Search

Background & Aims: Claudin-7 (Cldn7) is a tight junction (TJ) membrane protein located at the apical TJ and basolateral side of intestinal epithelial cells. Deletion of Cldn7 by gene targeting leads to the inflammatory bowel disease–like phenotype in mice, which includes weight loss, diarrhea, mucosa ulceration, and severe intestinal epithelial damage. In this study, we test our hypothesis that Cldn7 plays a critical role in regulating intestinal crypt stem cell functions. Methods: Gene expression microarray, quantitative reverse-transcription polymerase chain reaction, in situ hybridization, histologic examinations, immunoblotting, 3-dimensional organoid culture, and various treatments to rescue Cldn7-deficient organoid defects were conducted using global Cldn7 knockout mice and inducible, conditional Cldn7 knockout mice. Results: Gene deletion of Cldn7 in intestines showed significant alteration of expression profiles with striking down-regulation of intestinal crypt stem cell markers such as Olfm4, dislocated proliferative cells, and disrupted epithelial cell differentiation. In addition, the isolated Cldn7-deficient crypts where the stem cells reside were either unable to survive at all or formed defective spheroids, highlighting the functional impairment of crypt stem cells in the absence of Cldn7. Remarkably, the Cldn7-expressing organoids with buddings underwent rapid cell degeneration within days after turning off Cldn7 expression in the culture. We identified that activation of Wnt/β-catenin signaling rescued the organoid defects caused by Cldn7 deletion. Conclusions: In this study, we show that Cldn7 is indispensable in controlling Wnt/β-catenin signaling–dependent intestinal epithelial stem cell survival, self-renewal, and cell differentiation. This study could open a door to study roles of TJ proteins in stem cell regulations in other tissues and organs.

Background: We previously showed that lifelong rapamycin treatment of short-lived Apc Min/+ mice, a model for familial adenomatous polyposis, resulted in a normal lifespan. Apc Min/+ mice develop colon polyps with a low frequency but can be converted to a colon cancer model by dextran sodium sulfate (DSS) treatments (Apc Min/+ -DSS model)., Materials and Methods: We asked, what effect would pretreatment of Apc Min/+ mice with chronic rapamycin prior to DSS exposure have on survival and colonic neoplasia?, Results: Forty-two ppm enteric formulation of rapamycin diet exacerbated the temporary weight loss associated with DSS treatment in both sexes. However, our survival studies showed that chronic rapamycin treatment significantly extended lifespan of Apc Min/+ -DSS mice (both sexes) by reductions in colon neoplasia and prevention of anemia. Rapamycin also had prophylactic effects on colon neoplasia induced by azoxymethane and DSS in C57BL/6 males and females. Immunoblot assays showed the expected inhibition of complex 1 of mechanistic or mammalian target of rapamycin (mTORC1) and effectors (S6K→rpS6 and S6K→eEF2K→eEF2) in colon by lifelong rapamycin treatments. To address the question of cell types affected by chronic enteric rapamycin treatment, immunohistochemistry analyses demonstrated that crypt cells had a prominent reduction in rpS6 phosphorylation and increase in eEF2 phosphorylation relative controls., Conclusion: These data indicate that enteric rapamycin prevents or delays colon neoplasia in Apc Min/+ - DSS mice through inhibition of mTORC1 in the crypt cells., (Copyright © 2021. Published by Elsevier Inc.)

Background: Apc Min/+ mice model familial adenomatous polyposis (FAP), a disease that causes numerous colon polyps leading to colorectal cancer. We previously showed that chronic treatment of Apc Min/+ females with the anti-aging drug, rapamycin, restored a normal lifespan through reduced polyposis and anemia prevention. Lifespan extension by chronic rapamycin in wildtype UM-HET3 mice is sex-dependent with females gaining the most benefit. Whether Apc Min/+ mice have a similar sex-dependent response to chronic mTOR inhibition is not known., Methods: To address this knowledge gap and gain deeper insight into how chronic mTOR inhibition prevents intestinal polyposis, we compared male and female Apc Min/+ mice responses to chronic treatment with a rapamycin-containing diet. Animals were fed a diet containing either 42 ppm microencapsulate rapamycin or empty capsules, one group was used to determine lifespan and a second group with similar treatment was harvested at 16 weeks of age for cross-sectional studies., Results: We found that the survival of males is greater than females in this setting (P < 0.0197). To explore the potential basis for this difference we analyzed factors affected by chronic rapamycin. Immunoblot assays showed that males and females exhibited approximately the same level of mTORC1 inhibition using phosphorylation of ribosomal protein S6 (rpS6) as an indirect measure. Immunohistochemistry assays of rpS6 phosphorylation showed that rapamycin reduction of mTORC1 activity was on the same level, with the most prominent difference being in intestinal crypt Paneth cells in both sexes. Chronic rapamycin also reduced crypt depths in both male and female Apc Min/+ mice (P < 0.0001), consistent with reduced crypt epithelial cell proliferation. Finally, chronic rapamycin prevented anemia equally in males and females., Conclusions: In males and females, these findings link rapamycin-mediated intestinal polyposis prevention with mTORC1 inhibition in Paneth cells and concomitant reduced epithelial cell proliferation., Competing Interests: Potential financial conflict of interest: The University of Texas Health Science Center at San Antonio has applied for a patent, U.S. Patent Application No. 13/128,800, by inventors Zelton Dave Sharp, Randy Strong, and Paul Hasty for an encapsulated rapamycin formulation used in this paper. Under a licensing agreement between Emtora Biosciences (formerly Rapamycin Holdings, Inc.) and the University of Texas Health Science Center San Antonio, R. Strong, Z.D. Sharp, P. Hasty, and the University are entitled to milestone payments and royalty on sales of microencapsulated rapamycin. The university has a plan for managing conflicts of interest under its “Policy and Procedures for Promoting Objectivity in Research by Managing, Reducing, or Eliminating Conflicts of Interest.”