Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on <it>de novo </it>cholesterol synthesis

• Main Authors: Sonawane Nitin D, Previs Stephen F, Jiang Dechen, Ruddy Jennifer, Manson Mary E, West Richard H, Fang Danjun, Burgess James D, Kelley Thomas J
• Format: Article
• Language: English
• Published: BMC 2010-05-01
• Series: Respiratory Research
• Online Access: http://respiratory-research.com/content/11/1/61

<p>Abstract</p> <p>Background</p> <p>Previous observations demonstrate that <it>Cftr</it>-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased <it>de novo </it>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 <it>de novo </it>cholesterol synthesis.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>Membrane cholesterol measurements are elevated in both R117H and ΔF508 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 CFTR_inh-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. <it>De novo </it>cholesterol synthesis contributes to membrane cholesterol accessibility.</p> <p>Conclusions</p> <p>The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in <it>de novo </it>cholesterol synthesis to restore membrane content.</p>