Li L, Tucker R W, Hennings H, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892, USA.
Cell Growth Differ. 1995 Sep;6(9):1171-84.
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.
哺乳动物表皮的分化与基因表达在空间和时间上的协调变化相关，在此过程中，细胞从增殖性的基底细胞区室迁移至非增殖性的棘状细胞层和颗粒细胞层，在这些细胞层中完成成熟的终末阶段。先前的研究表明，体内表皮中Ca2+的梯度以及体外细胞外Ca2+的增加可诱导哺乳动物表皮角质形成细胞的分化。细胞内游离Ca2+的螯合可阻止这种Ca(2+)诱导的分化，但细胞内Ca2+的作用位点仍不明确。在本研究中，内质网Ca(2+)-ATP酶抑制剂毒胡萝卜素（Tg）和环匹阿尼酸（CPA）被用于评估细胞质Ca2+和储存Ca2+对培养的小鼠角质形成细胞Ca(2+)诱导的终末分化的相对贡献。细胞内游离Ca2+（Cai）和离子霉素敏感的Ca2+储存的持续增加与Ca(2+)诱导的角质形成细胞终末分化相关。Tg和CPA被用于改变游离Ca2+和储存Ca2+的这种协调调节。在无细胞外Ca2+的情况下，Tg和CPA均可短暂增加Cai并耗尽细胞内Ca2+储存；而在有细胞外Ca2+的情况下，Tg和CPA刺激Ca2+内流并导致Cai持续增加，同时耗尽储存的Ca2+。在有细胞外Ca2+的情况下，Tg（5至20 nM）和CPA（5至25 microM）可抑制Ca(2+)诱导的形态变化和分层，并阻止Ca2+对DNA合成的抑制作用。Tg和CPA还可抑制特定表皮棘状细胞标志物角蛋白1（K1）和角蛋白10（K10）的mRNA和蛋白质表达，阻止E-钙黏蛋白从弥漫性膜状模式重新分布至细胞间连接处的聚集，并抑制先前显示对Ca2+敏感的由K1增强子元件调控的报告基因的激活。Tg和CPA的这些作用可通过将细胞外Ca2+增加至部分恢复Ca2+储存的水平而逆转。相反，Tg和CPA可增强丝聚蛋白原和兜甲蛋白mRNA及蛋白质的表达，这些是颗粒细胞分化的标志物。Tg和CPA对角质形成细胞分化程序不同组分的这些不同作用表明，充足的细胞内Ca2+储存对于棘状细胞蛋白的表达和DNA合成的抑制很重要，而Cai的升高则刺激颗粒细胞分化标志物的表达。
