Department of Dermatology, University of Turku and Turku University Hospital, Turku, Finland; Department of Cell Biology and Anatomy, University of Turku, Turku, Finland.
Exp Dermatol. 2012 Aug;21(8):586-91. doi: 10.1111/j.1600-0625.2012.01520.x. Epub 2012 May 29.
Mutations in the ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes, in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by Western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mm) or high (1.2 mm) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudins 1 and 4 even in low calcium. ZO-1 did not, however, follow similar expression patterns. Protein levels of occludin, beta-catenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/desmocollin 3 and plakoglobin did not show marked changes in SPCA1-deficient keratinocytes. Indirect immunofluorescence labelling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes and increased intracellular pools of TJ and desmosomal components in SPCA1-inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD.
ATP2C1 基因突变导致编码 Ca(2+) /Mn(2+) ATP 酶 SPCA1 的基因发生突变，从而引起 Hailey-Hailey 病（HHD，OMIM 16960）。HHD 的特征是表皮棘层松解。我们试图使用正常角质形成细胞来建立 HHD 模型，通过小干扰 RNA（siRNA）方法下调 SPCA1 mRNA。qPCR 显示 siRNA 抑制显著下调了 SPCA1 mRNA，Western 分析显示 SPCA1 蛋白水平下降到无法检测的程度。然后，在低（0.06mm）或高（1.2mm）钙浓度下培养 SPCA1 缺陷和对照角质形成细胞，研究 SPCA1 缺陷和对照角质形成细胞中选定的桥粒、黏附连接和紧密连接（TJ）蛋白的表达。在从低钙浓度切换至高钙浓度时，未经处理的对照角质形成细胞培养物中大多数 TJ 成分的 mRNA 和蛋白水平上调。相比之下，即使在低钙浓度下，SPCA1 缺陷角质形成细胞中 TJ 蛋白 Claudin 1 和 Claudin 4 的水平也很高。ZO-1 则没有遵循类似的表达模式。Occludin、β-catenin、E-cadherin、desmoplakin、desmoglein 1-3、desmocollin 2/desmocollin 3 和 plakoglobin 的蛋白水平在 SPCA1 缺陷角质形成细胞中没有明显变化。间接免疫荧光标记显示，在 SPCA1 抑制的角质形成细胞中，桥粒中的 desmoplakin 和 desmoglein 3 发生了延迟转位，TJ 和桥粒成分在细胞内的池增多。结果表明，SPCA1 调节 Claudin 1 和 Claudin 4 的水平，但不影响桥粒蛋白水平，表明 TJ 蛋白的调节方式不同。结果还表明 Claudin 在 HHD 中可能发挥作用。
