Dataset Information

Core clock gene BMAL1 and RNA-binding protein MEX3A collaboratively regulate Lgr5 expression in intestinal crypt cells.

ABSTRACT: The intestinal epithelium is highly regenerative. Rapidly proliferating LGR5⁺ crypt base columnar (CBC) cells are responsible for epithelial turnover needed to maintain intestinal homeostasis. Upon tissue damage, loss of LGR5⁺ CBCs can be compensated by activation of quiescent +4 intestinal stem cells (ISCs) or early progenitor cells to restore intestinal regeneration. LGR5⁺ CBC self-renewal and ISC conversion to LGR5⁺ cells are regulated by external signals originating from the ISC niche. In contrast, little is known about intrinsic regulatory mechanisms critical for maintenance of LGR5⁺ CBC homeostasis. We found that LGR5 expression in intestinal crypt cells is controlled by the circadian core clock gene BMAL1 and the BMAL1-regulated RNA-binding protein MEX3A. BMAL1 directly activated transcription of Mex3a. MEX3A in turn bound to and stabilized Lgr5 mRNA. Bmal1 depletion reduced Mex3a and Lgr5 expression and led to increased ferroptosis, which consequently decreased LGR5⁺ CBC numbers and increased the number of crypt cells expressing +4 ISC marker BMI1. Together, these findings reveal a BMAL1-centered intrinsic regulatory pathway that maintains LGR5 expression in the crypt cells and suggest a potential mechanism contributing to ISC homeostasis.

SUBMITTER: Cheng LT

PROVIDER: S-EPMC10579233 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Core clock gene BMAL1 and RNA-binding protein MEX3A collaboratively regulate Lgr5 expression in intestinal crypt cells.

Cheng Li-Tzu LT Tan Grace Y T GYT Chang Fang-Pei FP Wang Cheng-Kai CK Chou Yu-Chi YC Hsu Pang-Hung PH Hwang-Verslues Wendy W WW

Scientific reports 20231016 1

The intestinal epithelium is highly regenerative. Rapidly proliferating LGR5+ crypt base columnar (CBC) cells are responsible for epithelial turnover needed to maintain intestinal homeostasis. Upon tissue damage, loss of LGR5+ CBCs can be compensated by activation of quiescent +4 intestinal stem cells (ISCs) or early progenitor cells to restore intestinal regeneration. LGR5+ CBC self-renewal and ISC conversion to LGR5+ cells are regulated by external s ...[more]

PMID: 37845346

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Project description:The intestinal exporter MRP2 plays an important role in disposition and elimination of a wide range of drugs. Here, we aimed to clarify the impact of circadian clock on intestinal MRP2, and to determine the molecular mechanisms for generation of diurnal MRP2 expression. Methods: The regulatory effects of Bmal1 on intestinal MRP2 expression were assessed using intestine-specific Bmal1 knockout (Bmal1iKO ) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Everted gut sac, cell viability and in situ intestinal perfusion experiments were performed to evaluate intestinal efflux of the MRP2 substrate methotrexate (MTX). Toxicity and pharmacokinetic experiments were performed with Bmal1iKO mice and control littermates (Bmal1fl/fl mice) after oral gavage of MTX. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays. Results: Bmal1iKO mice were generated by inter-crossing the mice carrying a Bmal1 exon 8 floxed allele (Bmal1fl/fl ) with Villin-Cre mice. Intestinal MRP2 expression exhibited a diurnal oscillation in Bmal1fl/fl mice with a zenith value at ZT6. Bmal1 ablation caused reductions in Mrp2 mRNA and protein levels [as well as in transport activity (measured by MTX)], and blunted their diurnal rhythms. Intestinal ablation of Bmal1 abrogated circadian time-dependency of MTX pharmacokinetics and toxicity. Bmal1/BMAL1 regulation of rhythmic Mrp2/MRP2 expression was also confirmed in the colon cancer CT26 and Caco-2 cells. Based on a combination of luciferase reporter, mobility shift and ChIP assays, we found that Dbp activated and E4bp4 repressed Mrp2 transcription via specific binding to a same D-box (-100/-89 bp) element in promoter region. Further, Bmal1 directly activated the transcription of Dbp and Rev-erbα through the E-boxes, whereas it negatively regulated E4bp4 via the transcriptional repressor Rev-erbα. Positive regulation of Mrp2 by Rev-erbα was also observed, and attained through modulation of E4bp4. Conclusion: Bmal1 coordinates temporal expressions of DBP (a MRP2 activator), REV-ERBα (an E4BP4 repressor) and E4BP4 (a MRP2 repressor), generating diurnal MRP2 expression.

Dataset Information

Core clock gene BMAL1 and RNA-binding protein MEX3A collaboratively regulate Lgr5 expression in intestinal crypt cells.

Publications

Core clock gene BMAL1 and RNA-binding protein MEX3A collaboratively regulate Lgr5 expression in intestinal crypt cells.

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