Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification
Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification
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UBIAD1在细胞内胆固醇代谢和血管细胞钙化中的作用
DOI：
10.1371/journal.pone.0149639
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发表时间：
2016-02-18
期刊：
影响因子：
3.7
通讯作者：
Liu, Wenhu
中科院分区：
文献类型：
--
作者：
Liu, Sha;Guo, Wang;Liu, Wenhu
血管钙化是慢性肾脏疾病患者死亡的重要危险因素。细胞内胆固醇代谢参与血管细胞钙化过程。在本研究中，我们研究了UbiA戊烯基转移酶结构域1 （UBIAD1）在细胞内胆固醇代谢和血管细胞钙化中的作用，并确定了其亚细胞位置。将原代人脐静脉平滑肌细胞（HUVSMCs）分别用生长培养基（1.4 mmol/L Pi）和钙化培养基（3.0 mmol/L Pi）培养。在CM处理下，HUVSMCs进一步与外源性胆固醇或甲基萘醌-4 （UBIAD1的产物）孵育。在HUVSMCs中转染质粒和小干扰RNA，改变UBIAD1的表达。测定基质钙含量、碱性磷酸酶活性、细胞内胆固醇水平和甲基萘醌-4水平。分析了几个参与胆固醇代谢的基因的表达。利用抗UBIAD1抗体、内质网标记物和高尔基体标记物，分析了UBIAD1在HUVSMCs中的亚细胞定位。CM增加基质钙、碱性磷酸酶活性和细胞内胆固醇水平，降低UBIAD1表达和甲基萘醌-4水平。添加胆固醇有助于增加基质钙化和碱性磷酸酶活性，并呈剂量依赖性。在CM处理的HUVSMCs中，升高UBIAD1或甲基萘醌-4的表达可显著降低细胞内胆固醇水平、基质钙化和碱性磷酸酶活性，但升高甲基萘醌-4水平。升高的UBIAD1或menaquinone-4的表达降低了固醇调控元件结合蛋白-2的基因表达，并增加了负责胆固醇合成和外排的ATP结合盒转运蛋白A1的基因表达。在huvsmc中，UBIAD1与内质网标记物和高尔基体标记物共定位。综上所述，细胞内高胆固醇含量有助于磷酸盐诱导的血管细胞分化和钙化。UBIAD1或甲基萘醌-4可以减少血管细胞分化和钙化，可能是通过其反向调节细胞胆固醇的有效作用。
Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1) in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs) were incubated with either growth medium (1.4 mmol/L Pi) or calcification medium (CM) (3.0 mmol/L Pi). Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and the Golgi marker in HUVSMCs. In conclusion, high intracellular cholesterol content contributes to phosphate-induced vascular cell differentiation and calcification. UBIAD1 or menaquinone-4 could decrease vascular cell differentiation and calcification, probably via its potent role of inversely modulating cellular cholesterol.