Li L, Tennenbaum T, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, Bethesda, MD 20892-4255, USA.
J Invest Dermatol. 1996 Feb;106(2):254-60. doi: 10.1111/1523-1747.ep12340654.
Modulating extracellular Ca2+ (Cao) and suspension culture are two frequently used methods to induce maturation of cultured human and mouse keratinocytes. To determine if the two methods share a common mechanism, changes in Ca2+ metabolism were studied in suspension cultures of mouse keratinocytes. Spontaneously detached and suspension- cultured keratinocytes in 0.05 microM Ca2+ medium express markers of suprabasal differentiation, while 0.05 microM Ca2+ is not permissive for marker expression by attached keratinocytes. Intracellular free Ca2+ (Cai) increased rapidly after placing keratinocytes in suspension in 0.05 microM Ca2+, reaching levels up to 3- to 4-fold higher than Cai in attached cells after 4-5 h. In suspended cells, the increase in Cai was associated with a 2- to 6- fold increase in Ca2+ transport across plasma membrane as well as depletion of intracellular Ca2+ -stores. Differentiation marker expression and terminal differentiation were inhibited in suspension-cultured keratinocytes by preventing the rise of Cai using either 1,2-bis (o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate intracellular Ca2+ or ethyleneglycol-bis (beta-aminoethyl ether)- N,N,N',N' -tetraacetic acid to reduce Cao. Together these results indicate that a rise in CAi is a common mechanism controlling differentiation in cultured mouse keratinocytes, and suspension of keratinocytes enhances Ca2+ transport and alters intracellular Ca2+ sequestration producing a rise in Cai.
调节细胞外钙离子（Ca₂⁺o）浓度和悬浮培养是诱导人及小鼠角质形成细胞成熟的两种常用方法。为确定这两种方法是否具有共同机制，我们研究了小鼠角质形成细胞悬浮培养过程中钙离子代谢的变化。在0.05微摩尔/升钙离子培养基中自发脱离并悬浮培养的角质形成细胞表达基底上层分化标志物，而0.05微摩尔/升钙离子浓度对于贴壁角质形成细胞的标志物表达并不适宜。将角质形成细胞置于0.05微摩尔/升钙离子的悬浮环境中后，细胞内游离钙离子（Ca₂⁺i）迅速增加，4 - 5小时后达到比贴壁细胞中Ca₂⁺i高3至4倍的水平。在悬浮细胞中，Ca₂⁺i的增加与质膜钙离子转运增加2至6倍以及细胞内钙离子储存的耗竭有关。使用1,2 - 双（邻氨基苯氧基）乙烷 - N,N,N',N' - 四乙酸螯合细胞内钙离子或乙二醇 - 双（β - 氨基乙基醚） - N,N,N',N' - 四乙酸降低Ca₂⁺o，通过阻止Ca₂⁺i升高来抑制悬浮培养角质形成细胞中的分化标志物表达和终末分化。这些结果共同表明，Ca₂⁺i升高是控制培养的小鼠角质形成细胞分化的共同机制，角质形成细胞的悬浮增强了钙离子转运并改变了细胞内钙离子螯合，导致Ca₂⁺i升高。
