Amplification of 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, and Russell A DeBose-Boyd

Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046

Corresponding Author: Russell.DeBose-Boyd{at}utsouthwestern.edu

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 4-fold in SRD-19 cells as compared to 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.

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