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FZD5 is an essential regulator of intestinal crypt homeostasis and surrogate Wnt agonist response

ABSTRACT: The rapid regeneration of the small intestinal epithelium is sustained by crypt intestinal stem cells (ISCs). Wnt/b-catenin signaling is essential for intestinal crypt homeostasis and maintenance of Lgr5+ ISC, and yet no single or combinatorial knockout of Frizzled (FZD) genes, representing Wnt receptors, has phenocopied the severe intestinal epithelial effects of Wnt signaling blockade. The elusive identification of specific Frizzled (Fzd) receptor(s) underlying homeostatic proliferation and ISC function would greatly inform therapeutic mucosal repair strategies. In prior pharmacologic studies, bioengineered antagonists that block Wnt binding to both FZD5 and FZD8 receptors induced lethal crypt and villus loss, implicating FZD5 and/or FZD8 as essential for ISCs maintenance. Here, the potential function of Fzd5 in during intestinal homeostasis was examined by epithelial-specific Fzd5 ko, which rapidly elicited lethal pan-intestinal crypt and villus loss, and by Lgr5-specific Fzd5 cKO, which strongly reduced Lgr5+ ISC while inducing their premature differentiation. In parallel, Fzd5 cKO potently repressed Wnt target gene expression, with phenotypic rescue by constitutive activation of b-catenin in vivo and confirmation upon in vitro organoid culture. Fzd5 cKO but not Fzd8 cKO in organoids ablated responsiveness to dual specificity bioengineered FZD5/FZD8-selective Wnt surrogate agonists, which reversed DSS-induced colitis phenotypes in both wild-type and Fzd8 cKO mice. Overall, our results implicate the FZD5 receptor as an essential regulator of crypt homeostasis, Lgr5+ ISCs and intestinal response to bioengineered Wnt surrogate agonists.

ORGANISM(S): Mus musculus

PROVIDER: GSE233260 | GEO | 2024/10/18

REPOSITORIES: GEO

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Project description:Background & Aims: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions. Methods: We generated mice with intestinal epithelial-specific disruption of Cul4b (pVillin-cre; Cul4bfn/Y), inducible disruption of Cul4b (Lgr5-creERT2; Cul4bfn/Y, CAG-creERT2; Cul4bfn/Y) and their control (Cul4bfn/Y). Intestinal tissues were analyzed by histology, immunofluorescence, RNA sequencing and mass spectrum. Intestinal organoids deprived from mice with pVillin-Cre; Cul4bfn/Y, Lgr5-Cre; Cul4bfn/Y, Tg-Cul4b and their controls were used in assays to measure intestinal self-renewal, proliferation and differentiation. Wnt signaling and intestinal markers were analyzed by immunofluorescence and immunoblot assays. Differential proteins upon Cul4b ablation or Cul4b-interacting proteins were identified by mass spectrometry. Results: Cul4b specifically located at ISCs zone. Block of Cul4b impaired intestinal homeostasis maintenance by reduced self-renewal and proliferation. Transcriptome analysis revealed that Cul4b-null intestine lose ISC characterization and showed disturbed ISC niche. Mechanistically, reactivated Wnt pathway could recover intestinal dysfunction of Cul4b knockout mice. Analysis of differential total and ubiquitylated proteins uncovered the novel targeting substrate of Cullin-Ring ubiquitin ligase 4b (CRL4b), immunity-related GTPase family M member 1 (Irgm1) in intestine. Decreased Irgm1 rescued abnormally interferon signaling, overemphasized autophagy and downstream phosphate proteins in Cul4b knockout mice. Conclusion: We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.

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FZD5 is an essential regulator of intestinal crypt homeostasis and surrogate Wnt agonist response

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