Mechanosensitive ATP release from hemichannels and Ca2+ influx through TRPC6 accelerate wound closure in keratinocytes

Cutaneous wound healing is accelerated by exogenous mechanical forces and is impaired in TRPC6-knockout mice. Therefore, we designed experiments to determine how mechanical force and TRPC6 channels contribute to wound healing using HaCaT keratinocytes. HaCaT cells were pretreated with hyperforin, a major component of a traditional herbal medicine for wound healing and also a TRPC6 activator, and cultured in an elastic chamber. At 3 h after scratching the confluent cell layer, the ATP release and intracellular Ca2+ increases in response to stretching (20%) were live-imaged. ATP release was observed only in cells at the frontier facing the scar. The diffusion of released ATP caused intercellular Ca2+ waves that propagated towards the rear cells in a P2Y-receptor-dependent manner. The Ca2+ response and wound healing were inhibited by ATP diphosphohydrolase apyrase, the P2Y antagonist suramin, the hemichannel blocker CBX and the TRPC6 inhibitor diC8-PIP2. Finally, the hemichannel-permeable dye calcein was taken up only by ATP-releasing cells. These results suggest that stretch-accelerated wound closure is due to the ATP release through mechanosensitive hemichannels from the foremost cells and the subsequent Ca2+ waves mediated by P2Y and TRPC6 activation.