The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca2+- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.

Stratum corneum, the outermost layer of the epidermis, consists of dead anucleated keratinocytes called corneocytes, functioning as a barrier. The exact molecular mechanisms by which the top layer of living keratinocytes (SG1 cells) die has been largely unknown. Here, we demonstrate via intravital imaging procedures combined with intracellular Ca 2+ -and pHresponsive fluorescent probes, we aimed to clarify the SG1 death process in vivo. We found that SG1 cells die after prolonged (w60 min) intracellular Ca 2+ elevation at neutral pH (phase I). Subsequently, irreversibly sustained rapid intracellular acidification occurs (phase II). In cultured primary SG1 cells, phase I was accompanied by increased cell membrane permeability, an indicator of cell death in general, whereas phase II was essential for disappearance of keratohyalin granules and nuclear DNA. Intravital Ca 2+ -and pH-imaging of dorsal and ear skin of TRPV3-deficient mice revealed that the duration of these phases was tightly regulated by the transient receptor potential cation channel V3 (TRPV3), suggesting that TRPV3 acts as a timekeeper for phase I and II conditions. Our combined various fluorescent probes and a newly-developed in vitro culture system clarified the molecular mechanisms of the unique SG1 cell death, termed corneoptosis.