatives identifies 3′,4′-dimethoxy-αNF (DiMNF) as a candidate SAhRM. Competitive ligand binding validates DiMNF as an AHR ligand, and DRE-dependent reporter assays with quantitative mRNA analysis of AHR target genes reveal minimal agonist activity associated with AHR binding. Consistent with loss of agonist activity, DiMNF fails to promote AHR binding to DRE probes as determined through electromobility shift assay. Importantly, mRNA analysis indicates that DiMNF retains the suppressive capacity of αNF regarding cytokine-mediated SAA1 expression in Huh7 cells. Interestingly, predictive docking modeling suggests that DiMNF adopts a unique orientation within the AHR ligand binding pocket relative to αNF and may facilitate the rational design of additional SAhRMs. Microarray studies with a non-DRE binding but otherwise functional AHR mutant identified complement factor C3 as a potential SAhRM target. We confirmed this observation in Huh7 cells using 10 μM DiMNF, which significantly repressed C3 mRNA and protein. These data expand the classes of AHR ligands exerting DRE-independent anti-inflammatory SAhRM activity, suggesting SAhRMs may have application in the amelioration of inflammatory disorders.