A unique mode of keratinocyte death requires intracellular acidification

Abstract

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.

Functional cell death, corneoptosis, requires temporally controlled intracellular acidification

“…Using these fluorescent reporters in the SG1 cells of live mice, they find that corneoptosis consists of two stereotyped and temporally distinct phases. Building off recent work (7), the authors show that SG1 cells in phase I show high [Ca 2+ ] with a neutral pH for about 60 min, followed closely by cells in phase II sustaining high [Ca 2+ ] with pH dropping rapidly, leading to an acidic pH (6).…”

“…Both keratins and SASPase may be pH sensitive and could explain the authors' observation that a rapid drop of pH in phase II leads to the degradation of KHGs (9)(10)(11). Additionally, Matsui et al (6) show that complete nuclear degradation, a step that must conclude before cells reach the SC layer, is dependent on the rapid drop in pH observed during phase II. The authors suggest this could be carried out by DNase1-like 2 (DNase1L2) and DN-ase2, which are DNases that have been shown to be optimal at acidic pH and active in SG1 cells (12)(13)(14).…”

“…However, the exact cellular and molecular mechanisms underpinning this unique process of cell death are still not fully understood. Matsui et al (6) combine elegant in vivo and in vitro experiments to dissect the process of SG1 cell death they coin "corneoptosis," the initial step in cornification.…”

“…Transglutaminases are calcium-dependent enzymes involved in the formation of the cornified envelope, an insoluble mix of cross-linked proteins and lipids that replaces the plasma membrane in corneocytes (1). Matsui et al (6) suggest that the sustained elevation in [Ca 2+ ] in phase I could be activating transglutaminases 3 and 5, leading to formation of the cornified envelope. In the future, it will be important to further understand exactly when and how phase I is triggered.…”

The Epidermis: Redox Governor of Health and Diseases

“…Microenvironmental cues, such as the calcium gradient [ 8 ], vitamin D [ 9 ], or vitamin A [ 10 , 11 ], profoundly affect cellular fate. The uppermost living layers, stratum granulosum (SG), where the functional cell death program [ 12 , 13 ] takes place, denote anatomical importance. Before being completely flattened and enucleated, the keratinocyte cell membrane is replaced by a specialized structure called cornified cell envelopes (CEs), which is a macromolecular structure composed of protein and lipids [ 14 ].…”