Inherent flaw of cholesterol processing in cell culture and in vivo models of cystic fibrosis

Cystic fibrosis (CF) is caused by a loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leading to lung infection and inflammation. Mechanisms that cause severe inflammatory responses in CF are not well established. It is hypothesized that CF systems possess a flaw in intracellular cholesterol transport initiating increased isoprenoid/cholesterol synthesis and leading to pro-inflammatory events. To verify the role of isoprenoid/cholesterol synthesis in cell signaling regulation a model system was explored. Niemann-Pick type C (NPC) disease is a lysosomal cholesterol storage disease due to improper intracellular cholesterol transport and increased isoprenoid/cholesterol synthesis. Evidence of signaling similarities with CF models make NPC a functionally relevant model. Additionally, models of CF exhibit increased unesterified cholesterol content as determined in NPC. CF epithelium of human airways further illustrates an accumulation of cholesterol compared to controls. Altered cholesterol homeostasis is indicated by an increase in membrane cholesterol content in CF cell culture and excised nasal epithelium. GC/MS analysis of deuterium incorporation of cholesterol in mouse tissue revealed that Cftr-/- mouse lung have an increase of newly made cholesterol compared to matched controls. Confirmation of these results was obtained utilizing microPET technology to image live mice with 11C-acetate labeling indicating greater acetate incorporation in CF mice, specifically the lung, compared to matched controls. These data establish an accumulation of intracellular cholesterol and an increase of isoprenoid/cholesterol synthesis.; Evidence of CFTR mediation of cholesterol processing was determined by examining different mouse models of CF disease severity. Comparison of CFTR mutations determined different phenotypic outcomes as measured by cholesterol parameters. A severe and mild mutation of CFTR, CftrDeltaF508/DeltaF508, (DeltaF/DeltaF) and Cftr R117H/R117H (R/R) respectively, were compared for rates of de novo cholesterol synthesis. DeltaF/DeltaF mice exhibited nearly two fold increases in newly made synthesis compared to R/R mouse lung. Greater acetate incorporation and excised nasal epithelium membrane cholesterol were exhibited in DeltaF/DeltaF mice compared to R/R mice. All parameters measured in CF were significantly above levels in matched littermates. These data demonstrate by multiple approaches an inherent lesion in cholesterol homeostasis in CF that correlates with CFTR genotype severity.