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.
Highlights
Tight spatial and temporal regulation of intracellular calcium ([Ca2+]i) signalling allows this ion to control a wide variety of physiological responses including cell growth and differentiation
nuclear factor of activated T‐cells (NFAT) as a functional readout of wound‐induced [Ca2+]i flux, we aimed to investigate the relative contribution of store‐operated calcium entry (SOCE), gap junctions and extracellular ATP signalling to wound‐induced NFAT activation
Our studies comparing responses in 0.06 and 1.2 mM [Ca2+]o showed no differences in [Ca2+]i flux response at the immediate wound edge suggesting that intracellular stores are the principle source for the initiation of the wound‐induced wave
Summary
Tight spatial and temporal regulation of intracellular calcium ([Ca2+]i) signalling allows this ion to control a wide variety of physiological responses including cell growth and differentiation. Cytosolic increases of calcium can arise essentially from extracellular influx through plasma membrane ion channels, entry of calcium from adjacent cells through gap junctions or [Ca2+]i release from intracellular stores. Calcium release from the ER is primarily triggered by local increases of the soluble mediator inositol 1,4,5‐trisphosphate (IP3) binding to its receptor on the ER and activating Ca2+ release through the IP3 receptor's intrinsic Ca2+ channel Both calcium and IP3 may pass through gap junctions (Boitano et al, 1992; Saez et al, 1989), the diffusibility of calcium is relatively limited, due in part to its propensity to interact with a variety of proteins. Gap junction signalling and release, followed by diffusion of ATP to adjacent cells, where it binds to G protein coupled receptors activating phospholipase C and generating IP3, have both been shown to play a role in mediating the wound‐ induced [Ca2+]i wave (Karvonen et al, 2000; Kobayashi et al, 2014). We designed experiments to establish the relative effects of gap‐junctional communication, extracellular ATP and SOCE on both wound‐induced NFAT transcriptional activation and keratinocyte migration to close the wound
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