Role for sterol regulatory element binding protein in the regulation of farnesyl diphosphate synthase and in the control of cellular levels of cholesterol and triglyceride: evidence from sterol regulation-defective cells.

S M Jackson,J Ericsson,J E Metherall,P A Edwards

doi:10.1016/s0022-2275(20)39114-8

S M Jackson, J Ericsson + Show 2 more

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https://doi.org/10.1016/s0022-2275(20)39114-8

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Abstract

In order to define the factors involved in the regulation of farnesyl diphosphate (FPP) synthase, we used sterol regulation-defective (SRD) cell lines that constitutively express either high (SRD-2) or low (SRD-6) levels of transcriptionally active sterol regulatory element binding protein (SREBP). FPP synthase mRNA levels were high in SRD-2 cells and low in SRD-6 cells and were unaffected by the addition or removal of sterols from the media. In contrast, the mRNA levels in parental CHO-7 cells were regulated by sterols. SRD-2, SRD-6, and CHO-7 cells were also transiently transfected with plasmids containing FPP synthase promoter-reporter genes. Reporter gene activity was significantly higher in SRD-2 cells than in either SRD-6 or CHO-7 cells, consistent with a higher rate of transcription of the reporter gene in SRD-2 cells. The high expression of the reporter gene in SRD-2 cells was not observed when the FPP synthase promoter contained a three base pair mutation within an SREBP binding site, termed sterol regulatory element-3 (SRE-3). These observations are consistent with the hypothesis that high levels of transcription of the FPP synthase gene are dependent on the availability of transcriptionally active SREBP. We also demonstrate that the incorporation of radioactive acetate into both cholesterol and fatty acids was enhanced in SRD-2 cells as compared to CHO-7 or SRD-6 cells. Finally, we demonstrate that the concentrations of cholesterol, cholesteryl ester, and triglyceride were all significantly elevated in SRD-2 cells. We conclude that SREBP is involved not only in the regulation of FPP synthase and cholesterogenesis but also in fatty acid and triglyceride synthesis.

Highlights

In order to define the factors involved in the regulation of farnesyl diphosphate (FPP) synthase, we used sterol regulation-defective (SRD) cell lines that constitutively express either high (SRD-2) or low (SRDB) levels of transcriptionally active sterol regulatory element binding protein (SREBP).FPP synthase mRNA levels were high in SRD-2 cells and low in SRD-6 cells and were unaffected by the addition or removal of sterols from the media
FPP synthase mRNA levels in parental Chinese hamster ovary (CHO)-7 cells were regulated 2- to 3-fold in response to the sterol content of the incubation media; the levels were high when the cells were incubated for 24 h in media supplemented with 10% lipoprotein-deficient serum (LPDS) and low when the cells were incubated in LPDS supplemented with cholesterol and 25-hydroxycholesterol(Fig. lA, lanes 1-3 vs. 4-6)
FPP synthase mRNA levels in SRD-2cells were unaffected by the sterol status of the media (Fig. lA, lanes 7-12); the levels were 3- and 10-fold higher than the corresponding levels observed in CHO-7 cells incubated under identical conditions (Fig. lA, lanes 7-12 vs. 1-6)