Effect of EGFR on Calcium Mobilization and Epithelial Repair in Gastric Organoids
Authors: Engevik Kristen , Aihara Eitaro , Matthis Andrea , Montrose Marshall 
Published: June 2021

Background

In vivo, addition of calcium (Ca2+) accelerates epithelial repair of damage; intracellular Ca2+ mobilization is known to be an essential factor for gastric epithelial repair. We are investigating upstream effectors that play a role in Ca2+ mobilization and repair. Epidermal growth factor receptor (EGFR) is implicated in repair as stimulation of EGFR and subsequent activation of its downstream MAP kinases (e.g. ERK) has been shown to occur in the healing gut mucosa. Additionally, ERK activation has been shown to affect intracellular Ca2+ signaling in several cell lines. However the connection between EGFR stimulation and Ca2+ during the gastric repair process has yet to be elucidated in native tissue, in part because of the limitations of working in vivo. In the present study, we utilize the 3D primary culture of gastric epithelial cells known as gastric organoids (gastroids), which mimic in vivo gastric epithelium, to test the role of EGFR in Ca2+‐driven restitution.

Methods

Gastroids were generated from isolated corpus tissue of yellow cameleon Nano15 (YC‐Nano) transgenic mice, and cultured 4–5 days prior to experiments. YC‐Nano is a fluorescent calcium reporter which utilizes Förster resonance energy transfer (FRET) from CFP to YFP to indicate changes in intracellular Ca2+ mobilization. Damage was induced in gastroids by 1–2s exposure of single epithelial cells to high intensity two‐photon 840 nm light (photodamage, PD), resulting in damage to the targeted cell, subsequent cell death, and repair of the epithelium over 10–15 min. Damage and repair progress was evaluated using confocal/2‐photon microscopy. YC‐Nano was excited by low intensity two‐photon 840 nm light and CFP and YFP (FRET) fluorescence imaged simultaneously. Ca2+ levels reported as YFP/CFP ratio (FRET ratio) were measured in the migrating cells that neighbor the damage area as well as intact cells 2–3 cells away from the site of damage.

Results

Single epithelial cell PD injury in gastroids resulted in the paired events of dead cell exfoliation and migration of undamaged neighboring cells to restore a continuous epithelium in the damaged site. Additionally, PD injury resulted in intracellular Ca2+ increase within 1–3 min after PD, selectively in migrating cells adjacent to the damage site. The observed peak FRET ratio corresponds to the time of maximum damage area. In contrast, PD did not affect intracellular Ca2+ level in 2–3 cells away from the site of damage. FRET ratio increased more in the lateral side of the cell adjacent to the damaged area versus whole cell FRET ratio values, suggesting localized Ca2+ mobilization at the site of cell migration. Gastroids supplemented with 200 nM AG1478 (EGFR inhibitor) resulted in delayed repair. AG1478 significantly decreased the observed FRET ratio peak within gastroids, suggesting the presence of EGFR within the gastroid model. In preliminary data, addition of 10 μM FR180204 (ERK1/2 inhibitor) also prevented prompt repair and affected the intracellular Ca2+ peak.

Conclusion

Results demonstrate that EGFR is required for efficient epithelial repair in gastroids. The parallel effects of AG1478 to decrease intracellular Ca2+ levels and delay repair suggests EGFR acts upstream in the pathway of calcium‐driven repair.

Support or Funding Information

Funded by R01 DK102551‐01 and F31 DK115126‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.