Li L, Tucker R W, Hennings H, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
J Cell Physiol. 1995 Apr;163(1):105-14. doi: 10.1002/jcp.1041630112.
The role of intracellular Ca2+ in the regulation of Ca(2+)-induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca2+ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca(2+) Intracellular BAPTA loaded by BAPTA/AM (15-30 microM) inhibited the expression of epidermal differentiation-specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation-associated redistribution of E-cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and loricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca2+, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca2+, the concentration of intracellular free Ca2+ (Cai) in BAPTA-loaded keratinocytes was examined by digital image analysis using the Ca(2+)-sensitive fluorescent probe fura-2, and Ca2+ influx was measured by 45Ca2+ uptake studies. Increase in extracellular Ca2+ (Cao) in the culture medium of keratinocytes caused a sustained increase in both Cai and Ca2+ localized to ionomycin-sensitive intracellular stores in keratinocytes. BAPTA lowered basal Cai concentration and prevented the Cai increase. After 12 hours of BAPTA treatment, the basal level of Cai returned to the control value, but the Ca2+ localized in intracellular stores was substantially decreased. 45Ca2+ uptake was initially (within 30 min) increased in BAPTA-loaded cells. However, the total 45Ca2+ accumulation over 24 hours in BAPTA-loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Cai and total cellular Ca2+ content in the presence of increased amount of intracellular Ca2+ buffer (e.g., BAPTA) by depleting intracellular Ca2+ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca(2+)-stores since this is the major change in intracellular Ca2+ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E-cadherin on the cell membrane may be more directly associated with Cai change.
使用细胞内钙离子螯合剂1,2 - 双（邻氨基苯氧基）乙烷 - N,N,N',N' - 四乙酸（BAPTA），研究了细胞内钙离子在调节小鼠角质形成细胞钙离子诱导的终末分化中的作用。一种细胞可渗透的乙酰氧基甲基（AM）酯衍生物BAPTA（BAPTA/AM）被加载到0.05 mM钙离子培养基中的原代小鼠角质形成细胞中，然后用含有0.12或0.5 mM钙离子的培养基诱导细胞分化。通过免疫印迹检测，BAPTA/AM加载的细胞内BAPTA（15 - 30 microM）抑制了表皮分化特异性蛋白角蛋白1（K1）、角蛋白10（K10）、丝聚蛋白和兜甲蛋白的表达。通过免疫荧光测定，E - 钙黏蛋白在细胞膜上与分化相关的重新分布被延迟但未被抑制。BAPTA还抑制了K1、K10和兜甲蛋白mRNA的表达。此外，BAPTA阻止了由0.12和0.5 mM钙离子诱导的DNA合成减少，表明该药物抑制分化但对角质形成细胞无毒。为了评估BAPTA对细胞内钙离子的影响，使用钙离子敏感荧光探针fura - 2通过数字图像分析检查了加载BAPTA的角质形成细胞中细胞内游离钙离子（Cai）的浓度，并通过45Ca2+摄取研究测量了钙离子内流。角质形成细胞培养基中细胞外钙离子（Cao）的增加导致Cai和定位于角质形成细胞中离子霉素敏感的细胞内储存库的钙离子持续增加。BAPTA降低了基础Cai浓度并阻止了Cai的增加。BAPTA处理12小时后，Cai的基础水平恢复到对照值，但细胞内储存库中定位的钙离子大幅减少。在加载BAPTA的细胞中，45Ca2+摄取最初（30分钟内）增加。然而，加载BAPTA的细胞在24小时内的总45Ca2+积累与对照值保持不变。这些结果表明，角质形成细胞可以通过长时间耗尽细胞内钙离子储存库，在细胞内钙离子缓冲剂（如BAPTA）量增加的情况下维持Cai和细胞总钙离子含量。BAPTA对角质形成细胞分化标志物表达的抑制可能是由于钙离子储存库的耗尽，因为这是在角质形成细胞表达分化标志物时检测到的细胞内钙离子的主要变化。相比之下，E - 钙黏蛋白在细胞膜上的重新分布可能与Cai变化更直接相关。
