Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Fang
Danjun
D
Department of Pediatrics and Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
West
Richard H
RH
Manson
Mary E
ME
Ruddy
Jennifer
J
Jiang
Dechen
D
Previs
Stephen F
SF
Sonawane
Nitin D
ND
Burgess
James D
JD
Kelley
Thomas J
TJ
D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000096:biosynthesis*; D002789:Cholesterol Oxidase / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D055664:Electrochemical Techniques / Q000295:instrumentation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D018181:Mice, Inbred CFTR; D008839:Microelectrodes; D009154:Mutation; D009297:Nasal Mucosa / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D020545:Respiratory Mucosa / Q000378:metabolism; D000077185:Resveratrol; D013267:Stilbenes / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology; D014162:Transfection
C482900:3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone; D001565:Benzoates; C505032:CFTR protein, human; D013267:Stilbenes; D053778:Thiazolidines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002784:Cholesterol; D002789:Cholesterol Oxidase; D006904:Hydroxymethylglutaryl-CoA Synthase; D000077185:Resveratrol