Differential gene regulation by the human and mouse aryl hydrocarbon receptor

Abstract

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.

FIG. 1.

AHR^Ttr mice express functional hAHR protein that is inducible with TCDD treatment. (A) Western blot using anti-AHR and control β-actin antibodies of total protein isolated from AHR^Ttr, Ahr^fx/fx, Ahr^fx/fx/Cre^Alb, and Ahr^b/b primary mouse hepatocytes. (B) Real-time RT-PCR: mRNA from whole livers of TCDD-treated (n = 3) AHR^Ttr, Ahr^fx/fx, and Ahr^fx/fx/Cre^Alb mice were subjected to RT-PCR using primers for Cyp1a1, Cyp1b1, and Cyp1a2. Mice were treated with 100 μg/kg TCDD or vehicle for 6 h via ip injection. (C) Real-time RT-PCR of Cyp1a1 mRNA expression in TCDD-treated primary hepatocytes isolated from AHR^Ttr, Ahr^b/b, and Ahr^fx/fx/Cre^Alb mice. Primary hepatocytes isolated from AHR^Ttr, Ahr^b/b, and Ahr^fx/fx/Cre^Alb mice were treated with 10nM TCDD for 6 h. Total RNA, isolated from liver and hepatocytes, was converted to cDNA, and gene expression was quantified using real-time RT-PCR and primers for Cyp1a1, Cyp1a2, and Cyp1b1 (Supplementary table 1). *p < 0.05, **p < 0.01, ***p < 0.001.

FIG. 2.

Number of genes induced or repressed in hepatocytes isolated from hAHR- and mAHR-expressing mouse primary hepatocytes in response to 10nM TCDD treatment for 6 h. DNA microarray data were normalized using the Probe Logarithmic Intensity Error (PLIER-MM) algorithm. Normalized DNA microarray data outputs from TCDD- and control-treated Ahr^b/band AHR^Ttr hepatocytes were compared for differential expression using SAM (Pan, 2002; Tusher et al., 2001) with 100 permutations, KNN-10. The total number of genes induced and repressed by the hAHR or mAHR^b in response to TCDD was then calculated from the SAM output gene lists and plotted graphically.

FIG. 3.

Functional annotation cluster analysis of the genes that are differentially induced or repressed by the mAHR and hAHR. hAHR and mAHR^b signal: Probe Logarithmic Intensity Error (PLIER-MM) normalized signal for TCDD-induced samples. Change fold induction: relative PLIER-MM normalized signals for TCDD-treated mAHR^b and hAHR samples. Gene clusters of differentially regulated genes were generated using DAVID Functional Annotation Clustering Tool (Dennis et al., 2003; Huang da et al., 2009). The number of genes for each functional cluster differentially regulated by each receptor was then calculated from the gene cluster lists and represented graphically.

FIG. 4.

Real-time RT-PCR of differentially expressed genes in primary hepatocytes derived from AHR^Ttr, Ahr^fx/fx, and Ahr^fx/fx/Cre^Alb mice. (A) Adi1 and Oxtr mRNA expression assessed using the cognate primers (Supplementary table 1) and total RNA isolated from AHR^Ttr, Ahr^fx/fx, and Ahr^fx/fx/Cre^Alb mice. (B) Real-time RT-PCR: Egf and Gsn mRNA expression was assayed using RNA isolated from AHR^Ttr, Ahr^fx/fx, and Ahr^fx/fx/Cre^Alb mice. (A–B) Primary hepatocytes isolated from AHR^Ttr, Ahr^b/b, and Ahr^fx/fx/Cre^Alb mice were treated with 10nM TCDD or DMSO vehicle control for 6 h. All experiments were repeated three times. Total RNA samples were generated in triplicate, with samples represented by the three genotypes (AHR^Ttr, Ahr^fx/fx, and Ahr^fx/fx/Cre^Alb) originating from hepatocytes isolated from one mouse each. RNA samples were generated using a new batch of control- and TCDD-treated hepatocytes that were generated independently of the hepatocytes RNA samples subjected to microarray analysis. For all experiments, total RNA, isolated from hepatocytes, was converted to cDNA and gene expression was quantified using the cognate primers (Supplementary table 1). Real-time RT-PCR data were analyzed using two-way ANOVA and Bonferroni posttests, and p values < 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.