Research ArticleGastroenterology Open Access | 10.1172/JCI170771

Rizwan Ahmad,1 Balawant Kumar,1 Ishwor Thapa,2 Raju Lama Tamang,1 Santosh K. Yadav,1 Mary K. Washington,3 Geoffrey A. Talmon,4 Alan S. Yu,5 Dhundy K. Bastola,2 Punita Dhawan,1,6,7 and Amar B. Singh1,6,7

1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

Find articles by Dhawan, P. in: JCI | PubMed | Google Scholar

1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2School of Interdisciplinary Informatics, University of Nebraska Omaha, Omaha, Nebraska, USA.

3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

4Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

5Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas, USA.

6Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

7VA Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

Address correspondence to: Amar B. Singh, Department of Biochemistry and Molecular Biology, Member, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA. Email: amar.singh@unmc.edu.

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Published October 10, 2023 - More info

Published in Volume 133, Issue 23 on December 1, 2023
J Clin Invest. 2023;133(23):e170771. https://doi.org/10.1172/JCI170771.
© 2023 Ahmad et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 10, 2023 - Version history
Received: March 22, 2023; Accepted: October 5, 2023 View PDF Abstract

Patients with inflammatory bowel disease (IBD) are susceptible to colitis-associated cancer (CAC). Chronic inflammation promotes the risk for CAC. In contrast, mucosal healing predicts improved prognosis in IBD and reduced risk of CAC. However, the molecular integration among colitis, mucosal healing, and CAC remains poorly understood. Claudin-2 (CLDN2) expression is upregulated in IBD; however, its role in CAC is not known. The current study was undertaken to examine the role for CLDN2 in CAC. The AOM/DSS-induced CAC model was used with WT and CLDN2-modified mice. High-throughput expression analyses, murine models of colitis/recovery, chronic colitis, ex vivo crypt culture, and pharmacological manipulations were employed in order to increase our mechanistic understanding. The Cldn2KO mice showed significant inhibition of CAC despite severe colitis compared with WT littermates. Cldn2 loss also resulted in impaired recovery from colitis and increased injury when mice were subjected to intestinal injury by other methods. Mechanistic studies demonstrated a possibly novel role of CLDN2 in promotion of mucosal healing downstream of EGFR signaling and by regulation of Survivin expression. An upregulated CLDN2 expression protected from CAC and associated positively with crypt regeneration and Survivin expression in patients with IBD. We demonstrate a potentially novel role of CLDN2 in promotion of mucosal healing in patients with IBD and thus regulation of vulnerability to colitis severity and CAC, which can be exploited for improved clinical management.

Graphical Abstract Introduction

Patients with inflammatory bowel disease (IBD) have two big fears: (a) developing colon cancer and (b) losing their colon by colectomy because of the heightened risk of colitis-associated cancer (CAC) (1, 2). CAC is driven by the continuous exposure to intestinal inflammation (3, 4). Thus, mechanism(s) that can prevent recurrent inflammation in IBD can also protect against CAC. Mucosal healing (MH), a critical endpoint in the clinical management of IBD, is associated with improved prognosis, including reduced risk of CAC (5). Epithelial cell survival, proliferation, and migration play integrated roles during MH and functional crypt formation. This phase is promoted by growth factors, including EGF and keratinocyte growth factor (KGF), and immune cytokines, such as IL-22 (6). However, details regarding the molecular regulation of MH and how it alters the outcomes for CAC remain poorly understood.

Claudin-2 (CLDN2) expression is upregulated in IBD (7–9). However, modeling of the pathological CLDN2 expression, as in IBD, protects mice from dextran sulfate sodium–induced (DSS-induced) or C. rodentium–induced colitis (10, 11). The KO of endogenous CLDN2 expression promotes colitis (11, 12). Interestingly, when subjected to the T cell colitis, the offspring of Villin-Cldn2TG and Rag1KO mice show severe colitis, while offspring from Cldn2KO and Rag1KO mice show resistance to colitis, though they die suddenly for unknown reasons (13). Together, these studies suggest a critical yet complex role of CLDN2 in colonic homeostasis and inflammation. Both epithelial and immune dysregulations underlie CAC susceptibility and progression (14, 15). Therefore, we wondered about the significance of CLDN2 upregulation in the susceptibility of patients with IBD to CAC, especially considering that CLDN2 is expressed at the crypt bottom; clusters with Cyclin-D1 and c-Myc proteins; and regulates intestinal epithelial cell (IEC) proliferation, differentiation, and migration as well as cancer stem cells in spontaneous colon cancer (CRC) (16, 17). Of note, CLDN2 expression is upregulated in and promotes CRC (16, 18–20).

In present study, we report the unexpected finding that Cldn2KO mice had significant inhibition of CAC despite severe colitis compared with WT littermates. Furthermore, we provide data that the protective role of CLDN2 against CAC stems from its currently unknown role in promoting MH. Our findings could increase current knowledge regarding the role of CLDN2 in IBD and clinical management of patients with IBD, including their disease progression and CAC risk.

Results

Loss of CLDN2 expression protects against CAC, despite promoting colitis. Cldn2KO mice showed, as reported, exacerbated colitis when subjected to DSS-induced colitis (Figure 1, B and C, and Supplemental Figure 1, A–C; supplemental material available online with this article; https://doi.org/10.1172/JCI170771DS1) (12, 21). We therefore hypothesized Cldn2KO mice to be susceptible to CAC. Interestingly, when subjected to azoxymethane (AOM)/DSS treatment, Cldn2KO mice showed a significantly lower tumor burden, despite higher colonic inflammation (Figure 1, A–K). In addition, tumors in Cldn2KO mice showed low-grade dysplasia compared with the high-grade dysplasia in WT mice (Figure 1L). Overall, above findings demonstrated an unexpected dichotomy in the effects of CLDN2 expression upon colitis and CAC.

Figure 1

Colitis-associated cancer is significantly inhibited in Cldn2KO mice. (A) Schematic of the experimental strategy to induce colitis-associated colon cancer. (B and C) Representative immunofluorescence and immunoblot analysis confirming loss of CLDN2 expression in Cldn2KO mice. (D) Representative images of colons and colon length in AOM/DSS-treated Cldn2KO and WT mice (n = 8/group). (E) Colon edema (g/cm; n = 8/group). (F–H) Percentage of colon involved by inflammation (n = 8/group), representative H&E images, and mucosal injury score (WT/Cldn2KO: n = 10/6 mice). (I and J) Tumor growth and representative H&E analysis of the colons from AOM/DSS-treated Cldn2KO (n = 10) and WT (n = 6) mice. (K and L) Size of the colon polyps and dysplasia in Cldn2KO (n = 6) versus WT (n = 10) mice. Data in D–F, H and I are presented as the mean ± SEM. *P < 0.05, **P < 0.01 by 2-tailed unpaired t test. Data in K and L are presented as percentage number. ****P < 0.0001 by χ2 and Fisher’s exact test. Scale bar: 100 μM (B and G); 200 μM (J).

Cldn2 loss promotes proinflammatory and proapoptotic programs during CAC. To understand why CLDN2 loss promotes colitis but not CAC, we examined global transcriptomic changes in CAC-challenged Cldn2KO colons versus WT mouse colons. Figure 2A shows the selected differentially expressed genes (DEGs). Notably, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, prominent pathways upregulated in Cldn2KO mice included TNF and necroapoptosis. The downregulated pathways included antigen processing and presentation, asthma, and rheumatoid arthritis (versus WT mice). Gene Ontology (GO) analysis showed that the biological functions affected by the loss of CLDN2 also included NF-κB, stress and immune responses, and regulation of cell killing (Figure 2, B–D). The downregulated pathways included antigen processing and presentation and catabolic pathways (Supplemental Figure 1, D and E). The heatmap analysis further highlighted genes linked with the cell cycle, inflammation, apoptosis, and immune regulations in Cldn2KO mice versus WT mice (Figure 2E). Furthermore, immunoblotting and RT-qPCR analyses showed significant upregulation of pStat3y705, pNF-kBs536, IL-1, and IL-6 expression in AOM/DSS-treated Cldn2KO mice (Figure 2, F and G, and Supplemental Figure 1F). In addition, significant increases in the expression of the markers of DNA damage and apoptosis (γH2AX and cleaved caspase-3) and contrasting downregulation of cell survival and proliferation proteins (Bcl2 and c-Myc) characterized these mice (Figure 2, F and H, and Supplemental Figure 1, G–I). IHC analysis using antibodies against Ki67 and cleaved caspase-3 further showed a significant inhibition of the proliferative index (ratio of Ki67/cleaved caspase-3) in Cldn2KO mice (Figure 2, I and J). Overall, above data showed significant impairment of the crypt proliferation and survival programs in AOM/DSS-treated Cldn2KO mice despite higher inflammation (versus WT mice; Figure 2K).

Figure 2

Cldn2 loss-of-expression promotes proinflammatory and proapoptotic transcriptional programs in mice subjected to colitis-associated cancer. (A) Scatter plot depicting the comparative transcriptomic profile (RNA-Seq) between Cldn2KO and WT mice subjected to colitis-associated cancer (CAC) (n = 3/group). (B–D) KEGG pathway and GO biological function analysis based on differentially expressed genes (DEGs) in RNA-Seq analysis using colon RNA from AOM/DSS-treated Cldn2KO and WT mice (n = 3/group). (E) Heatmap showing selected DEGs (n = 3/group). (F–H) Immunoblotting and densitometric analysis of proteins involved in inflammation, proliferation, cell survival, and apoptosis in AOM/DSS-treated Cldn2KO and WT mice (n = 4/group). (I and J) Representative images of Ki67 and cleaved caspase-3 expression, and proliferation index (Ki67/cleaved caspase-3 expression) in AOM/DSS-treated mice (n = 4/group; 3 images/mice). (K) Graphical modeling representing the overall outcome of Cldn2KO mice when subjected to CAC. Data in G, H, and J are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed unpaired t test. Scale bar: 100 μM.

Colitis regulates CLDN2 expression in context-specific manner. In light of above unexpected findings, we hypothesized a role for upregulated CLDN2 expression in IBD in promoting MH. We carefully examined CLDN2 regulation during colitis, as previous reports have shown variable CLDN2 expression in mouse models of colitis (10, 22). WT mice subjected to DSS-induced colitis, colitis/recovery, and chronic colitis were used (Supplemental Figure 2). Only distal colon, the principal site for the effects of DSS-induced colitis, was used (23). Interestingly, CLDN2 expression was downregulated in acute DSS-induced colitis colon samples although E-cadherin expression remained largely unaltered (Supplemental Figure 3A). Epithelium-enriched fractions from normal and colitis-subjected mouse colons showed similar outcomes (Supplemental Figure 3B). Coimmunofluorescence analysis for CLDN2 and β-catenin further showed robust β-catenin expression in inflamed colons lacking CLDN2, suggesting that it was not due to the loss of epithelium (Supplemental Figure 3C). Further analysis of the published microarray and RNA-Seq data sets from our and other laboratories from mice subjected to DSS-induced colitis validated Cldn2 downregulation in colitis versus naive WT mice (Supplemental Figure 3, D and E) (10, 24). RT-qPCR also confirmed the downregulation of Cldn2 expression in colitis (Supplemental Figure 3F).

However, the above findings contrast with those found with an upregulated CLDN2 expression in IBD. Hence, we determined if it represents a response to the inflammation-associated injury. CLDN2 expression was examined in naive mice, mice subjected to DSS-induced colitis, colitis/recovery, or chronic-colitis. Interestingly, immunoblotting and IHC analyses showed a biphasic regulation of CLDN2 expression during colitis where CLDN2 expression was downregulated during DSS-induced acute colitis, but upregulated during recovery from colitis and in chronic colitis (Figure 3, A–D). We found similar CLDN2 upregulation in mice subjected to C. rodentium colitis (data not shown). Taken together, above data demonstrated dynamic regulation of CLDN2 expression during colitis where CLDN2 upregulation associated with MH.

Figure 3

Colitis-mediated regulation of CLDN2 expression is context dependent and biphasic. (A and B) Epithelial enriched fractions of WT mouse colon that were untreated, subjected to DSS-induced colitis and recovery, or chronic DSS-induced colitis (n = 3/group). (C and D) Representative images and IHC intensity analysis using anti-CLDN2 antibody (n = 5/group). (E) Coimmunofluorescence image analysis for CLDN2 and SCA-1 in colon Swiss roll of mice subjected to acute DSS-induced colitis, DSS-induced colitis/recovery, or chronic DSS-induced colitis (n = 5/group). (F and G) Coimmunofluorescence and quantitative analysis using anti-CLDN2 and -Ki67 antibodies (n = 6/group). (H and I) MTT assay and RT-qPCR analysis using Caco2 cells subjected to DSS-induced injury and subsequent recovery (n = 3 independent experiments). (J) Immunoblot analysis for CLDN2, ECAD, c-Myc, and P27/Kip1 (n = 3 independent experiments). (K) Model depicting regulation of CLDN2 during colitis (injury phase) and recovery (repair/regeneration phase). Data in B, D, G, and H–J are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, $$$$P < 0.0001 by 1-way ANOVA with Tukey’s test. Scale bar: 100 μM (C, E, and F); 200 μM (C).

Colitis-associated CLDN2 upregulation correlates with crypt regeneration/repair. In face of above findings, we further examined transcriptomic changes associated with CLDN2 changes during colitis-associated MH. High-throughput RNA-Seq analysis was performed using RNA isolated from the distal colons of mice subjected to recovery after DSS-induced colitis. The KEGG and GO analysis demonstrated that the biological processes involved in inflammation and apoptosis were negatively enriched in WT mice recovering from colitis while processes associated positively with repair/regeneration were upregulated (Supplemental Figure 4A). In contrast, during DSS-induced colitis, the proinflammatory and proapoptotic pathways were upregulated while the cell cycle pathways were inhibited (Supplemental Figure 3, D and E). We further found that Cldn2 mRNA expression was downregulated during DSS-induced colitis; however, it was significantly upregulated during recovery along with Pcna and Mki67ip (markers of crypt proliferation). Expression of Vil1 and Cdkn1a (associated with inhibition of cell growth) were downregulated (Supplemental Figure 3D and Supplemental Figure 4B).

In light of above outcomes, we further examined whether expression of stem cell antigen-1 (SCA-1), associated positively with mucosal repair/regeneration, is upregulated in mice subjected to colitis/recovery and its potential correlation with CLDN2 expression (25). Coimmunofluorescence analysis was done using anti-CLDN2 and -SCA-1 antibodies using colons from the mice subjected to colitis/recovery. As shown in Figure 3E, an upregulated SCA-1 expression characterized the regenerative crypts, which also colocalized with CLDN2 in mice subjected to DSS-induced colitis/recovery and/or chronic colitis. Additional analysis also demonstrated a significant upregulation for Ki67+ cells during recovery from colitis, which aligned positively with CLDN2+ cells in the regenerative epithelium of mice subjected to DSS-induced colitis/recovery and/or chronic colitis (vs. naive mice or mice subjected to acute colitis) (Figure 3, F and G).

To determine if effects described above on CLDN2 expression were epithelial intrinsic, we developed an ex vivo model of colitis-induced IEC injury/repair (Supplemental Figure 4C), based on published reports (26). Effects on the markers of cell proliferation/survival (MTT assay, Ki67 and c-Myc) and cell cycle inhibition (P-21/Cip1, and P-27/Kip1) supported distinct phases of injury and repair (Figure 3, H–J). As shown in Figure 3J, CLDN2 expression was downregulated during the injury phase; however, it was significantly upregulated during the repair/regeneration phase. E-cadherin expression remained unaltered. Similar findings in HT-29 cells showed that the effects were not cell specific (Figure 3J).

We further determined the association of CLDN2 with MH using colonoscope-associated wound healing. Immunohistochemical analysis of the colon 24 hours after wounding with colonoscope biopsy forceps demonstrated sharp upregulation of CLDN2 and Ki67 in the crypts adjacent to the wounds (Supplemental Figure 5). Above results supported an association of CLDN2 upregulation with intestinal epithelial injury/repair (Figure 3K).

Cldn2KO mice show impaired recovery from colitis. To further determine a causal role for CLDN2 in colitis-associated MH, we subjected Cldn2KO mice and littermate WT mice to DSS-induced colitis and recovery (Figure 4A). As shown in Figure 4B, DSS treatment significantly lowered the body weight of WT mice, which reverted to the normal during the recovery. Cldn2KO mice exhibited significantly higher weight loss during colitis and further failed to recover the weight loss during recovery (Figure 4B). In addition, Cldn2KO mice died during recovery (Figure 4C). Colon thickness and mucosal injury were significantly high in Cldn2KO mice (versus WT mice; Figure 4, D and E). Histopathological analysis further revealed increased immune cell infiltration and a significant reduction in the regenerative potential (Figure 4, F and G). Colonoscope-assisted longitudinal mapping further supported progressive worsening of colitis in Cldn2KO mice (Figure 4H). In addition, expression of SCA-1 was significantly downregulated in Cldn2KO mice recovering from DSS-induced colitis (Figure 4I).

Figure 4

Cldn2KO mice show impaired recovery and mucosal healing following DSS-induced colitis. (A) Schematic illustration of experimental design. (B) Percentage weight change in Cldn2KO and WT mice subjected to DSS-induced colitis and recovery (Cldn2KO/WT: n = 8/7). (C) Km plot depicting mouse mortality during colitis/recovery (Cldn2KO/WT: n = 8/7). (D) Colon thickness (g/cm; Cldn2KO/WT: n = 8/7). (E and F) Mucosal injury score and representative H&E analysis (Cldn2KO/WT: n = 8/7). (G) Quantitation of epithelial regeneration by a pathologist in Cldn2KO and WT mice during recovery from colitis (Cldn2KO/WT: n = 8/7). (H) Representative images showing colonoscopic evaluation of colonic inflammation in Cldn2KO mice compared with WT mice. (I) Coimmunofluorescence image analysis for SCA-1 in colon Swiss roll of Cldn2KO and WT mice subjected to recovery from colitis. (J) Wound healing assay using colonoscope-assisted wounding. (K) Quantitative image analysis shows delayed mucosal healing in Cldn2KO mice compared with that in WT mice (n = 5/group). Data in B, D, E, G, and K are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed unpaired t test. Survival data in C was assessed by log-rank (Mantel-Cox) test. Scale bar: 100 μM.

We also examined relative healing of the colonoscope-assisted wounding in WT and Cldn2KO mice (Figure 4A). As shown in Figure 4, J and K, WT mice showed a substantial reduction in the ulcerated area on day 1. By day 3, the original lesion was almost indistinguishable from the non-injured colon. In Cldn2KO mouse colons, the reddish appearance of the tissue indicated incomplete healing (Figure 4, J and K). Overall, our findings demonstrated that the loss of CLDN2 expression impairs recovery/regeneration from mucosal injury.

Loss of Cldn2 expression promotes proinflammatory and proapoptotic responses during recovery from colitis. To determine why Cldn2KO mice show impaired recovery from colitis, we performed RNA-Seq analysis. The volcano plot (Figure 5A) shows contrasting transcriptomes in Cldn2KO versus WT mice based on the Euclidean distance between the DEGs (log2 fold change; P < 0.05). The top upregulated pathways in Cldn2KO mice (KEGG pathway analysis) were the proinflammatory and apoptotic pathways (Figure 5, B and D). The top biological functions (GO analysis) included the apoptotic process, cell death, cytokine production, and response to wounding, etc. (versus WT; Figure 5, C and D). In accordance, we found increased expression of IL-1 and IL-6 (Supplemental Figure 6A) as well as pNF-kBs536 and pStat3y705 in Cldn2KO mice recovering from colitis (Figure 5, F and G). The downregulated pathways in Cldn2KO mice included DNA replication and cell cycle (Figure 5, D and E). The metabolic related processes were also downregulated in Cldn2KO mice (Supplemental Figure 6B). Immunoblotting analysis further supported dysregulation of the DNA damage response (DDR) in Cldn2KO mice. In this regard, expression of γH2AX, P-21/Cip1 and pChk2t68, proteins associated with DDR, was significantly upregulated in Cldn2KO mice when subjected to recovery from DSS-induced colitis (vs. WT mice; Supplemental Figure 6, C and D). Immunoblotting using the same samples further showed significant downregulation of Bcl2 and C-Myc expressions in Cldn2KO mice while cleaved caspase-3 expression was upregulated (Figure 5, F and G). The above findings strongly support a role for CLDN2 expression in colitis-associated epithelial cell injury and thus restitution/repair.

Figure 5

Loss of Cldn2 results in a defective mucosal healing transcriptomic response, which differs from the conserved gene expression profile between WT mice recovering from IBD and patients with IBD. (A) Scatter graph showing differential gene expression between Cldn2KO and WT mice recovering from colitis (n = 3/group). (B and C) Most prominent KEGG pathways and GO biological processes based on gene expression upregulated in Cldn2KO mice versus WT mice during recovery from colitis (n = 3/group). (D) Heatmap depicting differential gene expression, which was primarily associated with cell cycle, apoptosis, inflammation, and immune homeostasis (Cldn2KO versus WT mice; n = 3/group). (E) Most prominent downregulated KEGG pathways in Cldn2KO mice compared with WT mice (n = 3/group). (F and G) Immunoblotting and densitometric analysis of proteins involved in inflammation, proliferation, survival, and apoptosis in mice recovering from colitis (Cldn2KO; n = 5) and WT (n = 4) mice. (H) The gene profile in Cldn2KO mice recovering from colitis differs from the conserved profile between mice and humans. (I) Significant dysregulation of GO biological processes that are associated with mucosal healing in Cldn2KO mice versus WT mice. (J and K) Immunohistochemical analysis for cleaved caspase-3 and Ki67, and proliferation index (WT/Cldn2KO: n = 4/5 mice; 3 fields in each mice Swiss role). (L) Schematics showing that loss of CLDN2 results in impaired mucosal healing. Data in G and K are presented as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 by 2-tailed unpaired t test. Scale bar: 100 μM.

CLDN2 deficiency associates with impaired MH-associated gene transcription in both colitis-challenged mice and patients with IBD. Having characterized that Cldn2 loss promotes proinflammatory and proapoptotic programs during colitis/recovery, we further examined its relevance to IBD. To this end, we compared the DEGs from WT mice subjected to recovery from colitis with DEGs in patients with IBD versus healthy humans (27). Among 4,413 DEGs from mice, 1,049 genes overlapped with the DEGs in patients with IBD. Of these, 420 genes had a similar tendency of regulation in mice recovering from colitis and patients with IBD (Figure 5H). Further determination of the potential overlap between these shared DEGs (colitis-challenged mice and patients with IBD) and the DEGs in Cldn2KO mice versus WT mice (recovering from colitis) identified 24 genes (14 up- and 10 downregulated; Figure 5H and Supplemental Table 1). The GO analysis, based on these 14 upregulated DEGs, identified response to wounding, wound healing, epithelial cell differentiation, and intrinsic apoptotic signaling. The downregulated biological functions included regulation of the cellular response to stress and cell death (Figure 5I). Furthermore, proliferative index, based on the ratio of proliferation/apoptosis, was significantly downregulated in Cldn2KO versus WT mice (Figure 5, J and K). Additional analysis of our published microarray data set for the DEGs in Villin-Cldn2TG versus WT mice (DSS-induced colitis) revealed contrasting upregulation of the cell proliferation pathways and downregulation of the inflammatory and apoptotic pathways (Supplemental Figure 7, A and B) (10). Overall, these results supported a role for CLDN2 in promoting colitis-associated MH (Figure 5L).

Inhibiting EGFR signaling inhibits recovery from colitis and associated CLDN2 upregulation. Having uncovered a potentially novel role for CLDN2 upregulation in inflammation-associated MH, we wondered about its pathological significance in face of the prosurvival signaling that promote MH (18, 28–30). EGFR signaling promotes colonic CLDN2 expression (18, 31). EGFR activation protects patients with IBD from colitis (29, 32, 33). Furthermore, our RNA-Seq analysis revealed significant increases in Egf and Ereg expression, ligands that activate EGFR signaling, along with Cldn2 (Figure 6A). Hence, we examined if colitis-induced CLDN2 upregulation depends on EGFR activation. Caco-2 cells recovering from DSS injury were subjected to the inhibitors of EGFR activation. Inhibiting EGFR signaling inhibited CLDN2 upregulation along with c-Myc and pERK1/2 expression (Figure 6B). To examine this in vivo, we inhibited EGFR signaling (gefitinib; 200 mg/kg body weight; oral gavage) in WT mice recovering from DSS-induced colitis. Inhibiting EGFR activation impaired recovery from colitis and resulted in mouse death (Figure 6, C and D). Histological evaluation showed increased inflammation, mucosal injury, and edema in EGFR-inhibited mice versus untreated mice (Figure 6, E and F). Inhibition of pERK1/2 supported the efficacy of the inhibition of EGFR activation (Figure 6G). Notably, recovery-associated CLDN2 upregulation was also inhibited in EGFR-inhibited mice (Figure 6H). Inhibiting EGFR signaling in mice recovering from colitis also promoted expression of pNF-κBs536 and pStat3y705 (Figure 3, I and K) while the proliferative index was significantly lower (Figure 6, J–L), similar to that in Cldn2KO mice. The regenerative index was also significantly lower in gefitinib-treated mice (Figure 6M). These results supported a critical role for CLDN2 expression in EGFR-mediated regulation of inflammation-associated MH (Figure 6N).