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

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.