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. 2014 Apr;63(4):622-34.
doi: 10.1136/gutjnl-2012-304241. Epub 2013 Jun 13.

Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling

Jillian L Pope  1 , Ajaz A Bhat, Ashok Sharma, Rizwan Ahmad, Moorthy Krishnan, Mary K Washington, Robert D Beauchamp, Amar B Singh, Punita Dhawan
Affiliations

Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling

Jillian L Pope et al. Gut. 2014 Apr.

Abstract

Objective: Claudin-1 expression is increased and dysregulated in colorectal cancer and causally associates with the dedifferentiation of colonic epithelial cells, cancer progression and metastasis. Here, we have sought to determine the role claudin-1 plays in the regulation of intestinal epithelial homeostasis.

Design: We have used a novel villin-claudin-1 transgenic (Cl-1Tg) mouse as model (with intestinal claudin-1 overexpression). The effect of claudin-1 expression upon colonic epithelial differentiation, lineage commitment and Notch-signalling was determined using immunohistochemical, immunoblot and real-time PCR analysis. The frequently used mouse model of dextran sodium sulfate (DSS)-colitis was used to model inflammation, injury and repair.

Results: In Cl-1Tg mice, normal colonocyte differentiation programme was disrupted and goblet cell number and mucin-2 (muc-2) expressions were significantly downregulated while Notch- and ERK1/2-signalling were upregulated, compared with the wild type-littermates. Cl-1Tg mice were also susceptible to colonic inflammation and demonstrated impaired recovery and hyperproliferation following the DSS-colitis. Our data further show that claudin-1 regulates Notch-signalling through the regulation of matrix metalloproteinase-9 (MMP-9) and p-ERK signalling to regulate proliferation and differentiation.

Conclusions: Claudin-1 helps regulate intestinal epithelial homeostasis through the regulation of Notch-signalling. An upregulated claudin-1 expression induces MMP-9 and p-ERK signalling to activate Notch-signalling, which in turn inhibits the goblet cell differentiation. Decreased goblet cell number decreases muc-2 expression and thus enhances susceptibility to mucosal inflammation. Claudin-1 expression also induces colonic epithelial proliferation in a Notch-dependent manner. Our findings may help understand the role of claudin-1 in the regulation of inflammatory bowel diseases and CRC.

Keywords: Differentiation; Inflammation; Mucin-2; Notch.

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Figures

Figure 1
Figure 1. Generation of Claudin-1 Transgenic mice
(A) Schematic of Villin-claudin-1 expression vector. (B) Immunoblot analysis using tissue lysates from wild type (WT) and Cl-1Tg mice were utilized to determine the expression of Claudin-1 expression. β-actin was used as loading control. MDCK cell lysate served as positive control. (C) Immunostaining for claudin-1 in the small intestine and colon of WT and Cl-1Tg mice. (D) Representative H&E staining for the colon of WT and Cl-1Tg mice. (E) Immunoblot analysis; and (F) Immunostaining for tight junctions and adherens junctions proteins in WT and Cl-1Tg mice.
Figure 2
Figure 2. Claudin-1 overexpression alters intestinal epithelial cell lineage and increases proliferation
(A) Immunostaining for PAS, Lysozyme, and Chromagranin-A in small intestine; (B) Immunostaining for PAS, Muc-2, Carbonic Anhydrase-I, and Chromagranin-A in colon; (C) Immunostaining of colon for BrdU incorporation and quantification; and (D) Immunoblot and immunostaining to determine p-ERK expression in WT and Cl-1Tg colons. ***p<0.001
Figure 3
Figure 3. Claudin-1 Tg mice are susceptible to DSS-colitis
WT and Cl-1Tg mice were given DSS (5% w/v) for 7-days and (A) percent change in body weight and (B) Colon weight/length (g/cm) in control and DSS-treated animals were monitored. Further, WT and Cl-1Tg mice were exposed to DSS (3.5% w/v) for 7-days and then allowed to recover for 5-days. Following parameters were then determined: (C) Percent change in body weight; (D) Representative H&E staining; and (E) Histopathologic scoring of the inflammation, depth of inflammation, and crypt damage. Data are represented as the percentage of mice per group with the indicated score. *p<0.05, ***p<0.001
Figure 4
Figure 4. Cl-1Tg mice have sustained inflammation during recovery from DSS-colitis
WT and Cl-1Tg mice exposed to either water alone, DSS (7 days), or DSS and then switched to regular water (5 days; recovery). These mice were then (A) Immunostained and quantitated for muc-2 expression using anti-muc-2 antibody; and (B) Immunostained and quantitated for T-cell infiltration using CD3, a T-cell marker, in the colon from control (water), DSS (day-7) and recovery (day-12), WT and Cl-1Tg mice. a compared to WT-control, b compared to Cl-1Tg-control, c compared to WT-DSS, d compared to WT-recovery (C) Quantitative RT-PCR analysis of TNFα, IFN-γ, K C a nd IL-10 expression. *p<0.05, **p<0.01, and ***p<0.001.
Figure 5
Figure 5. Proliferation and Apoptosis are altered in Cl-1Tg mice undergoing DSS treatment and recovery
(A) Proliferation was determined using immunohistochemical analysis to determine BrdU incorporation in the colon of WT and Cl-1Tg mice exposed to water, DSS and DSS-recovery; (B) Immunoblot analysis to determine the expression of pERK1/2 and total ERK1/2 in colon tissue samples. Quantification of (C) BrdU+ cells/crypt (50 crypts/mice, n#3), and (D) Cleaved caspase-3 positive cells in control, DSS and recovery samples. a compared to WT-control, b compared to Cl-1Tg control, c compared to WT-DSS, d compared to WT recovery. *p<0.05, **p<0.01, and ***p<0.001.
Figure 6
Figure 6. Increased Notch-signaling in Cl-1Tg mice and colon cancer cells during DSS injury and recovery
(A) Immunostaining for Hes1 expression to determine Notch activity during DSS-treatment and recovery. (B) Notch activity was assessed via immunoblotting for NICD expression and real-time PCR for Hes1 and Math1 in WT and Cl-1Tg colon tissue. **p<0.01, ***p<0.001. (C) Notch activity was assessed via immunoblotting for NICD and real-time PCR for Hes1 and Math1 expression in Claudin-1 overexpressing SW480 cells.
Figure 7
Figure 7. Claudin-1 Overexpression alters Notch signaling, proliferation and differentiation in colon cancer cells
(A) Notch activity was assessed via immunoblotting for NICD and real-time PCR for Hes1 and Math1 expression in goblet cell-like LS174T cells stably overexpressing claudin-1(LS174TClaudin-1). (B) LS174TClaudin-1 and control cells were assessed for changes in goblet cell differentiation by PAS immunostaining and qRT-PCR for TFF3 and KLF4. *p<0.05, ***p<0.001.(C) LS174TClaudin-1 cells were treated with a γ-secretase inhibitor, DAPT (100µM, 48h); (C) inhibition of NOTCH activity; (D) Cell proliferation-MTT assay; and differentiation was assessed via (E) PAS staining and qRT-PCR for differentiation markers TFF3 and KLF4.
Figure 8
Figure 8. Claudin-1 induced Notch signaling is mediated through the regulation of MMP-9
(A) Immunoblot and densitometric analysis for MMP-9 expression in the colon of WT and Cl-1Tg mice. (B) LS174TClaudin-1 cells were treated with a MMP inhibitor, GM6001 treatment (40µM, 48h) and effects on MMP-9 and NICD expression were determined in: (B) SW480 and SW480claudin-1 cells; (C) LS174T and LS174TClaudin-1 cells; (D) Cell proliferation-MTT assay; (E) differentiation (PAS staining and qRT-PCR for differentiation markers TFF3 and KLF4). *p<0.05, **p<0.01.
Figure 9
Figure 9. ERK signaling regulates Notch activation, apoptosis and differentiation in Claudin-1 overexpressing cells
Ls174Tclaudin-1 cells were treated with U0126 (10µM for 24 hrs); (A) Immunoblot analysis of MMP9, p-ERK, NICD and cleaved caspase-3; (B) cell proliferation-MTT assay; (C) PAS staining and real-time PCR analysis of differentiation specific markers TFF3 and KLF4. **p<0.01
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