Dominant effect of gap junction communication in wound‐induced calcium‐wave, NFAT activation and wound closure in keratinocytes

Hudson, Laura; Begg, Malcolm; Wright, Blythe; Cheek, Tim; Jahoda, Colin A.B.; Reynolds, Nick J.

Abstract

Wounding induces a calcium wave and disrupts the calcium gradient across the epidermis but mechanisms mediating calcium and downstream signalling, and longer-term wound healing responses are incompletely understood. As expected, live-cell confocal imaging of Fluo-4-loaded normal human keratinocytes showed an immediate increase in [Ca2+]i at the wound edge that spread as a calcium wave (8.3 µm/s) away from the wound edge with gradually diminishing rate of rise and amplitude. The amplitude and area under the curve of [Ca2+]i flux was increased in high (1.2 mM) [Ca2+]o media. 18α-glycyrrhetinic acid (18αGA), a gap-junction inhibitor or hexokinase, an ATP scavenger, blocked the wound-induced calcium wave, dependent in part on [Ca2+]o. Wounding in a high [Ca2+]o increased nuclear factor of activated T-cells (NFAT) but not NFkB activation, assessed by dual-luciferase receptor assays compared to unwounded cells. Treatment with 18αGA or the store-operated channel blocker GSK-7975A inhibited wound-induced NFAT activation, whereas treatment with hexokinase did not. Real-time cell migration analysis, measuring wound closure rates over 24 h, revealed that 18αGA essentially blocked wound closure whereas hexokinase and GSK-7975A showed relatively minimal effects. Together these data indicate that while both gap-junction communication and ATP release from damaged cells are important in regulating the wound-induced calcium wave, long-term transcriptional and functional responses are dominantly regulated by gap-junction communication.

• Journal Article Type: Article
• Acceptance Date: Jun 15, 2021
• Online Publication Date: Jun 27, 2021
• Publication Date: 2021-12
• Deposit Date: Jul 13, 2021
• Publicly Available Date: Jul 14, 2021
• Journal: Journal of Cellular Physiology
• Print ISSN: 0021-9541
• Electronic ISSN: 1097-4652
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 236
• Issue: 12
• Pages: 8171-8183
• DOI: https://doi.org/10.1002/jcp.30488
• Public URL: https://durham-repository.worktribe.com/output/1272302