Short Chain Fatty Acids Enhance Aryl Hydrocarbon (Ah) Responsiveness in Mouse Colonocytes and Caco-2 Human Colon Cancer Cells

Abstract

Aryl hydrocarbon receptor (AhR) ligands are important for gastrointestinal health and play a role in gut inflammation and the induction of T regulatory cells, and the short chain fatty acids (SCFAs) butyrate, propionate and acetate also induce similar protective responses. Initial studies with butyrate demonstrated that this compound significantly increased expression of Ah-responsive genes such as Cyp1a1/CYP1A1 in YAMC mouse colonocytes and Caco-2 human colon cancer cell lines. Butyrate synergistically enhanced AhR ligand-induced Cyp1a1/CYP1A1 in these cells with comparable enhancement being observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and also microbiota-derived AhR ligands tryptamine, indole and 1,4-dihydroxy-2-naphthoic acid (DHNA). The effects of butyrate on enhancing induction of Cyp1b1/CYP1B1, AhR repressor (Ahrr/AhRR) and TCDD-inducible poly(ADP-ribose)polymerase (Tiparp/TiPARP) by AhR ligands were gene- and cell context-dependent with the Caco-2 cells being the most responsive cell line. Like butyrate and propionate, the prototypical hydroxyamic acid-derived histone deacetylase (HDAC) inhibitors Panobinostat and Vorinostat also enhanced AhR ligand-mediated induction and this was accompanied by enhanced histone acetylation. Acetate also enhanced basal and ligand-inducible Ah responsiveness and histone acetylation, demonstrating that acetate was an HDAC inhibitor. These results demonstrate SCFA-AhR ligand interactions in YAMC and Caco-2 cells where SCFAs synergistically enhance basal and ligand-induced expression of AhR-responsive genes.

Figure 1

Butyrate modulates expression of Ah-responsive genes in YAMC and Caco-2 cells. Cells were treated with DMSO or 1–10 mM butyrate for 24 hr, and expression of AhR mRNA (A) and protein (B) were determined by real time PCR and western blots, respectively. Cells were treated with DMSO and 1–10 mM butyrate for 24 hr, and levels of Cyp1a1/CYP1A1 (C), Cyp1b1/CYP1B1 (D), Ahrr/AhRR (D) and Tiparp/TiPARP (F) mRNAs were determined by real time PCR. (G) Cells were treated with butyrate alone or in combination with CH223191, and Cyp1a1/CYP1A1 mRNA was determined by real time PCR. (H) YAMC-AhR-KO cells were treated with butyrate, and Cyp1a1 mRNA levels were determined by real time PCR. Results are expressed as means ± SE (3 replicated determination), and significantly (p < 0.05) induced (*) and inhibited (**) responses are indicated.

Figure 2

Butyrate enhances TCDD-induced gene expression YAMC and Caco-2 cells. Cells were treated with DMSO, TCDD and TCDD plus 5 mM butyrate, and effects on Cyp1a1/CYP1A1 (A), Cyp1b1/CYP1B1 (B), Ahrr/AhRR (C) and Tiparp/TiPARP (D) mRNA levels were determined by real time PCR. (E) Cells were treated with DMSO, TCDD, butyrate and their combination for 24 hr, and whole cell lysates were analyzed by western blots. (F) Cells were treated with DMSO, butyrate, TCDD and TCDD plus butyrate and also in combination with CH223191, and Cyp1a1/CYP1A1 mRNA levels were determined by real time PCR. (G) Cells were treated with DMSO, 5 mM butyrate, different concentrations of TCDD alone and TCDD plus butyrate, and Cyp1a1/CYP1A1 mRNA levels were determined by real time PCR. With the exception of AhRR in Caco-2 cells, TCDD significantly induced all other Ah-responsive genes, and significant (p < 0.05) enhancement by butyrate is indicated (*). Results are expressed as means ± SE for at least 3 separate determinations for each treatment group. Significant (p < 0.05) inhibition by CH223191 is indicated (**).

Figure 3

Butyrate enhances AhR ligand-induced gene expression in YAMC and Caco-2 cells. Cells were treated with DMSO, different AhR ligands alone or in combination with 5 mM butyrate and effects on expression of AhR (A), Cyp1a1/CYP1A1 (B), Cyp1b1/CYP1B1 (C), Ahrr/AhRR (D) and Tiparp/TiPARP (E) mRNA levels were determined by real time PCR. With the exception of AhRR mRNA, AhR ligands significantly induced all other Ah-responsive genes, and significant (p < 0.05) enhancement by butyrate is indicated (*). Results are expressed as means ± SE for at least 3 separate determinations for each treatment group.

Figure 4

Acetate and propionate enhances Cyp1a1/CYP1A1 expression in YAMC and Caco-2 cells. (A) Cells were treated with DMSO and different concentrations of acetate or propionate and expression of Cyp1a1/CYP1A1 and Cyp1b1/CYP1B1 mRNA levels was determined by real time PCR. (B) YAMA or Caco-2 cells were treated with DMSO, 10 nM TCDD alone and in combination with different concentrations of acetate or propionate, and expression of Cyp1a1/CYP1A1 (C) and Cyp1b1/CYP1B1 (D) mRNA levels was determined by real time PCR. (C) YAMC or Caco-2 cells were treated with DMSO, propionate and acetate alone and in combination with microbiota-derived AhR ligand, and Cyp1a1/CYP1A1 and Cyp1b1/CYP1B1 mRNA levels were determined by real time PCR. Significant (p < 0.05) induction of Cyps/CYPs by acetate or propionate alone and propionate/acetate-enhanced induction of Cyps/CYPs by AhR ligand is indicated (*). Results are expressed as means ± SE for at least 3 separate experiments for each treatment group.

Figure 5

HDAC inhibitors enhance TCDD-induced gene expression in YAMC and Caco-2 cells. Cells were treated with DMSO alone, TCDD alone, and TCDD in combination with different HDAC inhibitors, and Cyp1a1/CYP1A1 (A), Cyp1b1/CYP1B1 (B), Ahrr/AhRR (C) and Tiparp/TiPARP (D) mRNA levels were determined by real time PCR. Significant (p < 0.05) enhancement of TCDD-induced gene expression by HDAC inhibitors is indicated (*). Results are expressed as means ± SE for 3 separate determinations for each treatment group.

Figure 6

Effects of SCFAs and HDAC inhibitors on histone acetylation in ChIP assays. YAMC (A) and Caco-2 (B) cells were treated with DMSO, 10 nM TCDD alone or in combination with SCFAs and HDAC inhibitors for 24 hr, and whole cell lysates were analyzed by western blots. YAMC (C) and Caco-2 (D) cells were treated with DMSO, 10 nM TCDD and 5 mM butyrate alone or in combination for 4 hr, and ChIP assays were carried out as outlined in the Materials and Methods to detect interactions with the XRE-containing regions of the Cyp1a1 (mouse) and CYP1A1 (human) promoters.