Inhibitors of the intracellular Ca(2+)-ATPase in cultured mouse keratinocytes reveal components of terminal differentiation that are regulated by distinct intracellular Ca2+ compartments.

Differentiation of mammalian epidermis is associated with spatially and temporally coordinated changes in gene expression as cells migrate from the proliferative basal cell compartment through the nonproliferative spinous and granular cell layers where the terminal phase of maturation is completed. Previous studies have suggested that a gradient of Ca2+ in the epidermis in vivo and increased extracellular Ca2+ in vitro induce differentiation of mammalian epidermal keratinocytes. Chelation of intracellular free Ca2+ prevents this Ca(2+)-induced differentiation, but sites of action for intracellular Ca2+ remain undefined. In this study, thapsigargin (Tg) and cyclopiazonic acid (CPA), inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, were used to evaluate the relative contribution of cytoplasmic and stored Ca2+ to Ca(2+)-induced terminal differentiation of cultured mouse keratinocytes. A sustained increase of both intracellular free Ca2+ (Cai) and ionomycin-sensitive Ca2+ stores is associated with Ca(2+)-induced keratinocyte terminal differentiation. Tg and CPA was used to change this coordinated regulation of free and stored Ca2+. In the absence of extracellular Ca2+, both Tg and CPA transiently increase Cai and deplete intracellular Ca2+ stores; while in the presence of extracellular Ca2+, Tg and CPA stimulate Ca2+ influx and cause a sustained increase in Cai while depleting stored Ca2+. In the presence of extracellular Ca2+, Tg (5 to 20 nM) and CPA (5 to 25 microM) inhibit Ca(2+)-induced morphological changes and stratification and prevent the suppression of DNA synthesis by Ca2+. Tg and CPA also inhibit the expression of mRNA and protein for specific epidermal spinous cell markers, keratins 1 (K1) and 10 (K10), prevent the redistribution of E-cadherin from a diffuse membranous pattern to concentration at cell-cell junctions, and inhibit the activation of a reporter gene regulated by a K1 enhancer element shown previously to be Ca2+ sensitive. These effects of Tg and CPA can be reversed by increasing the extracellular Ca2+ to levels that partially restore Ca2+ stores. In contrast, Tg and CPA enhance the expression of profilaggrin and loricrin mRNA and protein, markers of granular cell differentiation. These divergent actions of Tg and CPA on distinct components of the keratinocyte differentiation program suggest that adequate intracellular Ca2+ stores are important for the expression of spinous cell proteins and inhibition of DNA synthesis, while elevation of Cai stimulates the expression of markers of granular cell differentiation.