SIRT2 Contributes to the Regulation of Intestinal Cell Proliferation and Differentiation

Abstract

Background and aims: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis.

Methods: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated.

Results: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/β-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression.

Conclusion: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-β-catenin signaling and is important for maintenance of IEC proliferation and differentiation.

Keywords: IEC; Intestinal Epithelial Cells; Intestinal Homeostasis; Mouse Model; Sirtuin; Wnt/β-Catenin.

Graphical abstract

Figure 1

Induction of SIRT2 is associated with intestinal cell differentiation. (A) Villus and crypt proteins were extracted from mouse small intestine. SIRT1, SIRT2, villin, proliferating cell nuclear antigen, and β-actin was detected by Western blotting. (B) Immunohistochemical analysis of SIRT2 protein expression in normal human colon and small intestine. Human normal colon and small intestine sections were fixed and stained with primary anti–human SIRT2 antibody. SIRT2 is specifically expressed in the more differentiated region of the small intestine (ie, villus) or colon (ie, upper crypt). Scale bars = 50 μm. The images are representative of 5 cases. (C) HT29 or Caco-2 cells were treated with NaBT at various dosages for 48 hours. (D) Caco-2 were incubated 3, 6 and 12 days after confluency to differentiation. Cells were lysed and Western blot analysis was performed using antibodies as indicated. The images are representative of 3 independent experiments. SIRT2 isoform 1, SIRT2 isoform 2, and SIRT1 signals from 3 separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin (n = 3, data represent mean ± SD; *P < .05 vs control).

Figure 2

Knockout of SIRT2 disturbs intestinal cell differentiation in vivo. (A, B) Small intestinal mucosal protein lysates were extracted for Western blot detection of MUC2, villin, Na,K-ATPase, LYZ, SIRT2 and β-actin protein expression. (A) Each well represents a different mouse from the relevant group. (B) Small intestinal mucosal protein lysates were extracted and IAP activity determined (n = 5, data represent mean ± SD; *P < .05 vs WT). (C) Total RNA was extracted from mucosa and IAP, MUC2, and LYZ mRNA expression was assessed by real-time RT-PCR (n = 5, data represent mean ± SD; *P < .05 vs WT). (D) Representative Fast Red staining of the small intestine revealed a decrease in IAP expression (arrows). (E) Representative AB staining of the small intestine revealed a decrease in mucinous goblet cells in SIRT2^–/– mice compared with WT mice (arrows). (F) Quantification of AB-positive cells in WT and SIRT2^–/– mice (n = 5, 3 villi per mouse, data represent mean ± SD; *P < .05 vs WT). (G) Representative IHC staining of the small intestine for LYZ showed the increase in Paneth cells (arrows) in SIRT2^–/– mice compared with WT mice. (H) Quantification of LYZ-positive cells in control and SIRT2^–/– mice (n = 5, 3 crypts per mouse, data represent mean ± SD; *P < .05 vs WT). Scale bars= 50 μm.

Figure 3

SIRT2 deficiency impairs intestinal enterocyte and goblet cell differentiation in an ex vivo model. Organoids were isolated from proximal small intestine and incubated for 3 days, followed by incubation with or without NaBT for 48 hours. (A) Microscopy analysis of organoids. Scale bars = 100 μm. (B) IAP activity and mRNA levels determined by real-time RT-PCR (n = 3). Expression of (C) LYZ and (D) MUC2 determined by real time RT-PCR (n = 3). Data represent mean ± SD; *P < .05 vs WT; ^#P < .05 vs NaBT plus WT. Data are from 1 of 3 independent experiments with similar results.

Figure 4

Intestinal SIRT2 deletion results in an increase in proliferation in the mature ileum. (A) Photograph of intestines from 3-month-old mice of the indicated genotypes. (B) Length of small intestine and colon from WT and SIRT2^–/– mice were measured (n = 5, data represent mean± SD; *P < .05, as compared with WT). (C, D) From hematoxylin and eosin–stained slides (C), villus and crypt length were determined (D), as indicated in the Materials and Methods (n = 5, 20 crypts per mouse, data represent mean ± SD; *P < .05 vs WT). (E, F) IHC staining for Ki67 (E) (arrows) reveals that the number of positive cells was increased after SIRT2 deletion (F) (n = 5, 3 crypts per mouse, data represent mean ± SD; *P < .05 vs WT). (G, H) IHC staining for cyclin D1 (G) (arrows) reveals that the number of positive cells was increased after SIRT2 deletion (H) (n = 5, 3 crypts per mouse, data represent mean ± SD; *P < .05 vs WT). Scale bars = 50 μm.

Figure 5

Intestinal epithelial SIRT2 deficiency results in the increased stemness. (A) Small intestinal organoids from WT and SIRT2^–/– mice during the first 5 days of culture. Representative images of crypt culture from each condition. (B) Colony-forming efficiency from WT and SIRT2^–/– mice after 3 days in culture; n = 3 mice per group. (C) Organoid structural complexity of experiment shown in panel A (n = 3). Expression of ISC markers in (D) organoids and (E) small intestinal crypts from WT and SIRT2^–/– mice detected by real-time RT-PCR; n = 3 (data represent mean ± SD; *P < .05 vs WT). (F) Quantification (left) and representative images (right) of OLFM4+ stem cells by IHC. (n = 5, 15 crypts per mouse, data represent mean ± SD; *P < .05 vs WT). Scale bars = 50 μm.

Figure 6

SIRT2 deficiency results in enhanced Wnt/β-catenin signaling. (A) Loss of SIRT2 results in the increased β-catenin protein expression in crypt organoids (left panel). β-catenin protein signals from 3 separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin (right panel). (B) mRNA levels of Wnt/β-catenin target genes in crypt organoids (n = 3) from WT and SIRT2^–/– mice after 5 days in culture. (C) Crypt organoids were isolated from WT mice and treated with SIRT2 inhibitor AGK2 for 48 hours. mRNA levels of Wnt/β-catenin target genes were detected (n = 3). Levels of β-catenin protein, acetylated β-catenin, and its (D) targets and (E) quantification of β-catenin protein and acetylated β-catenin in IECs (n = 3). (F) mRNA levels of Wnt/β-catenin target genes in IECs (n = 3) from WT and SIRT2^–/– mice. (G) RKO cells were treated with SIRT2 inhibitor AGK2 (5 μM) for 48 hours. (H) RKO cells were transfected with nontargeting control small interfering RNA (siRNA) or SIRT2 siRNA. Inhibition or knockdown of SIRT2 increased acetylation of β-catenin and activation of β-catenin signaling in RKO cells. (I) Flag-β-catenin was isolated from 293T cells treated with trichostatin A/nicotinamide and then incubated with recombinant human GST-SIRT2 protein, followed by analysis of Flag-β-catenin (upper panel). Acetylated β-catenin signals from 3 separate experiments were quantitated densitometrically and expressed as fold change with respect to total β-catenin (lower panel). The images are representative of 3 independent experiments. Results are representative of 3 experiments (n = 3, data represent mean ± SD; *P < .05). (J) Model proposed to explain the role of SIRT2 in intestinal cell proliferation and differentiation.

Figure 7

Intestinal epithelial SIRT2 deficiency is associated with colitis in humans. (A) The mRNA levels of SIRT2 are reduced in human IBD patients. mRNA from purified colonic epithelium of human IBD patients and controls were analyzed by real RT-PCR (n = 7–8, *P < .05). (B) IHC staining of SIRT2 protein in the colon and small intestine from control individuals and IBD patients. All these experiments were repeated in human tissue samples obtained from 5 control individuals or patients with IBD and showed similar results. Treatment with TNF repressed SIRT2 protein expression in (C) HIEC6, (D) HT29, and (E) mouse small intestinal organoids. Cells were treated with TNF for 24 hours. SIRT2 expression was determined by Western blot. The images are representative of 3 independent experiments. SIRT2 signal from 3 separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin (n = 3, data represent mean ± SD; *P < .05 vs control).