Objectives: To investigate the role of pyruvate kinase isozyme type M2 (PKM2) in regulating the expression of matrix metalloproteinase-1 (MMP-1) in human umbilical vein endothelial cells (HUVECs) under high glucose exposure and the underlying mechanisms.
Methods: HUVECs were exposed to concentration gradient (5.5, 15.0, 30.0 mmol/L) of D-glucose for 72 h. Western blotting was used to detect the expression of PKM2, p-PKM2 Y105 as well as its upstream and downstream proteins. The formation of PKM2 tetramer/dimer was examined. Immunofluorescence was used to detect the translocation of PKM2. The specific siRNAs, TEPP-46, and rapamycin were used to analyze the regulatory relations among PKM2, mammalian target of rapamycin (mTOR) complex, and MMP-1. Co-immunoprecipitation was used to detect the interaction of mTOR complex and PKM2.
Results: High glucose exposure upregulated the level of p-PKM2 Y105, and promoted PKM2 dimer formation and nuclear translocation as well as the expression of MMP-1 and Rictor in HUVECs. Down-regulation of PKM2 expression or the treatment of TEPP-46 reversed the high glucose induced-upregulation of MMP-1. Inhibition of mTOR singnal pathway by rapacymin reversed the phosphorylation of PKM2 on tyrosine 105 and the expression of MMP-1 in high glucose-treated cells. Knockdown the expression of Rictor decreased the phosphorylation of PKM2 on tyrosine 105, while knockdown the expression of Raptor upregulated the phosphorylation of PKM2 on tyrosine 105 under high glucose condition. Co-immunoprecipitation indicated the direct interaction between Rictor and PKM2.
Conclusions: The phosphorylation of PKM2 promotes high glucose-induced upregulation of MMP-1, and mTORC2 participates in the upregulation of PKM2 phosphorylation under high glucose condition.
目的: 探讨高糖条件下M2型丙酮酸激酶(pyruvate kinase isozyme type M2，PKM2)对内皮细胞基质金属蛋白酶-1(matrix metalloproteinase-1，MMP-1)表达的影响及其机制。方法: 用低、中、高浓度(5.5，15.0，30.0 mmol/L)的D-葡萄糖孵育人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVECs)72 h后，采用蛋白质印迹法检测PKM2，p-PKM2 Y105及其上下游分子的表达，检测PKM2四聚体/二聚体状态，结合免疫荧光分析检测PKM2核转位情况。通过使用siRNAs，PKM2抑制剂TEPP-46和哺乳动物雷帕霉素受体(mammalian target of rapamycin，mTOR)抑制剂雷帕霉素研究mTOR复合体，PKM2和MMP-1之间的调控关系。使用免疫共沉淀技术验证mTOR复合体和PKM2的相互作用。结果: 高糖上调HUVECs中p-PKM2 Y105，MMP-1，PKM2四聚体，Rictor的蛋白质表达，促进PKM2核转位。沉默PKM2的表达或使用PKM2抑制剂TEPP-46可以阻止高糖引起的MMP-1的蛋白质表达上调。雷帕霉素逆转高糖引起的p-PKM2 Y105和MMP-1的蛋白质表达上调。在高糖情况下，沉默Rictor后p-PKM2 Y105的蛋白质表达下调，而沉默Raptor后p-PKM2 Y105的表达上调。免疫共沉淀结果表明Rictor与PKM2存在直接结合。结论: 高糖条件下磷酸化的PKM2促进内皮细胞MMP-1表达，mTORC2参与了高糖诱导的PKM2磷酸化。.
Keywords: human umbilical vein endothelial cells; mammalian target of rapamycin; matrix metalloproteinase-1; pyruvate kinase isozyme type M2.