Amplification of the gene for SCAP, coupled with Insig-1 deficiency, confers sterol resistance in mutant Chinese hamster ovary cells

Peter C W Lee, Pingsheng Liu, Wei Ping Li, Russell A. DeBose-Boyd

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

The endoplasmic reticulum membrane proteins Insig-1 and Insig-2 limit cholesterol synthesis, in part through their sterol-dependent binding to sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). This binding prevents proteolytic processing of SREBPs, membrane-bound transcription factors that enhance cholesterol synthesis.We report here the characterization of mutant Chinese hamster ovary (CHO) cells, designated SRD-19, that are resistant to 25-hydroxycholesterol, a potent inhibitor of SREBP processing. SRD-19 cells were produced by mutagenesis of Insig-1-deficient SRD-14 cells, followed by selection in high levels of 25-hydroxycholesterol. 25-Hydroxycholesterol fails to suppress SREBP processing in SRD-19, even though they express normal levels of Insig-2. The number of copies of the gene encoding SCAP was found to be increased by 4-fold in SRD-19 cells compared with wild-type CHO cells, leading to the overproduction of SCAP mRNA and protein. Our data indicate that overproduced SCAP saturates the remaining Insig-2 in SRD-19 cells, thus explaining their resistance to 25-hydroxycholesterol. Consistent with this conclusion, regulated SREBP processing is restored in SRD-19 cells upon transfection of plasmids encoding either Insig-1 or Insig-2. These results highlight the importance of SCAP/Insig ratios in normal sterol-regulated processing of SREBPs in cultured cells.

Original languageEnglish (US)
Pages (from-to)1944-1954
Number of pages11
JournalJournal of lipid research
Volume48
Issue number9
DOIs
StatePublished - Sep 2007

Keywords

  • Cholesterol homeostasis
  • Endoplasmic reticulum-Golgi translocation
  • Mutagenesis
  • Somatic cell genetics
  • Sterol-regulatory element binding protein cleavage-activating protein
  • Sterol-sensing domain
  • Ubiquitination

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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