Comparative analysis of AhR-mediated TCDD-elicited gene expression in human liver adult stem cells

Abstract

Time course and dose-response studies were conducted in HL1-1 cells, a human liver cell line with stem cell-like characteristics, to assess the differential gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compared with other established models. Cells were treated with 0.001, 0.01, 0.1, 1, 10, or 100nM TCDD or dimethyl sulfoxide vehicle control for 12 h for the dose-response study, or with 10nM TCDD or vehicle for 1, 2, 4, 8, 12, 24, or 48 h for the time course study. Elicited changes were monitored using a human cDNA microarray with 6995 represented genes. Empirical Bayes analysis identified 144 genes differentially expressed at one or more time points following treatment. Most genes exhibited dose-dependent responses including CYP1A1, CYP1B1, ALDH1A3, and SLC7A5 genes. Comparative analysis of HL1-1 differential gene expression to human HepG2 data identified 74 genes with comparable temporal expression profiles including 12 putative primary responses. HL1-1-specific changes were related to lipid metabolism and immune responses, consistent with effects elicited in vivo. Furthermore, comparative analysis of HL1-1 cells with mouse Hepa1c1c7 hepatoma cell lines and C57BL/6 hepatic tissue identified 18 and 32 commonly regulated orthologous genes, respectively, with functions associated with signal transduction, transcriptional regulation, metabolism and transport. Although some common pathways are affected, the results suggest that TCDD elicits species- and model-specific gene expression profiles.

FIG. 1.

Basal AhR mRNA and protein expression in HL1-1 cells. (A) AhR mRNA levels were measured by QRT-PCR and normalized to several house keeping (HK) genes. HL1-1: human liver stem cell, HepG2: human hepatoma cell, Hepa1c1c7: mouse hepatoma cell, and Mm liver: C57BL/6 mouse liver. Error bars represent the SEM for the average. (B) AhR protein expression in HL1-1 cells, HepG2 cells, Hepa1c1c7 cells, and C57BL/6 mouse liver detected by Western analysis. The mw is estimated to be 112 and 95 kDa for human and rodent AhR, respectively.

FIG. 2.

QRT-PCR verification of CYP1A1 mRNA levels from the dose-response (12 h) (A) and time course (10nM TCDD) (B) studies in HL1-1 cells treated with TCDD. The EC₅₀ value for CYP1A1 expression was 8.3nM. Error bars represent the SEM for the average fold change. The asterisk (*) indicates p < 0.05.

FIG. 3.

Number of genes exhibiting differential expression in the dose-response (12 h) (A) and time course studies (10nM TCDD) (B). Differentially expressed genes clustered according to early (2-4 h), mid- (8–12 h), and late- (24–48 h) time points (Supplementary Fig. 4)

FIG. 4.

QRT-PCR verification of CYP1B1, ALDH1A3, and SLC7A5 microarray results in the time course (10nM TCDD) and dose-response (12 h) studies. Fold changes were calculated relative to time-matched vehicle controls. Bar (left axis) and lines (right axis) represent QRT-PCR and cDNA microarray data, respectively. EC₅₀ values were calculated from QRT-PCR and cDNA microarray (parenthesized value) data, respectively. Results are represented as the average of three biological replicates. QRT-PCR data error bars represent the SEM for the average fold change. The asterisk (*) indicates p < 0.05 for QRT-PCR.

FIG. 5.

Putative primary TCDD-responsive genes from CHX study. Microarray analysis identified 78 and 203 TCDD-responsive genes at 4 and 12 h, respectively. CHX cotreatment identified 47 and 53 genes classified as putative primary responsive genes at 4 and 12 h, respectively. A total of 78 unique putative primary responses were identified at both time points.

FIG. 6.

Comparative analysis of HL1-1, HepG2, Hepa1c1c7 and C57BL/6 hepatic tissue gene expression profiles. (A) The number of genes represented on the human and mouse cDNA arrays. In total 5505 orthologs were represented on both platforms. (B) Comparative analysis of the TCDD-elicited temporal gene expression of HL1-1, HepG2, Hepa1c1c7, and C57BL/6 mice hepatic tissue (Mm liver) studies. Collectively, 251, 1057, 770, and 1465 differentially expressed genes were identified from HL1-1, HepG2, Hepa1c1c7 and mouse liver TCDD time course study, respectively, using relaxed filtering criteria (P1(t) > 0.999 and |fold change| > 1.3) to include responses on the margins. A total of 74, 18, and 32 differentially expressed genes were identified when comparing HL1-1 cells to HepG2 cells, Hepa1c1c7 cells, and C57BL/5 hepatic tissue, respectively. Further analysis identified 13 conserved genes between HL1-1, HepG2, and mouse liver, three conserved genes between HL1-1, HepG2, and Hepa1c1c7, and eight conserved genes between HL1-1, Hepa1c1c7, and mouse liver.