Integration of wound-induced calcium signals to transcriptional activation and regulation of cutaneous wound healing responses

Abstract

Wounding is a major clinical problem. Calcium is a common secondary messenger eliciting a range of responses through spatial/temporal regulation. In keratinocytes, intracellular calcium (Ca2+i) plays a key role in growth and differentiation. An epidermal calcium gradient exists in skin which is disrupted post-wounding and wounding of monolayer human keratinocytes results in an intercellular calcium wave. Additionally, regulation of the calcium wave is known to be dependent on purinergic signalling and/or gap-junctions. However, their relative contribution in mediating the calcium flux post-wounding in primary human keratinocytes have not been well characterised. Furthermore, the importance of this calcium flux to downstream transcriptional and functional responses is not fully understood. To address this knowledge gap, the effect of store-operated calcium entry (SOCE), gap-junctions and extracellular ATP was investigated using primary human keratinocyte monolayers loaded with the calcium dye Fluo4-AM. Scratch wounding was performed in 0.06mM and 1.2mM [Ca2+]o and images captured using confocal microscopy. Calcium add-back experiments and the use of specific inhibitors were used to characterise the calcium responses. Results showed that, as expected, wounding caused an increase in Ca2+i within cells at the wound edge, which then travelled back as a wave. Both gap-junction inhibition (18αGA) and the removal of extracellular ATP (hexokinase) reduced Ca2+i flux and prevented the spread of the calcium wave following wounding. Nuclear factor of activated T-cells (NFAT) is a transcription factor activated by an increase in Ca2+i and known to be involved in keratinocyte differentiation. NFAT firefly luciferase was used to investigate activation in response to wounding. Results show NFAT transcriptional activation post-wounding in 1.2mM but not 0.06mM [Ca2+]o. Additionally, 18αGA, and the SOCE inhibitor GSK-7975A significantly reduced wound-induced NFAT activation. Perhaps surprisingly, hexokinase had no effect. Finally, the functional consequence of these signalling pathways were investigated using scratch wound migration assays. Wounds closed at a faster rate when wounding was performed in 1.2mM [Ca2+]o compared to 0.06mM. Manipulation of all three signalling pathways inhibited wound closure. However, gap-junction blockade completely prevented wound closure. Together these data indicate that, whilst purinergic and gap-junction signalling regulate the Ca2+i flux and wave post-wounding, there is a dominant effect of gap-junctions in the activation of NFAT and cell migration.