Department of Pediatrics and Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
Respir Res. 2010 May 20;11(1):61. doi: 10.1186/1465-9921-11-61.
Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.
Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.
Membrane cholesterol measurements are elevated in both R117H and DeltaF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.
The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.
先前的观察结果表明，与野生型对照相比，Cftr 缺失细胞和组织的胆固醇处理发生改变，包括核周胆固醇积累、从头合成增加以及质膜胆固醇可及性增加。本研究的假设是，膜胆固醇可及性与 CFTR 基因型相关，部分受从头胆固醇合成的影响。
使用带有改良铂线圈的毛细管微电极电化学检测质膜上的胆固醇，该改良铂线圈可接受胆固醇氧化酶的共价附着。缺乏胆固醇氧化酶的改良电极用作基线对照。胆固醇合成通过随时间推移将氘掺入脂质来确定。通过质谱分析专门确定掺入胆固醇。本研究中使用的所有小鼠均为 C57Bl/6 背景，年龄在 6 至 8 周之间。
与年龄匹配的 wt 对照相比，R117H 和 DeltaF508 小鼠鼻上皮中的膜胆固醇测量值均升高，表明膜胆固醇检测与基因型相关。wt CFTR 在 CF 上皮细胞中的表达将膜胆固醇恢复至 WT 水平，进一步证明 CFTR 对这些过程的影响。在 wt 上皮细胞中，添加 CFTR 抑制剂 Gly H101 或 CFTRinh-172 24 小时后，膜胆固醇测量值最初下降，然后在 72 小时后反弹，这表明反馈机制可能是导致膜胆固醇增加的原因。从头胆固醇合成有助于膜胆固醇可及性。
本研究中的数据表明，CFTR 影响胆固醇向质膜的转运，当胆固醇耗尽时，会导致从头胆固醇合成增加以恢复膜含量。
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