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.
Dahl
N K
NK
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Gutheil
W G
WG
Liscum
L
L
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D004951:Esterification; D006888:Hydroxycholesterols / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008798:Mevalonic Acid / Q000494:pharmacology; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism
D006888:Hydroxycholesterols; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C007997:25-hydroxycholesterol; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid