Nord M, Låg M, Cassel T N, Randmark M, Becher R, Barnes H J, Schwarze P E, Gustafsson J A, Lund J
Department of Medical Nutrition, Novum Karolinska Institute, Huddinge, Sweden.
DNA Cell Biol. 1998 May;17(5):481-92. doi: 10.1089/dna.1998.17.481.
The Clara-cell secretory protein (CCSP) is a cell-specific differentiation marker for the bronchiolar Clara cell. Isolated rat Clara and alveolar type 2 cells kept in primary culture proliferate and dedifferentiate, providing the opportunity to study differentiation-dependent mechanisms. In freshly isolated Clara cells, high levels of CCSP and the corresponding mRNA were detected. During culture in vitro, these levels decreased. In the type 2 cell fraction, low levels of CCSP were detected, which decreased further during culture. A promoter fragment of the rat CCSP gene encompassing the sequence from -188 to +53 was able to drive high-level expression of reporter genes in transfected Clara cells. Reporter gene expression in transfected type 2 cells was markedly lower, and no expression could be detected in alveolar macrophages. Expression of transcription factors previously described to stimulate CCSP expression appeared not to parallel CCSP levels in the primary Clara cells. However, expression of the transcription factor C/EBP alpha correlated with the CCSP expression pattern. In electrophoretic mobility shift assays, we were able to demonstrate binding of C/EBP alpha from rat Clara cell nuclear extracts to an element located 85 bp upstream of the start site of transcription. Overexpression of C/EBP alpha increased expression from the CCSP -188 promoter fragment up to fivefold in NCI-H441-cells and 30-fold in A549-cells, establishing the functional importance of C/EBP alpha. Our results show that primary cultures of Clara cells constitute a useful model for investigating terminal airway differentiation and suggest a role for C/EBP-factor(s) in this process.
克拉拉细胞分泌蛋白（CCSP）是细支气管克拉拉细胞的一种细胞特异性分化标志物。原代培养的分离大鼠克拉拉细胞和Ⅱ型肺泡细胞会增殖并去分化，这为研究分化依赖机制提供了机会。在新鲜分离的克拉拉细胞中，检测到高水平的CCSP及相应的mRNA。在体外培养过程中，这些水平下降。在Ⅱ型细胞组分中，检测到低水平的CCSP，其在培养过程中进一步下降。大鼠CCSP基因的一个启动子片段，包含从-188到+53的序列，能够在转染的克拉拉细胞中驱动报告基因的高水平表达。在转染的Ⅱ型细胞中报告基因的表达明显较低，在肺泡巨噬细胞中未检测到表达。先前描述的刺激CCSP表达的转录因子的表达似乎与原代克拉拉细胞中的CCSP水平不平行。然而，转录因子C/EBPα的表达与CCSP表达模式相关。在电泳迁移率变动分析中，我们能够证明大鼠克拉拉细胞核提取物中的C/EBPα与转录起始位点上游85 bp处的一个元件结合。在NCI-H441细胞中，C/EBPα的过表达使CCSP -188启动子片段的表达增加了5倍，在A549细胞中增加了30倍，确立了C/EBPα的功能重要性。我们的结果表明，克拉拉细胞的原代培养构成了研究终末气道分化的有用模型，并提示C/EBP因子在此过程中发挥作用。
