High glucose up-regulates MMP-1 expression in endothelial cells via mTORC2-PKM2 pathway.

Abstract

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. 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. 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. 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.