TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metal loproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP
TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metal loproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP
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DOI：
10.1042/bj20030557
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发表时间：
2003-09-14
影响因子：
4.1
通讯作者：
Fridman, R
中科院分区：
文献类型：
--
作者：
Bernardo, MM;Fridman, R
基质金属蛋白酶（MMP）-2在细胞外基质降解中起关键作用，与肿瘤转移和血管生成有关。在金属蛋白酶组织抑制剂（TIMP）-2调节的过程中，潜伏形式pro-MMP-2在细胞表面上被膜系膜I型（MT 1）-MMP激活。活性MT 1-MMP和TIMP-2的复合物结合pro-MMP-2形成三元复合物，其允许pro-MMP-2被不含TIMP-2的相邻MT 1-MMP激活。目前尚不清楚MMP-2活性在细胞周围空间的TIMP-2的存在下是如何调节。为了解决这个问题，在活细胞及其分离的质膜部分中研究了TIMP-2对细胞外间隙中MMP-2活性的影响，所述活细胞及其分离的质膜部分被工程化以控制MT 1-MMP和TIMP-2表达的相对水平。我们发现，自由和抑制MMP-2中检测到的介质，和净MMP-2活性与TIMP-2的表达水平。在具有活性MMP-2的纯化系统中置换MT 1-MMP结合的TIMP-2的研究显示，在实验条件下，由于TIMP-2和MT 1-MMP之间的高亲和力相互作用，抑制剂的置换最小。因此，细胞外间隙中MMP-2活性的抑制不太可能仅仅是TIMP-2从其与MT 1-MMP的复合物中解离的结果。一致的，质膜的免疫印迹分析，和表面生物素化实验表明，TIMP-2的表面缔合的水平是独立的MT 1-MMP的表达，因此，TIMP-2的低亲和力结合到不同的网站MT 1-MMP可能有调节MMP-2活性的三元复合物产生的细胞外空间的作用。
The matrix metalloproteinase (MMP)-2 has a crucial role in extracellular matrix degradation associated with cancer metastasis and angiogenesis. The latent form, pro-MMP-2, is activated on the cell surface by the membrane-tethered membrane type I (MT1)-MMP, in a process regulated by the tissue inhibitor of metalloproteinase (TIMP)-2. A complex of active MT1-MMP and TIMP-2 binds pro-MMP-2 forming a ternary complex, which permits pro-MMP-2 activation by a TIMP-2-free neighbouring MT1-MMP. It remains unclear how MMP-2 activity in the pericellular space is regulated in the presence of TIMP-2. To address this question, the effect of TIMP-2 on MMP-2 activity in the extracellular space was investigated in live cells, and their isolated plasma membrane fractions, engineered to control the relative levels of MT1-MMP and TIMP-2 expression. We show that both free and inhibited MMP-2 is detected in the medium, and that the net MMP-2 activity correlates with the level of TIMP-2 expression. Studies to displace MT1-MMP-bound TIMP-2 in a purified system with active MMP-2 show minimal displacement of inhibitor, under the experimental conditions, due to the high affinity interaction between TIMP-2 and MT1-MMP. Thus inhibition of MMP-2 activity in the extracellular space is unlikely to result solely as a result of TIMP-2 dissociation from its complex with MT1-MMP. Consistently, immunoblot analyses of plasma membranes, and surface biotinylation experiments show that the level of surface association of TIMP-2 is independent of MT1-MMP expression, Thus low-affinity binding of TIMP-2 to sites distinct to MT1-MMP may have a role in regulating MMP-2 activity in the extracellular space generated by the ternary complex.