Bigg H F, Morrison C J, Butler G S, Bogoyevitch M A, Wang Z, Soloway P D, Overall C M
Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Cancer Res. 2001 May 1;61(9):3610-8.
The tissue inhibitors of metalloproteinases 1-4 (TIMPs) have discrete regulatory roles in the activation of matrix metalloproteinase (MMP)-2 (gelatinase A), an important basement membrane-degrading MMP pivotal to tumor metastasis and angiogenesis. TIMP-2 binds to both the hemopexin C domain of progelatinase A and the active site of membrane type-1 (MT1) MMP. This trimeric complex presents the cell surface-bound gelatinase A zymogen to a free MT1-MMP molecule for activation. To investigate the role of TIMP-4 in the activation process, we developed a new procedure for the expression and purification of recombinant human TIMP-4 from baby hamster kidney cells. The recombinant TIMP-4 was a potent inhibitor of gelatinase A (apparent K(i) [Ki(app.)] < or = 9 pM; k(on) (association rate constant), 4.57 +/- 0.13 x 10(6) M(-1)s(-1)) and was less dependent upon hemopexin C domain interactions than TIMP-2 in its mode of binding and inhibition. Unlike TIMP-1, TIMP-4 strongly inhibited MT1-MMP (Ki(app.) < or = 100 pM; k(on), 3.49 +/- 0.34 x 10(6) M(-1)s(-1)) and blocked the concanavalin A-induced cellular activation of progelatinase A. In concanavalin A-stimulated homozygous Timp2 -/- fibroblasts or unstimulated MT1-MMP-transfected Timp2 -/- cells, which cannot activate progelatinase A, activation was restored by the addition of 0.3-5 nM TIMP-2 but not by TIMP-4, unequivocally showing the TIMP-2 dependency of MT1-MMP-induced activation of gelatinase A and the fact that TIMP-4 cannot support activation. The dominance of TIMP-2 in the activation process was further supported by the preferential binding of TIMP-2 compared with TIMP-4 to the hemopexin C domain of progelatinase A in inhibitor mixtures and by the ability of TIMP-2 to displace TIMP-4 from the hemopexin C domain. Hence, TIMP-4 regulates gelatinase A activity by efficient inhibition of MT1-MMP-mediated activation and by inhibiting the activated enzyme and, thus, is a tumor resistance factor in the peritumor stroma.
金属蛋白酶组织抑制剂1 - 4（TIMPs）在基质金属蛋白酶（MMP）-2（明胶酶A）的激活过程中具有不同的调节作用，MMP-2是一种重要的基底膜降解MMP，对肿瘤转移和血管生成至关重要。TIMP-2与前明胶酶A的血红素结合蛋白C结构域以及膜型-1（MT1）MMP的活性位点都能结合。这种三聚体复合物将细胞表面结合的明胶酶A酶原呈递给游离的MT1-MMP分子以进行激活。为了研究TIMP-4在激活过程中的作用，我们开发了一种从幼仓鼠肾细胞中表达和纯化重组人TIMP-4的新方法。重组TIMP-4是明胶酶A的有效抑制剂（表观K(i) [Ki(app.)]≤9 pM；k(on)（结合速率常数），4.57±0.13×10(6) M(-1)s(-1)），并且在其结合和抑制模式上比TIMP-2对血红素结合蛋白C结构域相互作用的依赖性更小。与TIMP-1不同，TIMP-4强烈抑制MT1-MMP（Ki(app.)≤100 pM；k(on)，3.49±0.34×10(6) M(-1)s(-1)）并阻断伴刀豆球蛋白A诱导的前明胶酶A的细胞激活。在伴刀豆球蛋白A刺激的纯合Timp2 -/-成纤维细胞或未刺激的MT1-MMP转染的Timp2 -/-细胞中，这些细胞无法激活前明胶酶A，加入0.3 - 5 nM的TIMP-2可恢复激活，但TIMP-4不能，这明确显示了MT1-MMP诱导的明胶酶A激活对TIMP-2的依赖性以及TIMP-4不能支持激活这一事实。抑制剂混合物中TIMP-2与TIMP-4相比优先结合前明胶酶A的血红素结合蛋白C结构域，以及TIMP-2能够将TIMP-4从血红素结合蛋白C结构域上置换下来，进一步支持了TIMP-2在激活过程中的主导地位。因此，TIMP-4通过有效抑制MT1-MMP介导的激活以及抑制活化酶来调节明胶酶A的活性，因此是肿瘤周围基质中的一种肿瘤抵抗因子。
