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A 25-hydroxycholesterol-resistant cell line deficient in acyl-CoA: cholesterol acyltransferase
J E Metherall1, N D Ridgway, P A Dawson
1University of Texas Southwestern Medical Center, Department of Molecular Genetics, Dallas 75235.
Abstract:
We describe a line of mutant Chinese hamster ovary cells, SRD-4 cells, that lacks acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and fails to synthesize cholesteryl esters when stimulated with 25-hydroxycholesterol or low density lipoprotein. The cells also have a partial defect in their ability to repress transcription of three sterol-regulated genes, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and the low density lipoprotein receptor. The cells were selected by mutagenesis followed by growth in the presence of 25-hydroxycholesterol, which kills the parental cells by cholesterol depletion, owing to an inhibition of cholesterol synthesis and a stimulation of cholesterol esterification. Treatment of parental cells with compound 58-035 (3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1- phenylethyl]propanamide), an inhibitor of ACAT, abolished cholesterol esterification but did not reproduce the defect in gene repression seen in the SRD-4 cells, and it only partially reproduced the resistance to the killing effect of 25-hydroxycholesterol. We conclude that the SRD-4 cells most likely have two independent defects, one in ACAT and the other in a factor that mediates sterol-dependent transcriptional repression. The SRD-4 cells thus resemble a line of hamster cells previously isolated (Cadigan, K.M., Heider, J.G., and Chang, T.-Y. (1988) J. Biol. Chem. 263, 274-282), which has similar independent defects. The results raise the possibility that a partial resistance to sterol repression provides a growth advantage to cells that lack ACAT.
Insights
Newly identified SRD-4 cells exhibit a dual defect in acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and sterol-regulated gene repression, impacting cholesterol metabolism and cellular response to sterols.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Chinese hamster ovary (CHO) cells are crucial models for studying cellular lipid metabolism.
- Acyl-coenzyme A:cholesterol acyltransferase (ACAT) plays a key role in cholesterol esterification.
- Sterol regulatory element-binding proteins (SREBPs) control the transcription of genes involved in cholesterol homeostasis.
Purpose of the Study:
- To characterize a novel mutant cell line, SRD-4, with defects in cholesterol metabolism.
- To investigate the relationship between ACAT activity, cholesteryl ester synthesis, and sterol-regulated gene expression.
- To elucidate the mechanisms underlying cellular resistance to 25-hydroxycholesterol.
Main Methods:
- Mutagenesis of CHO cells followed by selection using 25-hydroxycholesterol.
- Assay of acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity.
- Measurement of cholesteryl ester synthesis.
- Analysis of the transcriptional repression of sterol-regulated genes (HMG-CoA synthase, HMG-CoA reductase, LDL receptor).
- Pharmacological inhibition of ACAT using compound 58-035.
Main Results:
- SRD-4 cells exhibit a complete loss of ACAT activity and fail to synthesize cholesteryl esters.
- SRD-4 cells display a partial defect in the repression of three key sterol-regulated genes.
- Pharmacological inhibition of ACAT in parental cells partially mimics the resistance to 25-hydroxycholesterol but does not replicate the gene repression defect.
- SRD-4 cells possess two independent defects: one in ACAT and another in sterol-dependent transcriptional repression.
Conclusions:
- The SRD-4 cell line presents a valuable model for dissecting the distinct roles of ACAT and sterol-mediated transcriptional regulation in cholesterol homeostasis.
- The findings suggest that a partial defect in sterol repression may confer a growth advantage to cells lacking ACAT activity.
- These results highlight the complex interplay between cholesterol esterification and the regulation of sterol-responsive genes.