Baricault L, Garcia M, Cibert C, Sapin C, Geraud G, Codogno P, Trugnan G
Unité de Recherches sur la Biologie et la Physiopathologie des Cellules Digestives, INSERM U239, Paris, France.
Exp Cell Res. 1993 Dec;209(2):277-87. doi: 10.1006/excr.1993.1312.
In a previous work, we showed that the differentiation-dependent expression of dipeptidyl peptidase IV (DPP IV) in Caco-2 cells, a human colon adenocarcinoma cell line, was controlled at the mRNA level (D. Darmoul et al. J. Biol. Chem., 1992, 267, 4824-4833). Whether post-translational events may contribute to the final control of DPP IV cell surface expression was explored here by studying the potential effect of forskolin (FK), a drug known to permanently stimulates adenylyl cyclase and to strongly perturbs glucose metabolism in fully differentiated Caco-2 cells. FK treatment reduces by about 50% the amount of active DPP IV present at the brush border membrane, whereas the total amount of active DPP IV remains unchanged. Biosynthesis and maturation of DPP IV were measured using [35S]methionine labeling and were shown to be essentially unaffected by FK treatment. Pulse-chase experiments demonstrate that up to 50% of the neosynthesized DPP IV do not appear at the apical membrane after FK treatment. To get further insight into this phenomenon, we have used confocal laser scanning microscopy. We demonstrate that the blockade of DPP IV transport is associated with the accumulation of this protein in intracellular vesicles. Double-staining experiments demonstrate that these vesicles are not labeled with a monoclonal antibody directed against the Golgi apparatus but display a strong staining with a polyclonal antibody raised against lamp-1, a lysosomal membrane protein. Using a newly developed image analysis procedure, we have been able to quantitate the relative distribution of lamp-1 and DPP IV labels in both control and forskolin-treated cells. We show that the overlap of the two labels dramatically increases in FK-treated Caco-2 cells. These results suggest that, beside the transcriptional level, post-translational events may be involved in the final control of the apical expression of a differentiation-dependent hydrolase.
在之前的一项研究中，我们发现二肽基肽酶IV（DPP IV）在人结肠腺癌细胞系Caco-2细胞中的分化依赖性表达在mRNA水平受到调控（D. 达穆尔等人，《生物化学杂志》，1992年，第267卷，4824 - 4833页）。本文通过研究福斯可林（FK）的潜在作用，探讨了翻译后事件是否可能对DPP IV细胞表面表达的最终调控产生影响。福斯可林是一种已知能永久刺激腺苷酸环化酶并强烈干扰完全分化的Caco-2细胞中葡萄糖代谢的药物。FK处理使刷状缘膜上活性DPP IV的量减少了约50%，而活性DPP IV的总量保持不变。使用[35S]甲硫氨酸标记法测定了DPP IV的生物合成和成熟过程，结果表明其基本不受FK处理的影响。脉冲追踪实验表明，FK处理后，多达50%新合成的DPP IV未出现在顶端膜上。为了进一步深入了解这一现象，我们使用了共聚焦激光扫描显微镜。我们证明DPP IV转运的阻断与该蛋白在细胞内囊泡中的积累有关。双重染色实验表明，这些囊泡未被针对高尔基体的单克隆抗体标记，但用针对溶酶体膜蛋白lamp-1的多克隆抗体进行染色时显示出强烈的染色。使用新开发的图像分析程序，我们能够定量lamp-1和DPP IV标记在对照细胞和福斯可林处理细胞中的相对分布。我们表明，在FK处理的Caco-2细胞中，两种标记的重叠显著增加。这些结果表明，除了转录水平外，翻译后事件可能参与了一种分化依赖性水解酶顶端表达的最终调控。
