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Journal of Lipid Research, Vol. 44, 1581-1590, August 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology

Low-temperature effect on the sterol-dependent processing of SREBPs and transcription of related genes in HepG2 cells

Ishaiahu Shechter^1,*, Peihua Dai^*, Mark A. Roseman^*, Sita D. Gupta^*, Bert B. Boyer and Guimin Guan^*

^* Department of Surgery, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799
Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775

¹ To whom correspondence should be addressed. e-mail: ishechter{at}usuhs.mil

Lowering the growth temperature of HepG2 cells from 37°C to 20°C results in a 73% reduction in human squalene synthase (HSS) protein, a 76% reduction in HSS mRNA, and a 96% reduction in promoter activity of a secreted alkaline phosphatase-HSS reporter gene. A similar decrease in either mRNA or protein levels is observed for 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl diphosphate synthase, the LDL receptor, and fatty acid synthase. All these proteins and mRNAs show either a decrease or a complete loss of sterol-dependent regulation in cells grown at 20°C. In contrast, sterol regulatory element binding proteins (SREBPs)-1 and -2 exhibit a 2- to 3-fold increase in mRNA levels at 20°C. The membrane-bound form of the SREBPs is dramatically increased, but the proteolytic processing to the nuclear (N-SREBP) form is inhibited under these conditions. Overexpression of the N-SREBP or SREBP cleavage-activating protein (SCAP), but not site-1 or site-2 proteases, restores the activation of the HSS promoter at 20°C, most likely by liberating the SCAP-SREBP complex so that it can move to the Golgi for processing.

These results indicate that the cholesterol synthesizing machinery is down-regulated at low temperatures, and points to the transport of the SCAP-SREBP complex to the Golgi as the specific down-regulated step.

Abbreviations: CMV, cytomegalovirus; FAS, fatty acid synthase; FPPS, farnesyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; HSS, human squalene synthase; LDLR, low density lipoprotein receptor; pCMV-ßGAL, pCMV-ß-galactosidase; S1P, site-1 protease; S2P, site-2 protease; SCAP, SREBP cleavage-activating protein; SQS, squalene synthase; SRE, sterol regulatory element; SREBP, sterol regulatory element binding protein

Supplementary key words sterol regulatory element binding protein • transcriptional regulation • cholesterol metabolism

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