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

Abstract

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 ␣-naphthoflavone (␣NF) mediates the suppressive, non-DRE dependent effects on SAA1 expression and partial DRE-mediated CYP1A1 induction. These observations suggest that ␣NF may be structurally modified to a derivative exhibiting only SAhRM activity. A screen of ␣NF derivatives identifies 3Ј,4Ј-dimethoxy-␣NF (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 ␣NF 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 ␣NF 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 M 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.

The aryl hydrocarbon receptor ligand omeprazole inhibits breast cancer cell invasion and metastasis

“…Knockdown of the AHR alone also significantly increased MDA-MB-231 cell invasion, suggesting that the receptor alone inhibited invasion. Similar results were observed in MDA-MB-231 cells cotreated with omeprazole and the AHR antagonists 3′,4′-methoxy-α-naphthoflavone (Figure 2B) [29] and 3′-methoxy-4′-nitroflavone (Figure 2C) [30], further confirming a role for the AHR in mediating the inhibitory effects of omeprazole on MDA-MB-231 breast cancer cell invasion. As a positive control, we also showed that AHR knockdown and the AHR antagonists also inhibited induction of CYP1A1 by TCDD and omeprazole (Figure 2D).…”

Comparisons of differential gene expression elicited by TCDD, PCB126, βNF, or ICZ in mouse hepatoma Hepa1c1c7 cells and C57BL/6 mouse liver

“…TCDD-induced enzyme expression and/or activity (AHH, EROD, and Cyp1a1 expression) (Astroff and Safe, 1989; Astroff et al, 1988; Bannister et al, 1989; Harris et al, 1989; Kim et al, 2006), AhR binding (Gasiewicz and Rucci, 1991), ER binding down-regulation (Romkes et al, 1987), and TCDD-induced porphyria (Yao and Safe, 1989), immunotoxicity (Davis and Safe, 1988; Dickerson et al, 1990), cleft palate, and hydronephrosis (Bannister et al, 1989; Biegel et al, 1989) can be antagonized by SAhRMs. More recently, SAhRMS that modulate hematopoietic progenitor expansion (Boitano et al, 2010), repress cytokine-mediated induction of complement factor genes (Murray et al, 2011), antagonize cytokine-mediated inflammatory signaling (Murray et al, 2010), and inhibit tumor growth (Jin et al, 2012; Safe et al, 1999; Yin et al, 2012) in absence of canonical DRE-elicited transcription have also been developed. Genome-wide AhR ChIP-chip and two-hybrid studies also suggest that structurally diverse ligands such as 3-methylcholanthrene (3-MC), PeCDD, PeCDF, TCDF, and PCB126 elicit DRE-dependent AhR-mediated selective modulation of gene expression (Pansoy et al, 2010; Zhang et al, 2008).…”

Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis

“…However, this point of view has been challenged by multiple studies. Ligandspecific biological responses have been observed in some instances of AhR-dependent transcription, including that of T cell differentiation and the effects of selective AhR modulators (18,(24)(25)(26)(27)(53)(54)(55)(56)(57). To account for these ligand-specific differences, modifications of the classical pathway have been suggested, with most involving ligand-selective binding of the AhR complex to unconventional DRE sequences (24,25,58) or binding by different subsets of coactivators (27).…”