Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation

Activation of endothelial cells, important in processes such as angiogenesi s, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosph orylation, turnover, or proteolytic release of a soluble extracellular doma in. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through p rotein kinase C and a Ca2+-dependent protease. In this study, the release o f soluble tie-1 was shown to be stimulated by inflammatory cytokines and va scular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor cw (TGF alpha). Release of soluble tie by tumor necrosis factor alpha (TNF alpha) or VEGF occurred within 10 minutes of stimulation and reached maximal level s within 60 minutes. Specificity was shown by fluorescence-activated cell s orting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TN F alpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E-749/S-750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-de pendent inhibition of cytokine-, PMA-. and VEGF-stimulated shedding, sugges ting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membr anes, (2) specifically activated in TNF alpha-treated cells, and (3) inhibi ted by BB-24. Additionally, proliferation of endothelial cells in response to VEGF. but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cell s is stimulated by inflammatory cytokines and VEGF through the activation o f an endothelial membrane-associated metalloprotease. (C) 1999 by The Ameri can Society of Hematology.