Storey M K, Byers D M, Cook H W, Ridgway N D
Department of Pediatrics, Atlantic Research Centre, Dalhousie University, 5849 University Avenue, Halifax, Nova Scotia, Canada, B3H 4H7.
Biochem J. 1998 Nov 15;336 ( Pt 1)(Pt 1):247-56. doi: 10.1042/bj3360247.
Sphingomyelin (SM) and cholesterol content is positively correlated in cellular membranes, and in several pathological and experimental conditions there is evidence for coregulation. The potential role of oxysterols and oxysterol binding protein (OSBP) in mediating the coregulation of cholesterol and SM was examined using Chinese hamster ovary (CHO) and cholesterol auxotrophic, sterol regulatory defective (SRD) 6 cells. SRD 6 cells grown in the presence or absence of cholesterol for 24 h displayed a 30-50% reduction in SM synthesis compared with control CHO 7 cells. SM synthesis in CHO 7 and cholesterol-supplemented SRD 6 cells was stimulated 2-fold by 25-hydroxycholesterol, but cholesterol-starved SRD 6 cells were unresponsive. Basal and 25-hydroxycholesterol-stimulated SM synthesis was also inhibited in lovastatin-treated wild-type CHO-K1 cells. Lack of 25-hydroxycholesterol activation of SM synthesis in cholesterol-starved SRD 6 and lovastatin-treated CHO-K1 cells was correlated with dephosphorylation of OSBP. In SRD 6 cells, this was evident after 12 h of cholesterol depletion, it occurred equally at all phosphorylation sites and was exacerbated by 25-hydroxycholesterol. Unlike CHO 7 cells, where OSBP was observed in small vesicles and the cytoplasm, OSBP in cholesterol-starved SRD 6 cells was constitutively localized in the Golgi apparatus. Supplementation with non-lipoprotein cholesterol promoted redistribution to vesicles and the cytoplasm. Similarly, OSBP in CHO-K1 cells grown in delipidated serum was predominantly in the Golgi apparatus. Low-density lipoprotein (LDL) supplementation of CHO-K1 cells caused the redistribution of OSBP to the cytoplasm and small vesicles, and this effect was blocked by pharmacological agents ¿3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one and progesterone¿, which inhibited LDL cholesterol efflux from lysosomes. The results showed that localization of OSBP between the Golgi apparatus and a cytoplasmic/vesicular compartment was responsive to changes in cholesterol content and trafficking. In cholesterol depleted SRD 6 cells, this was accompanied by dephosphorylation of OSBP and attenuation of 25-hydroxycholesterol activation of SM synthesis.
鞘磷脂（SM）与胆固醇在细胞膜中的含量呈正相关，并且在一些病理和实验条件下有共同调节的证据。利用中国仓鼠卵巢（CHO）细胞以及胆固醇营养缺陷型、甾醇调节缺陷（SRD）6细胞，研究了氧化甾醇和氧化甾醇结合蛋白（OSBP）在介导胆固醇和SM共同调节中的潜在作用。与对照CHO 7细胞相比，在有或无胆固醇存在的情况下培养24小时的SRD 6细胞，其SM合成减少了30 - 50%。25 - 羟基胆固醇使CHO 7细胞和补充了胆固醇的SRD 6细胞中的SM合成增加了2倍，但胆固醇饥饿的SRD 6细胞无反应。在洛伐他汀处理的野生型CHO - K1细胞中，基础和25 - 羟基胆固醇刺激的SM合成也受到抑制。胆固醇饥饿的SRD 6细胞和洛伐他汀处理的CHO - K1细胞中25 - 羟基胆固醇对SM合成缺乏激活作用，这与OSBP的去磷酸化相关。在SRD 6细胞中，胆固醇耗竭12小时后这种现象明显，所有磷酸化位点均同等发生，并且25 - 羟基胆固醇会使其加剧。与在小泡和细胞质中观察到OSBP的CHO 7细胞不同，胆固醇饥饿的SRD 6细胞中的OSBP组成性地定位于高尔基体。补充非脂蛋白胆固醇会促使其重新分布到小泡和细胞质中。同样，在无脂血清中生长的CHO - K1细胞中的OSBP主要位于高尔基体。向CHO - K1细胞补充低密度脂蛋白（LDL）会导致OSBP重新分布到细胞质和小泡中，并且这种作用被药物3 - β - [2 - （二乙氨基）乙氧基]雄甾 - 5 - 烯 - 17 - 酮和孕酮阻断，这两种药物抑制了LDL胆固醇从溶酶体的流出。结果表明，OSBP在高尔基体与细胞质/小泡区室之间的定位对胆固醇含量和运输的变化有反应。在胆固醇耗竭的SRD 6细胞中，这伴随着OSBP的去磷酸化以及25 - 羟基胆固醇对SM合成激活作用的减弱。
