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.
Maguire
Jean Ann
JA
Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Reagan
Jerry W
JW
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