缺氧对细胞膜ABCA1降解的影响及其机制研究

Hypoxia enhances degradation of plasma membrane ABCA1 via increased calpain activity

目的探讨缺氧对细胞膜三磷酸腺苷结合盒转运体A1(ABCA1)降解的影响及其与钙蛋白酶的相关机制。方法肝X受体激动剂TO-901317刺激RAW264.7细胞24 h,实时定量聚合酶链反应检测ABCA1 m RNA表达水平。生物素标记法提取细胞膜蛋白,Western blot检测细胞膜ABCA1蛋白水平表达。Western blot检测在放线菌酮存在或不存在条件下,TO-901317干预的RAW264.7细胞经过12 h缺氧(1%氧气O_2)处理后细胞膜ABCA1蛋白水平,Suc-LLVY-氨基虫荧光素法检测缺氧细胞内钙蛋白酶活性。RAW264.7细胞给予钙蛋白酶抑制剂ALLN干预,检测细胞膜ABCA1蛋白表达及钙蛋白酶活性。结果 TO-901317上调ABCA1 mRNA及细胞膜蛋白水平表达。无论放线菌酮存在或不存在情况下,缺氧均能降低细胞膜ABCA1蛋白水平,升高钙蛋白酶活性。钙蛋白酶抑制剂ALLN能部分逆转缺氧诱导细胞膜ABCA1蛋白水平降低。结论缺氧可能通过增加钙蛋白酶活性,从而加速细胞膜ABCA1蛋白降解。

Objective To investigate the effect of hypoxia on the degradation of plasma membrane ATP binding cassette transporter A1（ABCA1） and its calpain-related mechanism. Methods q RT-PCR was used to detect ABCA1 mRNA level in RAW264.7 cells stimulated by liver X receptor agonist TO-901317 for 24 h.Plasma membrane protein was extracted by the biotin labeling method of surface proteins, and membrane ABCA1 protein level was determined by Western blot. Western blot was applied to detect the plasma membrane ABCA1 protein level from TO-901317-treated RAW264.7 cells after 12-h hypoxia（1% O_2） treatment in the presence or absence of Cycloheximide. Furthermore, the calpain activity in the hypoxic cells was assayed by the method of Suc-LLVY-aminoluciferin. Finally, plasma membrane ABCA1 protein and calpain activity were measured in the RAW264.7 cells after intervention with calpain inhibitor ALLN. Results LXR agonist TO-901317 up-regulated ABCA1 m RNA and membrane protein levels. Hypoxia reduced plasma membrane ABCA1 protein in the RAW264.7 cells and increased the calpain activity in the absence or presence of Cycloheximide. Calpain inhibitor ALLN, in part, reversed the decrease of plasma membrane ABCA1 induced by hypoxia. Conclusions Hypoxia might accelerate the degradation of plasma membrane ABCA1 via enhancement of calpain activity.