Yabkowitz R, Meyer S, Black T, Elliott G, Merewether L A, Yamane H K
Departments of Mammalian Cell Molecular Biology, Experimental Hematology, Protein Structure, and Protein Chemistry, Amgen Inc, Thousand Oaks, CA, USA.
Blood. 1999 Mar 15;93(6):1969-79.
Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (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 TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited 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 cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.
内皮细胞的激活在诸如血管生成等过程中很重要，它受细胞表面受体调控，包括酪氨酸激酶（RTK）家族中的受体。反过来，受体活性可通过磷酸化、更新或可溶性细胞外结构域的蛋白水解释放来调节。此前，我们证明佛波酯肉豆蔻酸酯乙酸酯（PMA）介导内皮细胞释放可溶性tie-1受体是通过蛋白激酶C和一种钙依赖性蛋白酶实现的。在本研究中，可溶性tie-1的释放显示受炎性细胞因子和血管内皮生长因子（VEGF）刺激，但不受碱性成纤维细胞生长因子（bFGF）或转化生长因子α（TGFα）等生长因子刺激。肿瘤坏死因子α（TNFα）或VEGF刺激后10分钟内即可发生可溶性tie的释放，并在60分钟内达到最高水平。荧光激活细胞分选（FACS）分析显示了其特异性；内皮细胞对TNF反应时细胞表面tie-1表达显著降低，而表皮生长因子受体（EGF-R）和CD31的表达则稳定。相反，巨核母细胞UT-7细胞上的tie-1表达不受PMA或TNFα影响。对裂解受体的序列分析表明，tie-1在近端跨膜结构域的E749/S750肽键处被蛋白水解。此外，异羟肟酸衍生物BB-24显示出对细胞因子、PMA和VEGF刺激的脱落具有剂量依赖性抑制作用，表明tie-1蛋白酶是一种金属蛋白酶。tie-1肽裂解试验中的蛋白酶活性（1）与内皮细胞膜相关，（2）在TNFα处理的细胞中特异性激活，（3）被BB-24抑制。此外，BB-24抑制了内皮细胞对VEGF而非bFGF的增殖反应，表明可溶性tie-1受体的释放参与了VEGF介导 的增殖过程。本研究表明，炎性细胞因子和VEGF通过激活一种内皮膜相关金属蛋白酶刺激内皮细胞释放可溶性tie-1。
