Failure of cAMP agonists to activate rescued ΔF508 CFTR in CFBE41o- airway epithelial monolayers
Failure of cAMP agonists to activate rescued ΔF508 CFTR in CFBE41o- airway epithelial monolayers
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DOI：
10.1113/jphysiol.2005.096669
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发表时间：
2005-12-01
影响因子：
5.5
通讯作者：
Clancy, JP
中科院分区：
文献类型：
--
作者：
Bebok, Z;Collawn, JF;Clancy, JP
囊性纤维化跨膜传导调节剂（CFTR）是一种环amp调控的氯离子通道。CFTR基因突变导致囊性纤维化（CF）。最常见的突变Delta F508导致CFTR的内质网相关降解（ERAD）。Delta F508 CFTR被描述为一种温度敏感突变，在27℃下生长后可以获救。为了研究野生型和获救型Delta F508 CFTR在非极化和极化条件下在细胞表面的加工和功能，我们建立了表达Delta F508或野生型CFTR的稳定细胞系。cfbe410（-）是一种人气道上皮细胞系，在可渗透载体上培养时能够形成高阻力、极化单层，而HeLa细胞通常在非极化条件下生长。在27℃下培养48小时后，免疫沉淀、细胞表面生物素化、免疫荧光和功能分析证实，两种细胞系的细胞表面都存在Delta F508 CFTR。然而，野生型CFTR的刺激剂，如福斯科林、β(2)-肾上腺素能或A(2B)-腺苷受体激动剂，未能激活cfbe410（-）单层中拯救的Delta F508 CFTR。获救的Delta F508 CFTR可以用染料木黄酮刺激，而不依赖于cAMP信号激动剂的预处理。有趣的是，在HeLa细胞中获救的Delta F508 CFTR可以被福斯克林或染料木素有效地刺激以促进Cl-运输。这些结果表明，当在cfbe410（-）人气道上皮细胞中获救时，Delta F508 CFTR对已知调节野生型CFTR的信号通路反应较差。此外，两种细胞系中Delta F508 CFTR修复和激活的差异表明，Delta F508 CFTR加工的细胞类型特异性差异可能会使识别Delta F508缺陷的增强因子和/或校正因子的工作复杂化。
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, Delta F508, results in endoplasmic reticulum-associated degradation (ERAD) of CFTR. Delta F508 CFTR has been described as a temperature-sensitive mutation that can be rescued following growth at 27 degrees C. In order to study the processing and function of wild-type and rescued Delta F508 CFTR at the cell surface under non-polarized and polarized conditions, we developed stable cell lines expressing Delta F508 or wild-type CFTR. CFBE41o(-) is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non-polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of Delta F508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27 degrees C. However, stimulators of wild-type CFTR such as forskolin, beta(2)-adrenergic or A(2B)-adenosine receptor agonists failed to activate rescued Delta F508 CFTR in CFBE41o(-) monolayers. Rescued Delta F508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued Delta F508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl- transport. These results indicate that Delta F508 CFTR, when rescued in CFBE41o(-) human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild-type CFTR. Furthermore, the differences in rescue and activation of Delta F508 CFTR in the two cell lines suggest that cell-type specific differences in Delta F508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the Delta F508 defect.