Coordinate regulation of phosphatidylcholine, sphingomyelin, cholesterol and fatty acid metabolism in sterol regulatory defective Chinese hamster ovary cells

We investigated the mechanism for covariance of phosphatidylcholine (PtdCho), sphingomyelin (SM), and cholesterol observed in various pathological and experimental conditions using three sterol regulatory defective (SRD) Chinese hamster ovary (CHO) cell lines. SRD 6 cells are cholesterol auxotrophs, SRD 2 possess constitutive cholesterol synthesis and SRD 4 cells display increased cholesterol synthesis and lack cholesterol esterification. In SRD 6 cells, [3H]choline incorporation into PtdCho was decreased 50% due to of inhibition of phosphocholine conversion to CDP-choline (catalyzed by CTP:phosphocholine cytidylyltransferase, CT). In SRD 6 cells, CT mass was increased in the cytosol fraction, but membrane-bound CT activity and content were similar to controls. SRD 6 cells exhibited decreased synthesis of fatty acid, a known activator of CT. Accordingly, PtdCho synthesis in SRD 6 cells was normalized by treatment with oleate. In SRD 2 and SRD 4 cells, PtdCho synthesis was similar to CHO 7 values, but [3H]glycerophosphocholine levels were increased indicating stimulated PtdCho catabolism. Results suggest that SREBP activation and resultant effects on fatty acid biosynthesis were the primary factors regulating PtdCho synthesis in the SRD 6 cells. In addition to aberrant PtdCho metabolism, basal and 25-OH cholesterol-stimulated SM synthesis were affected. In SRD 6 and wild-type CHO-K1 cells treated with cholesterol synthesis and transport inhibitors, basal SM synthesis was decreased. 25-OH cholesterol-stimulation of SM synthesis was inhibited in cholesterol-depleted SRD 6 cells, but cholesterol supplementation restored this stimulation. Regulation of SM by oxysterols and cholesterol may occur through the regulation of the oxysterol binding protein (OSBP). Conditions which inhibit oxysterol stimulation of SM also caused dephosphorylation of OSBP. In the case of cholesterol-depleted SRD 6 cells, this was accompanied by constitutive Golgi localization. Results from these studies demonstrated that cholesterol levels influenced the phosphorylation and intracellular localization of OSBP and affected its ability to alter SM synthesis in response to 25-OH cholesterol.