Regulation of phosphatidylcholine metabolism in Chinese hamster ovary cells by the sterol regulatory element-binding protein (SREBP)/SREBP cleavage-activating protein pathway

Abstract

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 [3H]choline incorporation into PtdCho and 10-15-fold increase in intracellular [3H]glycerophosphocholine. [3H]Phosphocholine levels in SRD 4 cells were reduced by over 10-fold, suggesting enhanced activity of CTP:phosphocholine cytidylyltransferase α (CCTα). CHO-SCAP and CHO-SCAP D443N cells displayed modest increases in [3H]choline incorporation into PtdCho (2-fold) and only a 2-fold reduction in [3H]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, CCTα protein, mRNA, and in vitro activity were reduced in SRD 4 cells and unchanged in SCAP transfected cells. Unlike control and SCAP transfected cells, CCTα 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 CCTα 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 CCTα by fatty acids or a fatty acid-derived signal.