A KDM4-DBC1-SIRT1 Axis Contributes to TGF-b Induced Mesenchymal Transition of Intestinal Epithelial Cells

Abstract

Intestinal fibrosis is one of the common pathophysiological processes in inflammatory bowel diseases (IBDs). Previously it has been demonstrated that epithelial-mesenchymal transition (EMT) can contribute to the development of intestinal fibrosis. Here we report that conditional ablation of SIRT1, a class III lysine deacetylase, in intestinal epithelial cells exacerbated 2, 4, 6-trinitro-benzene sulfonic acid (TNBS) induced intestinal fibrosis in mice. SIRT1 activity, but not SIRT1 expression, was down-regulated during EMT likely due to up-regulation of its inhibitor deleted in breast cancer 1 (DBC1). TGF-β augmented the recruitment of KDM4A, a histone H3K9 demethylase, to the DBC1 promoter in cultured intestinal epithelial cells (IEC-6) leading to DBC1 trans-activation. KDM4A depletion or inhibition abrogated DBC1 induction by TGF-β and normalized SIRT1 activity. In addition, KDM4A deficiency attenuated TGF-β induced EMT in IEC-6 cells. In conclusion, our data identify a KDM4-DBC1-SIRT1 pathway that regulates EMT to contribute to intestinal fibrosis.

Keywords: SIRT1; epigenetics; epithelial-mesenchymal transition; histone demethylase; intestinal fibrosis; transcriptional regulation.

FIGURE 1

Intestinal epithelial deletion of SIRT1 exacerbates TNBS induced intestinal fibrosis. Intestine-conditional SIRT1 knockout (CKO) mice and wild type littermates were induced to develop colitis and intestinal fibrosis by TNBS injection. (A) Expression levels of pro-fibrogenic molecules in the small intestines were examined by qPCR. (B) Expression levels of pro-fibrogenic molecules in the colons were examined by qPCR. (C) Paraffin sections were stained with picrosirius red. Scale bar, 100 mm. (D) Expression levels of epithelial marker genes in the small intestines were examined by qPCR. (E) Expression levels of epithelial marker genes in the colons were examined by qPCR. N = 5 mice for each group.

FIGURE 2

SIRT1 activity is down-regulated by TNBS in vivo and by TGF-β in epithelial cells in vitro. (A–C) Colitis was induced in C57B/6 mice by TNBS as described in section “Materials and Methods.” SIRT1 activity was determined by a colorimetric kit. Expression levels of SIRT1 and DBC1 were examined by qPCR and Western. N = 5 mice for each group. (D–F) IEC-6 cells were treated with TGF-β (2 ng/ml) and harvested at indicated time points. SIRT1 activity was determined by a colorimetric kit. Expression levels of SIRT1 and DBC1 were examined by qPCR and Western. All experiments were performed in triplicate wells and repeated three times. One representative experiment is shown.

FIGURE 3

DBC1 contributes to TGF-β induced EMT by repressing SIRT1 activity. (A–C) IEC-6 cells were transfected with siRNAs targeting DBC1 or scrambled siRNAs followed by treatment with TGF-β (2 ng/ml). Expression levels of SIRT1 and DBC1 were examined by qPCR and Western. SIRT1 activity was determined by a colorimetric kit. (D,E) IEC-6 cells were transfected with indicated siRNAs followed by treatment with TGF-β (2 ng/ml) for 48 h. Gene expression levels were examined by qPCR and Western. (F,G) IEC-6 cells were transfected with indicated siRNAs followed by treatment with TGF-β (2 ng/ml) in the presence or absence of EX527 (10 mM) for 48 h. Gene expression levels were examined by qPCR and Western. All experiments were performed in triplicate wells and repeated three times. One representative experiment is shown.

FIGURE 4

TGF-β promotes KDM4A recruitment to the DBC1 promoter. (A–I) IEC-6 cells were treated with TGF-β(2 ng/ml) and collected at indicated time points. ChIP assays were performed anti-acetyl H3, anti-trimethyl H3K4, anti-trimethyl H3K9, anti-trimethyl H3K27, anti-trimethyl H4K20, anti-KDM4A, anti-KDM4B, anti-KDM4C, or anti-KDM4D. All experiments were performed in triplicate wells and repeated three times. One representative experiment is shown.

FIGURE 5

KDM4A mediates TGF-β induced DBC1 transcription. (A–D) IEC-6 cells were transfected with siRNAs targeting KDM4A or scrambled siRNAs followed by treatment with TGF-β (2 ng/ml). DBC1 expression levels were examined by qPCR and Western. ChIP assay was performed with anti-trimethyl H3K9. SIRT1 activity was determined by a colorimetric kit. (E–H) IEC-6 cells were treated with TGF-β (2 ng/ml) in the presence or absence of CP2 (1 μM). DBC1 expression levels were examined by qPCR and Western. ChIP assay was performed with anti-trimethyl H3K9. SIRT1 activity was determined by a colorimetric kit. All experiments were performed in triplicate wells and repeated three times. One representative experiment is shown.

FIGURE 6

KDM4A silencing or inhibition attenuates TGF-β induced EMT in a SIRT1-dependent manner. (A,B) IEC-6 cells were transfected with indicated siRNAs followed by treatment with TGF-β (2 ng/ml) for 48 h. Gene expression levels were examined by qPCR and Western. (C,D) IEC-6 cells were transfected with indicated siRNAs followed by treatment with TGF-β (2 ng/ml) in the presence or absence of EX527 (10 μM) for 48 h. Gene expression levels were examined by qPCR and Western. All experiments were performed in triplicate wells and repeated three times. One representative experiment is shown. (E) A schematic model.