Upregulated nicotinic ACh receptor signaling contributes to intestinal stem cell function through activation of Hippo and Notch signaling pathways

Abstract

Backgrounds: Recent studies have shown that various mammalian non-neuronal cells synthesize acetylcholine (ACh) in situ and operate cholinergic signaling via nicotinic and muscarinic ACh receptors (nAChRs and mAChRs). Understanding the mechanisms that control intestinal stem cell (ISC) function through activation of nAChR signaling is critical for developing therapeutic interventions for diseases such as inflammatory bowel disease (IBD). Previously, by conducting RNA sequencing (RNA-Seq) analysis using crypt-villus organoid cultures, we found that the Hippo signaling pathway, a stem cell regulating network, is upregulated in ISCs after treatment with nicotine. Here, we explored the roles of nAChR signaling through activation of the Hippo signaling pathway.

Methods: RNA-Seq data were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. β4-knock-in mice were generated, and experiments using the knock-in mice and their intestinal organoids were carried out.

Results: RNA-Seq and qRT-PCR analyses demonstrated that the expression of YAP1/TAZ and Notch1/Dll1 was upregulated after treatment with nicotine. However, a nAChR antagonist, mecamylamine, strongly inhibited the expression of these genes. Notably, we found that in β4-knock-in mouse small intestines, expression of YAP1 and Notch1 was significantly reduced, but not that of TAZ and Dll1, suggesting that Hippo and Notch signaling pathways are putative targets for nAChR signaling. Furthermore, fluorescent signals were detected in Paneth cells that interact with ISCs at the crypt bottom, indicating an interaction between Paneth cells and ISCs via nAChR signaling through the activation of Hippo and Notch signaling pathways.

Conclusion: Our results indicate that upregulated nAChR signaling contributes to the maintenance of ISC activity and balances differentiation through activation of Hippo and Notch signaling pathways.

Keywords: Hippo signaling; Inflammatory bowel disease (IBD); Intestinal stem cell (ISC); Nicotinic acetylcholine receptor (nAChR); Non-neuronal ACh; Notch signaling; Organoid; Yes-associated protein 1 (YAP1).