Leppimäki P, Kronqvist R, Slotte J P
Department of Biochemistry and Pharmacy, Abo Akademi University, P.O. Box 66, FIN-20521 Turku, Finland.
Biochem J. 1998 Oct 15;335 ( Pt 2)(Pt 2):285-91. doi: 10.1042/bj3350285.
Plasma membrane sphingomyelin (SM) is known to affect the cellular distribution of cholesterol. The aim of this work was to examine how SM homoeostasis in human skin fibroblasts is affected by alterations in the level of cholesterol in the cell. The cellular cholesterol level was decreased by exposing cells to 2-hydroxypropyl-beta-cyclodextrin, and increased by exposing cells to cholesterol-methyl-beta-cyclodextrin inclusion complexes. A lowering of the cellular unesterified cholesterol content by 20% was shown to increase the incorporation of [14C]palmitic acid into SM by 70%. Subsequently, the cellular SM mass was shown to be increased (24% increase after a 24 h period). Since l-cycloserine completely abolished the increased incorporation of [14C]palmitic acid into SM in cholesterol-depleted cells, we concluded that the de novo synthesis of the sphingosine backbone of SM was activated in cholesterol-depleted cells. This conclusion was further verified by performing a cell-free assay of serine C-palmitoyltransferase (SPT) in cholesterol-depleted cells, which showed that the activity of the enzyme was increased by 30% after cholesterol depletion. Most of the newly synthesized SM in cholesterol-depleted cells was susceptible to degradation by sphingomyelinase, indicating that it was transported efficiently to the cell surface. Loading of fibroblasts with cholesterol had essentially the opposite effects on SM homoeostasis to those of cholesterol depletion, i.e. 20-30% decreased incorporation of [14C]palmitic acid into SM and decreased activity of SPT. The results of this study show that cellular cholesterol levels have marked effects on the homoeostasis of SM.
已知质膜鞘磷脂（SM）会影响胆固醇的细胞分布。本研究的目的是探究细胞内胆固醇水平的改变如何影响人皮肤成纤维细胞中SM的稳态。通过将细胞暴露于2-羟丙基-β-环糊精来降低细胞胆固醇水平，通过将细胞暴露于胆固醇-甲基-β-环糊精包合物来提高细胞胆固醇水平。结果显示，细胞内未酯化胆固醇含量降低20%会使[14C]棕榈酸掺入SM的量增加70%。随后，细胞SM含量增加（24小时后增加24%）。由于L-环丝氨酸完全消除了胆固醇缺乏细胞中[14C]棕榈酸掺入SM的增加，我们得出结论，胆固醇缺乏细胞中SM鞘氨醇骨架的从头合成被激活。通过对胆固醇缺乏细胞中的丝氨酸C-棕榈酰转移酶（SPT）进行无细胞测定进一步验证了这一结论，结果显示胆固醇缺乏后该酶的活性增加了30%。胆固醇缺乏细胞中大多数新合成的SM易被鞘磷脂酶降解，这表明它被有效地转运到了细胞表面。用胆固醇加载成纤维细胞对SM稳态的影响与胆固醇缺乏基本相反，即[14C]棕榈酸掺入SM的量减少20 - 30%，且SPT活性降低。本研究结果表明，细胞胆固醇水平对SM的稳态有显著影响。
