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

• Published in: Journal of cellular physiology 2021-12, Vol.236 (12), p.8171-8183
• Main Authors: Hudson, Laura, Begg, Malcolm, Wright, Blythe, Cheek, Tim, Jahoda, Colin A. B., Reynolds, Nick J.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amplitudes; Animals; ATP; ATP signalling; Calcium; Calcium (extracellular); Calcium (intracellular); Calcium - metabolism; calcium flux; Calcium ions; Calcium Signaling - physiology; Calcium signalling; Cell interactions; Cell migration; Cell Movement - physiology; Cells, Cultured; Communication; Epidermis; Gap Junctions - metabolism; Hexokinase; Humans; Keratinocytes; Keratinocytes - metabolism; NF-AT protein; NF-κB protein; NFATC Transcription Factors - metabolism; store‐operated‐calcium‐entry; Transcription; Wound healing; Wound Healing - physiology
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.30488

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. In this study, we analysed specific parameters of the wound‐induced calcium wave in human keratinocytes and delineated the relative the contribution of extracellular ATP signalling, gap‐junctional communication, and extracellular calcium influx providing insight into the mechanisms of wave transmission. By inhibiting the specific components contributing to wave transmission, we established that gap‐junctional communication and store‐operated calcium entry regulated wound‐induced nuclear factor of activated T‐cells transcriptional activation and keratinocyte migration to close the wound. Gap‐junctional communication exerted the most profound effect and completely prevented wound closure with relatively small contributions from extracellular ATP.