Intracellular cholesterol transport.

The intracellular movement of cholesterol in mammalian cells may involve complex pathways by which the sterol moves to various cellular sites and mediates transcriptional regulation, enzyme activation, and protein degradation. Current evidence indicates that there are three distinct pathways modulating intracellular cholesterol trafficking. The movement of endogenously synthesized cholesterol from the endoplasmic reticulum appears to be distinct from movement of exogenous, low density lipoprotein (LDL)-derived cholesterol to the plasma membrane. In addition, steroidogenic cells possess a third mechanism by which cholesterol is transported to the mitochondria to initiate steroid hormone synthesis. In this review, we have outlined the current knowledge of cholesterol transport mechanisms and pathways and have described approaches that may help define cholesterol trafficking mechanisms in molecular detail. The use of genetic and molecular biologic techniques can potentially reveal gene products that are involved in intracellular cholesterol transport and regulation as well as those that may secondarily affect this process.

A second complementation class of cholesterol transport mutants with a variant Niemann-Pick type C phenotype.

We previously isolated Chinese Hamster ovary cell mutants that were defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol (Dahl, N.K., K.L. Reed, M.A. Daunais, J.R. Faust, and L. Liscum. 1992 J. Biol. Chem. 267: 4889-4896). Several of the mutants exhibited the same biochemical phenotype as classical Niemann-Pick type C (NPC) fibroblasts. Complementation analysis between these mutants and other cholesterol transport mutants with a variant biochemical phenotype has defined two complementation classes. One class is characterized by expression of the classical NPC phenotype and may represent a true cholesterol transport mutant, while the second is characterized by expression of a variant NPC phenotype and may represent a signaling defect in LDL-sensitive homeostatic responses.

Abnormal regulation of low density lipoprotein-sensitive events in a cholesterol transport mutant.

We have isolated and characterized Chinese hamster ovary cell mutants defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol (Dahl, N. K., Reed, K. L., Daunais, M. A., Faust, J. R., and Liscum, L. (1992) J. Biol. Chem. 267, 4889-4896). Mutant 2-2, which exhibits a cholesterol transport defect indistinguishable from the Niemann-Pick C phenotype, shows impaired but not absent LDL-mediated suppression of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. In parental cells, LDL suppression of HMG-CoA reductase is modulated by two mechanisms, decreased gene transcription and accelerated protein turnover. Using the chimeric protein HMGal as a reporter protein for LDL-mediated turnover and Northern blot analysis to monitor HMG-CoA reductase mRNA levels, we have dissected the contributions of these two regulatory responses to LDL-mediated suppression of HMG-CoA reductase activity. Kinetic modeling using the kinlsq program showed the following. Mutant 2-2 exhibits normal LDL-mediated acceleration of HMGal degradation, coupled with relatively abnormal regulation of mRNA. This suggests that the LDL-cholesterol signaling pathway to the nucleus is defective relative to the signal that results in HMG-CoA reductase turnover. In addition, LDL-mediated acceleration of HMGal turnover occurs well before LDL stimulation of cholesterol esterification in mutant 2-2, whereas these events occur synchronously in the parental cell line. This suggests that more than one pathway or mechanism exists for LDL-cholesterol signaling to the endoplasmic reticulum.

Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.

We have isolated clones of an established cell line which express defects in intracellular cholesterol metabolism. Chinese hamster ovary cells were mutagenized, and clones unable to mobilize low density lipoprotein (LDL)-derived cholesterol to the plasma membrane were selected. Biochemical analysis of two mutant clones revealed a phenotype characteristic of the lysosomal storage disease, Niemann-Pick type C. The mutant cell lines were found to be defective in the regulatory responses elicited by LDL-derived cholesterol. LDL-mediated stimulation of cholesterol esterification was grossly defective, and LDL suppression of 3-hydroxy-3-methylglutaryl-CoA reductase was impaired. However, the mutants modulated these activities normally in response to 25-hydroxycholesterol or mevalonate. The LDL-specific defects were predicated by the inability of these mutants to mobilize LDL-derived cholesterol from lysosomes. Cell fractionation studies showed that LDL-derived, unesterified cholesterol accumulated in the lysosomes of mutant cells to significantly higher levels than normal, commensurate with defective movement of cholesterol to other cellular membranes. Characterization of cell lines defective in intracellular cholesterol transport will facilitate identification of the gene(s) required for intracellular cholesterol movement and regulation.