Regulation of phosphatidylcholine metabolism in Chinese hamster ovary cells by the sterol regulatory element-binding protein (SREBP)/SREBP cleavage-activating protein pathway.
Sterol regulation-defective (SRD) 4 cells expressing a mutant sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP D443N) and Chinese hamster ovary (CHO) cells stably expressing SCAP (CHO-SCAP) and SCAP D443N (CHO-SCAP-D443N) have increased cholesterol and fatty acid synthesis because of constitutive processing of SREBPs. We assessed whether constitutive activation of SREBPs also influenced the CDP-choline pathway for phosphatidylcholine (PtdCho) biosynthesis. Relative to control CHO 7 cells, SRD 4 cells displayed increased PtdCho synthesis and degradation as indicated by a 4-6-fold increase in [(3)H]choline incorporation into PtdCho and 10-15-fold increase in intracellular [(3)H]glycerophosphocholine. [(3)H]Phosphocholine levels in SRD 4 cells were reduced by over 10-fold, suggesting enhanced activity of CTP:phosphocholine cytidylyltransferase alpha (CCTalpha). CHO-SCAP and CHO-SCAP D443N cells displayed modest increases in [(3)H]choline incorporation into PtdCho (2-fold) and only a 2-fold reduction in [(3)H]phosphocholine. Elevated PtdCho metabolism in SRD 4, compared with SCAP-overexpressing cells, was correlated with fatty acid synthesis. Inhibition of fatty acid synthesis by cerulenin resulted in almost complete normalization of PtdCho synthesis and choline metabolite profiles in SRD 4 cells, indicating that fatty acids or a fatty acid-derived metabolite was responsible for up-regulation of PtdCho synthesis. In contrast to apparent activation in vivo, CCTalpha protein, mRNA, and in vitro activity were reduced in SRD 4 cells and unchanged in SCAP transfected cells. Unlike control and SCAP transfected cells, CCTalpha in SRD 4 cells was localized by immunofluorescence to the nuclear envelope, suggesting that residual enzyme activity in these cells was in an active membrane-associated form. Translocation of CCTalpha to the nuclear envelope was reproduced by treatment of CHO 7 cells with exogenous oleate. We conclude that the SREBP/SCAP pathway regulates PtdCho synthesis via post-transcriptional activation of nuclear CCTalpha by fatty acids or a fatty acid-derived signal.
Lagace
T A
TA
Atlantic Research Center and the Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
Storey
M K
MK
Ridgway
N D
ND
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000378:metabolism*; D002798:Diacylglycerol Cholinephosphotransferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010713:Phosphatidylcholines; D012333:RNA, Messenger; C497387:SREBF1 protein, human; C102196:SREBP cleavage-activating protein; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D019916:Choline-Phosphate Cytidylyltransferase; D002798:Diacylglycerol Cholinephosphotransferase