Loss of liver-specific and sexually dimorphic gene expression by aryl hydrocarbon receptor activation in C57BL/6 mice

<div><p>The aryl hydrocarbon receptor (AhR) is a highly conserved transcription factor that mediates a broad spectrum of species-, strain-, sex-, age-, tissue-, and cell-specific responses elicited by structurally diverse ligands including 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD). Dose-dependent effects on liver-specific and sexually dimorphic gene expression were examined in male and female mice gavaged with TCDD every 4 days for 28 or 92 days. RNA-seq data revealed the coordinated repression of 181 genes predominately expressed in the liver including albumin (3.7-fold), α-fibrinogen (14.5-fold), and β-fibrinogen (17.4-fold) in males with corresponding AhR enrichment at 2 hr. Liver-specific genes exhibiting sexually dimorphic expression also demonstrated diminished divergence between sexes. For example, male-biased <i>Gstp1</i> was repressed 3.0-fold in males and induced 4.5-fold in females, which were confirmed at the protein level. Disrupted regulation is consistent with impaired GHR-JAK2-STAT5 signaling and inhibition of female specific CUX2-mediated transcription as well as the repression of other key transcriptional regulators including <i>Ghr</i>, <i>Stat5b</i>, <i>Bcl6</i>, <i>Hnf4a</i>, <i>Hnf6</i>, <i>Foxa1/2/3</i>, <i>and Zhx2</i>. Attenuated liver-specific and sexually dimorphic gene expression was concurrent with the induction of fetal genes such as alpha-fetoprotein. The results suggest AhR activation causes the loss of liver-specific and sexually dimorphic gene expression producing a functionally “de-differentiated” hepatic phenotype.</p></div

AhR-mediated changes in sexually dimorphic regulator expression.

<p>(A) GHR-JAK2-STAT5 signaling cascade interactions with liver-specific gene expression regulators [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref016" target="_blank">16</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref018" target="_blank">18</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref019" target="_blank">19</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref043" target="_blank">43</a>]. GH-mediated activation of GHR induces STAT5 phosphorylation promoting male-specific gene expression, and induction of female-specific CUX2 (red outline) which regulates female-specific gene expression. CUX2 and STAT5 compete for binding sites resulting in sexually dimorphic gene expression. BCL6, HNF4α, HNF6, and FOXA also regulate sexually dimorphic and liver-specific gene regulation. Blue identifies repressed genes while white represents genes unaffected by TCDD. (B) Heatmap of sexually dimorphic and liver-specific gene expression regulators (A) in males (28d) and females (28 and 92d). Genes in blue were repressed while red indicates induction. STAT5 protein levels were determined in (C) female and (D) male mice gavaged with 30 μg/kg TCDD every 4 days for 28 days. Bars represent mean ± SEM for at least 3 animals (N = 3–4). Asterisks (*) indicate a significant difference compared to vehicle control as determined by Mann-Whitney U-test.</p

AhR enrichment and motif analyses.

<p>Comparison of AhR enrichment at liver-specific genes in male and female mice. (A) Values represent the number of unique genes in each segment of the Venn diagrams while numbers in parentheses show the number of AhR enriched regions containing a pDRE (matrix similarity score ≥ 0.85) for liver-specific genes. (B) Sequences beneath AhR enriched regions were queried for over-represented transcription factor binding motifs using Pscan-ChIP (Zambelli et al. 2013).</p

Immunohistochemical evaluation of hepatic AFP.

<p>Representative photomicrographs for AFP stained liver sections of (A) sesame oil vehicle treated females, (B) sesame oil vehicle treated males, (C) 30 μg/kg TCDD treated females, and (D) 30 μg/kg TCDD treated males. Scale bar represents 50 μm. The portal vein is designated by the letter V, bile ducts with the letter b, and AFP positive stained regions by solid black arrows.</p

Gene expression changes of liver related genes.

<p>Gene set enrichment analysis (GSEA) of 181 liver specific-genes in (A) male and (B) female mice gavaged with 30 μg/kg TCDD every 4 days for 28 days, or (C) female mice gavaged with 30 μg/kg TCDD every 4 days for 92 days. The 181 liver-specific genes were identified using published microarray datasets representing 96 tissues/cell types [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref034" target="_blank">34</a>]. Identification of liver-specific genes is described in materials and methods. TCDD elicited gene expression changes were ranked from most induced (left—red) to most repressed (right). Vertical black line represents identified liver-specific genes. The top panel (green line) represents a running-sum statistic (enrichment score) based on the lower panel, increasing when a gene is a member of the liver-specific gene set and decreasing when it is not. Enrichment scores increased most dramatically on the right indicating most of the liver-specific genes were repressed by TCDD. (D) Heat map of liver-specific gene expression changes elicited by TCDD. (E) Heatmap of TCDD-elicited repression of hepatokines. For heatmaps (D and E) blue indicates repression while red represents induction. The presence of pDREs (MSS ≥ 0.856) and hepatic AhR enrichment peaks (FDR ≤ 0.05) at 2h are shown as green boxes. Read count represents the maximum raw number of aligned reads to each transcript where yellow represents a lower level of expression (≤ 500 reads) and pink represents a higher level of expression (≥ 10,000).</p

Albuminoid genomic region.

<p>Albuminoid genomic region including <i>Alb</i>, <i>Afp</i>, and <i>Afm</i>. UCSC genome browser tracks show (1) the scale, (2) male AhR ChIP-seq peaks at 2 h, (3) male AhR enriched sites (FDR ≤ 0.05), (4) female AhR ChIP-seq peaks, (5) female AhR enriched sites (FDR ≤ 0.05), (6) location of pDREs (diagonal line indicates pDREs with a matrix similarity score ≥0.85), and (7) location of <i>Alb</i>, <i>Afp</i> and <i>Afm</i> genes within the albuminoid genomic region. <i>Gc</i>, the fourth albuminoid, is located 1 Mb upstream of <i>Alb</i> (not shown). Tracks are available for visualization at <a href="http://dbzach.fst.msu.edu/index.php/publications/supplementary-data/" target="_blank">http://dbzach.fst.msu.edu/index.php/publications/supplementary-data/</a>.</p

Hepatic protein levels changes by TCDD.

<p>Hepatic protein levels in male and female mice gavaged with sesame oil vehicle or 30 μg/kg TCDD every 4 days for 28 days were measured using the ProteinSimple Wes system. Bars represent mean log₂fold-change + SEM for at least 4 animals (N = 4–5). Asterisks (*) indicate a significant difference (<i>P</i> ≤ 0.05) compared to vehicle control determined by Mann-Whitney U-test.</p

Gene expression changes of sexually dimorphic genes.

<p>Gene set enrichment analysis (GSEA) of sexually dimorphic genes in (A-B) male and (B-C) female mice gavaged with TCDD every 4 days for 28 days. GSEA was performed using male-enriched and female-enriched gene sets determined as described in materials and methods. Gene expression changes were ranked from largest positive to largest negative. The top panel (green line) represents a ‘running-sum statistic’ which increases when a gene is in a gene set (denoted by black bars), and decreasing when it is not. TCDD repressed the majority of male-specific dimorphic genes (A) while inducing many female-specific genes (B) in male mice. Conversely, TCDD induced many male-specific genes (C) while repressing a majority of the female-specific genes (D) in female mice. (E) Heat map demonstrates the gene expression changes in male-specific and female-specific genes at 28 days.</p

Lipidomic Evaluation of Aryl Hydrocarbon Receptor-Mediated Hepatic Steatosis in Male and Female Mice Elicited by 2,3,7,8-Tetrachlorodibenzo‑<i>p</i>‑dioxin

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) induces hepatic steatosis mediated by the aryl hydrocarbon receptor. To further characterize TCDD-elicited hepatic lipid accumulation, mice were gavaged with TCDD every 4 days for 28 days. Liver samples were examined using untargeted lipidomics with structural confirmation of lipid species by targeted high-resolution MS/MS, and data were integrated with complementary RNA-Seq analyses. Approximately 936 unique spectral features were detected, of which 379 were confirmed as unique lipid species. Both male and female samples exhibited similar qualitative changes (lipid species) but differed in quantitative changes. A shift to higher mass lipid species was observed, indicative of increased free fatty acid (FFA) packaging. For example, of the 13 lipid classes examined, triglycerides increased from 46 to 48% of total lipids to 68–83% in TCDD treated animals. Hepatic cholesterol esters increased 11.3-fold in male mice with moieties consisting largely of dietary fatty acids (FAs) (i.e., linolenate, palmitate, and oleate). Phosphatidylserines, phosphatidylethanolamines, phosphatidic acids, and cardiolipins decreased 4.1-, 5.0-, 5.4- and 7.4-fold, respectively, while ceramides increased 6.6-fold. Accordingly, the integration of lipidomic data with differential gene expression associated with lipid metabolism suggests that in addition to the repression of <i>de novo</i> fatty acid synthesis and β-oxidation, TCDD also increased hepatic uptake and packaging of lipids, while inhibiting VLDL secretion, consistent with hepatic fat accumulation and the progression to steatohepatitis with fibrosis