Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids

Abstract

Key pointsr Determining the signalling cascade of epithelial repair, using murine gastric organoids, allows definition of regulatory processes intrinsic to epithelial cells, at the same time as validating and dissecting the signalling cascade with more precision than is possible in vivo r Following single cell damage, intracellular calcium selectively increases within cells adjacent to the damage site and is essential for promoting repair.r Trefoil factor 2 (TFF2) acts via chemokine C-X-C receptor 4 and epidermal growth factor receptor signalling, including extracellular signal-regulated kinase activation, to drive calcium mobilization and promote gastric repair.r Sodium hydrogen exchanger 2, although essential for repair, acts downstream of TFF2 and calcium mobilization.Abstract The gastric mucosa of the stomach is continually exposed to environmental and physiological stress factors that can cause local epithelial damage. Although much is known about the complex nature of gastric wound repair, the stepwise process that characterizes epithelial restitution remains poorly defined. The present study aimed to determine the effectors that drive gastric epithelial repair using a reductionist culture model. To determine the role of trefoil factor 2 (TFF2) and intracellular calcium (Ca 2+ ) mobilization in gastric restitution, gastric organoids were derived from TFF2 knockout (KO) mice and yellow Cameleon-Nano15 (fluorescent calcium reporter) transgenic mice, respectively. Inhibitors and recombinant protein were used to determine the upstream and downstream effectors of gastric restitution following photodamage (PD) to single cells within the gastric organoids. Single cell PD resulted in parallel events of dead cell exfoliation and migration of intact neighbouring cells to restore a continuous epithelium in the damage site. Under normal conditions following PD, Ca 2+ levels increased Kristen Engevik is a PhD candidate at the University of Cincinnati College of Medicine. Kristen's research focuses on investigating the mechanism behind gastric restitution. Her expertise lies in culturing three-dimensional gastric organoids and the use of two-photon light microscopy to induce photodamage with respect to examining the pathways involved in gastric repair. K. A. Engevik and othersJ Physiol 597.10 within neighbour migrating cells, peaking at ß1 min, suggesting localized Ca 2+ mobilization at the site of cell protrusion/migration. TFF2 KO organoids exhibit delayed repair; however, this delay can be rescued by the addition of exogenous TFF2. Inhibition of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK)1/2 or a TFF2 receptor, chemokine C-X-C receptor 4 (CXCR4), resulted in significant delay and dampened Ca 2+ mobilization. Inhibition of sodium hydrogen exchanger 2 (NHE2) caused significant delay but did not affect Ca 2+ mobilization. A similar delay was observed in NHE2 KO organoids. In TFF2 KO gastric organoids, the addition of exogenous TFF2 in the presence of EGFR or CXCR4 inhibition was u...

Vitamin D differentially regulates colon stem cells in patient‐derived normal and tumor organoids

“…Moreover, we observed in mouse, due the lack of the equivalent human signature, that calcitriol induces a gene expression profile that is related to the Mex3a high /Lgr5 high signature of slow‐dividing intestinal stem cells . Additionally, calcitriol inhibits the activator of EGF pathway TFF2 gene and regulates the expression of the pro‐tumorigenic gene TNS4 and of the negative regulators of colon carcinoma cell proliferation RARRES1 (also known as TIG1 ) and its target GRK5 . Conceivably, these effects may contribute to the antiproliferative action of calcitriol in crypt stem cells.…”

Trefoil factor 2–C‐X‐C chemokine receptor 4‐mediated calcium signalling is implicated in gastric epithelial restitution

“…In a recent issue of The Journal of Physiology , Engevik et al . () utilize a gastric organoid culture systems to uncover the convergence of the TFF2, EGFR and Ca 2+ signalling pathways in gastric epithelium repair.…”

“…Additionally, Engevik et al . () made use of the in‐bred sodium–hydrogen exchanger 2 (NHE2) KO model derived from FVB/N mice. Both male and female mice, between the ages of 2 and 4 months, were used.…”

“…In the case of the methodology of Engevik et al . (), isolation and culturing native murine gastric tissue for organoid development creates a risk of de‐differentiation during the culturing process. In contrast, the methods of McCraken et al .…”

“…The system used by Engevik et al . () reflects a significant advancement in the precise measurement of secondary messengers through the use of fluorescent reporters in tandem with inhibitor assays.…”