OBJECTIVE: The angiopoietin (Ang)-Tie-2 system plays a critical role during fetal and adult angiogenesis. Herein, we explored the Tie-2 shedding-related molecular mechanisms and the pathophysiological significance. METHODS AND RESULTS: By using a mouse hindlimb ischemia model, we observed dissociated expression between the full-length Tie-2 (fTie-2) protein and Tie-2 mRNA in thigh muscles 1 day after an ischemic operation, suggesting that fTie-2 expression was modified through the posttranscriptional regulation in vivo. A soluble form of Tie-2 produced in human umbilical vein endothelial cells was dramatically suppressed by treatment with siRNA-matrix metalloproteinase (MMP) 14 or tissue inhibitor of metalloproteinase 3, resulting in an increase in cellular fTie-2 and thereby enhancing Ang-1-dependent Akt phosphorylation and Akt-dependent endothelial functions, such as Ang-2 downregulation or an increase of endothelial viability. Phorbol-12-myristate-13 acetate (PMA) upregulates MMP-14 mRNA via protein kinase C-extracellular signal-regulated kinase pathways, and enhanced soluble Tie-2 production in an MMP-14-dependent manner, resulting in a reduction of cellular fTie-2. In addition, the PMA-induced soluble Tie-2 was mediated by the protein kinase C-extracellular signal-regulated kinase signaling pathways. Finally, downregulation of tissue inhibitor of metalloproteinase 3 and upregulation of MMP-14 mRNA were confirmed in ischemic thigh muscles 1 day after the operation. CONCLUSIONS: An autocrine linkage between the endothelial protein kinase C-MMP-14 axis and Tie-2 shedding was shown to be a novel regulatory mechanism for the Ang-Tie-2 system and may play a role in modulating endothelial function during angiogenesis.