Low-temperature effect on the sterol-dependent processing of SREBPs and transcription of related genes in HepG2 cells.
Lowering the growth temperature of HepG2 cells from 37 degrees C to 20 degrees 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 degrees C. In contrast, sterol regulatory element binding proteins (SREBPs)-1 and -2 exhibit a 2- to 3-fold increase in mRNA levels at 20 degrees 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 degrees 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.
Shechter
Ishaiahu
I
Department of Surgery, F Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA. ischechter@usuhs.mil
Dai
Peihua
P
Roseman
Mark A
MA
Gupta
Sita D
SD
Boyer
Bert B
BB
Guan
Guimin
G
D015152:Blotting, Northern; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000096:biosynthesis; D003080:Cold Temperature; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008055:Lipids / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000494:pharmacology*; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D008055:Lipids; D012333:RNA, Messenger; D011973:Receptors, LDL; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; D002784:Cholesterol; D013186:Farnesyl-Diphosphate Farnesyltransferase