Abstract
Intestinal stem cells (ISCs) play a crucial role in the continuous self-renewal and recovery of the intestinal epithelium. In previous studies, we have revealed that the specific absence of Claudin-7 (Cldn-7) in intestinal epithelial cells (IECs) can lead to the development of spontaneous colitis. However, the mechanisms by which Cldn-7 maintains homeostasis in the colonic epithelium remain unclear. Therefore, in the present study, we used IEC- and ISC-specific Cldn-7 knockout mice to investigate the regulatory effects of Cldn-7 on colonic Lgr5+ stem cells in the mediation of colonic epithelial injury and repair under physiological and inflammatory conditions. Notably, our findings reveal that Cldn-7 deletion disrupts the self-renewal and differentiation of colonic stem cells alongside the formation of colonic organoids in vitro. Additionally, these Cldn-7 knockout models exhibited heightened susceptibility to experimental colitis, limited epithelial repair and regeneration, and increased differentiation toward the secretory lineage. Mechanistically, we also established that Cldn-7 facilitates the proliferation, differentiation, and organoid formation of Lgr5+ stem cells through the maintenance of Wnt and Notch signalling pathways in the colonic epithelium. Overall, our study provides new insights into the maintenance of ISC function and colonic epithelial homoeostasis.
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Introduction
Adult stem cells play a pivotal role in maintaining homoeostasis and facilitating tissue repair through their capacity for self-renewal and differentiation. Notably, the intestinal epithelium possesses an enhanced capacity for self-renewal, occurring approximately every 4–5 days [1]. This rapid turnover is driven by intestinal stem cells (ISCs) located at the base of epithelial crypts [2]. Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), a target gene in Wnt signalling, serves as a widely recognised marker for gastrointestinal stem cells [3]. Notably, +4 label-retaining cells are located specifically at the +4 position of the crypt, which is marked by the expression of Bmi1, Hopx, mTert, and Lrig1 [1, 4,5,6,7,8].
Stem cells give rise to transit-amplifying cells, which in turn differentiate into various cell lineages, including nutrient-absorbing enterocytes, protective mucus-secreting goblet cells, antimicrobial compound- and growth factor-producing Paneth cells, hormone-secreting enteroendocrine cells, and immune response-mediating chemosensory Tuft cells [9]. REG4/cKit-labelled deep crypt secretory cells in the colon are functionally similar to Paneth cells, providing specific support for Lgr5+ colonic stem cells [10, 11]. These cells migrate toward the intestinal lumen, undergo apoptosis, and are shed into the intestinal lumen, resulting in a continuous turnover of epithelial cells [12]. Lineage-tracing experiments have revealed that Lgr5+ ISCs can differentiate into all types of intestinal cells, form intact intestinal crypts, and produce organoids in vitro, which continuously maintain intestinal epithelial homoeostasis [3, 13].
The intestinal epithelium also rapidly regenerates in response to acute injury. Once damaged, the intestinal epithelium undergoes epithelial repair, in which ISCs activate, proliferate, and differentiate to restore the epithelium [14,15,16]. Acute inflammation has been shown to eradicate Lgr5+ ISCs in the small intestine and colon; this depletion of stem cells can delay or inhibit the regeneration of the damaged intestinal epithelium [17, 18]. Despite the robust regenerative capacity of ISCs, severe injury can disrupt intestinal epithelial homoeostasis, leading to inflammatory bowel disease (IBD), such as Crohn’s disease or ulcerative colitis (UC) [19, 20].
Maintaining the integrity of the intestinal barrier is crucial for host intestinal homoeostasis, which relies on the normal morphology and function of intestinal epithelial cells (IECs) and the formation of tight junctions [21, 22]. Claudin-7 (Cldn-7), a tight junction protein, is expressed in the apical, lateral, and basal membranes of IECs, with widespread expression in intestinal crypt stem cells [23]. Our previous research highlighted a significant down-regulation of Cldn-7 expression in UC; additionally, Cldn-7 deficiency led to spontaneous colitis, increasing susceptibility to dextran sulphate sodium (DSS)-induced epithelial injury [24]. Moreover, Cldn-7 has been found to contribute to the maintenance of ISCs homoeostasis in the small intestine [1.
Western blot analysis
Total protein was extracted from frozen colonic tissues using PIRA lysates supplemented with protease inhibitors; protein quantification was then performed using a BCA kit (Solaibao Life Science). Equal amounts of total protein were separated on 4–12% SDS-PAGE gels before being transferred to nitrocellulose membranes. the membranes were blocked with 5% non-fat dry milk and incubated with primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with secondary antibodies for 2 h at room temperature. Finally, the immunoreactive bands were imaged using the Ll-COR Odyssey Imaging System (LI-COR). Western blotting was repeated at least three times; representative images have been presented in corresponding figures. The antibodies used in western blotting are listed in Supplementary Table 2. All uncropped immunoblots are shown in Supplementary Fig. 6.
Statistical analyses
All experiments in this study were independently repeated a minimum of three times, with the number of animals in each experiment being specified in the figure legends. Data visualisation and statistical analyses were performed using GraphPad Prism 8.0.2 software. All data are presented as the mean ± standard error of the mean (SEM). Unpaired Student’s t-test (two-tailed) or nonparametric Mann–Whitney U test was used for comparisons between the two groups. Changes in body weight over time were analysed using repeated-measures ANOVA. Histopathological examination and statistical analyses were carried out by two authors blinded to experimental design. A p value < 0.05 was considered statistically significant.
Data availability
The authors declare that all data supporting the findings of this work are available within the main text and the supplementary information files, or from the corresponding author upon reasonable request.
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Acknowledgements
We would like to thank Professor Yong Tian (Institute of Biophysics, Chinese Academy of Sciences) for the kind gift of Lgr5-EGFP-IRES-CreERT2 mice. We thank Yu Luo and Pengfei Wu for the advice and technical assistance. This work was supported by grants from the National Natural Science Foundation of China (Grant # 82170525) and Bei**g Natural Science Foundation (Grant # 7192087).
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K.W. and Y.L. designed performed the experiments and wrote the manuscript; K.W. generated and characterised the knockout mice; H.M.L. and X.Q.L. assisted with mouse intraperitoneal injection and organoid culture; Y.L. and M.D.H. provided technical assistance with RNA-seq data analysis; D.J.Y. contributed to histological staining and analysis; L.D. led the conceptualisation and provided valuable infrastructure, guidance, and additional funding for the project.
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All procedures involving animals were followed the ARRIVE guidelines and approved by the Animal Ethics Committee of Bei**g Shijitan Hospital Institutional Review Board (sjtkyll-1x-2021(105)).
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Wang, K., Liu, Y., Li, H. et al. Claudin-7 is essential for the maintenance of colonic stem cell homoeostasis via the modulation of Wnt/Notch signalling. Cell Death Dis 15, 284 (2024). https://doi.org/10.1038/s41419-024-06658-x
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DOI: https://doi.org/10.1038/s41419-024-06658-x
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