Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures
Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures
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DOI：
10.1152/ajplung.00016.2009
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发表时间：
2010-03-01
影响因子：
4.9
通讯作者：
Gentzsch, Martina
中科院分区：
文献类型：
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作者：
Cholon, Deborah M.;O'Neal, Wanda K.;Gentzsch, Martina
Cholon DM，O'Neal WK，Randell SH，Riordan JR，Gentzsch M.原代人气道上皮细胞培养物中CFTR的内吞运输和顶端稳定性的调节。Am J Physiol Lung Cell Mol Physiol 298：L304-L314，2010.首次发表于2009年12月11日; doi：10.1152/ajplung.00016.2009.- CFTR是上皮细胞的高度调节的顶端氯离子通道，其在囊性纤维化（CF）中突变。在这项研究中，我们表征了野生型和突变型CFTR在其天然环境中的顶端稳定性和细胞内运输，即，高度分化的原代人气道上皮（HAE）培养物。我们标记了CFTR的顶端池，随后在细胞内隔室中观察蛋白质。CFTR从顶端表面移动到内体，然后有效地再循环回到表面。CFTR内吞作用在极化的HAE细胞或极化的上皮细胞系中比在非极化的HAE细胞中发生得更慢。CF中最常见的突变，Delta F508 CFTR，通过低温孵育从内质网滞留中获救，但从顶膜过渡到内吞隔室的速度更快，回收效率低于野生型CFTR。用小分子校正剂孵育导致在顶端膜处的Delta F508 CFTR，但没有恢复顶端稳定性。为了稳定突变蛋白在顶端膜，我们发现，发动蛋白抑制剂Dynasore和胆固醇提取剂环糊精显着降低内化的三角洲F508，而蛋白酶体抑制剂MG-132完全阻断内吞的三角洲F508。在检查CFTR的内在特性，可能会影响其顶端的稳定性，我们发现，N-连接的寡糖是不必要的运输到顶端的膜，但需要有效的顶端再循环，因此，影响营业额的表面CFTR。因此，CFTR在其天然环境中的顶端稳定性受到蛋白质性质和内吞运输调节的影响。
Cholon DM, O'Neal WK, Randell SH, Riordan JR, Gentzsch M. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures. Am J Physiol Lung Cell Mol Physiol 298: L304-L314, 2010. First published December 11, 2009; doi:10.1152/ajplung.00016.2009.-CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, Delta F508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wildtype CFTR. Incubation with small-molecule correctors resulted in Delta F508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of Delta F508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of Delta F508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking.