Silencing of the mutant SCAP allele accounts for restoration of a normal phenotype in CT60 cells selected for NPC1 expression.
The sterol regulatory element binding protein (SREBP)/SREBP cleavage-activating protein (SCAP) complex regulates the transcription of numerous genes involved in cellular cholesterol metabolism. The CHO mutant, CT60, and its parental cell line, 25RA, possess a gain-of-function mutation in one allele of the SCAP gene that renders the cells resistant to sterol-mediated suppression of cholesterol synthesis and uptake. In addition, CT60 cells do not express a functional Niemann-Pick type C1 (NPC1) protein, which leads to lysosomal accumulation of free cholesterol. Correction of the NPC1 defect by expression of a yeast artificial chromosome (YAC) containing the NPC1 genetic interval restored normal mobilization of cholesterol from the lysosomal compartment. Unexpectedly, the YAC-containing cell lines have overall cellular cholesterol concentrations that are comparable to wild-type levels, despite the assumed presence of the SCAP mutation. This phenotypic change results from a reduction in endogenous sterol synthesis, LDL receptor message, and HMG-CoA reductase message. Genetic analysis of the SCAP gene revealed that the YAC-expressing CT60 cells have normal regulation of these sentinel cholesterogenic genes as a result of selective silencing of the mutant SCAP allele, which appears to be independent of functional NPC1 expression.
Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Jean Ann
JA
Maguire
Jerry W
JW
Reagan
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002784:Cholesterol / Q000378:metabolism; D018244:Chromosomes, Artificial, Yeast; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry; D004951:Esterification; D020868:Gene Silencing; D006706:Homeostasis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D000077296:Niemann-Pick C1 Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; C102196:SREBP cleavage-activating protein; D002784:Cholesterol