Lagace T A, Byers D M, Cook H W, Ridgway N D
Atlantic Research Centre, and the Departments of Pediatrics and Biochemistry, Dalhousie University, 5849 University Avenue, Halifax, Nova Scotia, Canada B3H 4H7.
J Lipid Res. 1999 Jan;40(1):109-16.
25-Hydroxycholesterol negatively regulates cholesterol synthesis and activates cholesterol esterification in a variety of cultured cells. Concurrent with these effects, 25-hydroxycholesterol also stimulates the synthesis of sphingomyelin in Chinese hamster ovary (CHO)-K1 cells. The role of oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol, in activation of SM synthesis was assessed by overexpression in CHO-K1 cells. When compared to mock transfected controls, three CHO-K1 clones overexpressing OSBP by 10- to 15-fold displayed a 2- to 3-fold enhancement of [3H]serine incorporation into sphingomyelin when treated with 25-hydroxycholesterol. Closer examination of one of these clones (CHO-OSBP cells) revealed a >8.5-fold stimulation of sphingomyelin synthesis after a 6-h treatment with 25-hydroxycholesterol compared to 3.5-fold in controls, slightly higher basal levels of sphingomyelin synthesis, and a more rapid response to 25-hydroxycholesterol. [3H]serine incorporation into phosphatidylserine, phosphatidylethanolamine, ceramide, or glucosylceramide was affected by <15%. Synthesis of sphingomyelin from exogenous [3H]sphinganine-labeled ceramide was enhanced in overexpressing cells treated with 25-hydroxycholesterol. However, in vitro activities of sphinganine N-acyltransferase, sphingomyelin synthase, and serine palmitoyltransferase were not affected by OSBP overexpression or 25-hydroxycholesterol. Overexpression of OSBP or 25-hydroxycholesterol did not significantly affect the ceramide content of Golgi-enriched fractions from control or overexpressing cells. However, diglyceride mass was reduced in Golgi-enriched fractions from overexpressing cells and by treatment with 25-hydroxycholesterol. Results from overexpressing cells show that OSBP potentiates the stimulatory effects of 25-hydroxycholesterol on sphingomyelin synthesis. 25-Hydroxycholesterol promotes translocation of OSBP to the Golgi apparatus where it appears to stimulate conversion of ceramide to sphingomyelin.
25-羟基胆固醇对胆固醇合成具有负调控作用，并能在多种培养细胞中激活胆固醇酯化。伴随着这些效应，25-羟基胆固醇还能刺激中国仓鼠卵巢（CHO）-K1细胞中鞘磷脂的合成。通过在CHO-K1细胞中过表达，评估了25-羟基胆固醇的高亲和力受体——氧化固醇结合蛋白（OSBP）在鞘磷脂合成激活中的作用。与mock转染对照相比，三个将OSBP过表达10至15倍的CHO-K1克隆在用25-羟基胆固醇处理时，[3H]丝氨酸掺入鞘磷脂的量增加了2至3倍。对其中一个克隆（CHO-OSBP细胞）进行更仔细的研究发现，与对照组的3.5倍相比，用25-羟基胆固醇处理6小时后，鞘磷脂合成受到了>8.5倍的刺激，鞘磷脂合成的基础水平略高，并且对25-羟基胆固醇的反应更快。[3H]丝氨酸掺入磷脂酰丝氨酸、磷脂酰乙醇胺、神经酰胺或葡糖神经酰胺的量受到的影响小于15%。在用25-羟基胆固醇处理的过表达细胞中，由外源性[3H]鞘氨醇标记的神经酰胺合成鞘磷脂的过程增强。然而，鞘氨醇N-酰基转移酶、鞘磷脂合酶和丝氨酸棕榈酰转移酶的体外活性不受OSBP过表达或25-羟基胆固醇的影响。OSBP的过表达或25-羟基胆固醇并未显著影响对照细胞或过表达细胞中富含高尔基体组分的神经酰胺含量。然而，过表达细胞中富含高尔基体的组分以及用25-羟基胆固醇处理后，甘油二酯的量减少。过表达细胞的结果表明，OSBP增强了25-羟基胆固醇对鞘磷脂合成的刺激作用。25-羟基胆固醇促进OSBP向高尔基体的转运，在高尔基体中它似乎刺激神经酰胺向鞘磷脂的转化。
