ROLE OF COLLAGENOLYTIC METALLOPROTEINASES IN METASTASES

为了探讨IV型胶原酶在肿瘤侵袭中的作用 和转移，我们专注于多层次的调控， 酵素 这些研究表明，与其他成员相比， 胶原酶家族，72 kDa IV型胶原酶mRNA水平 对TGF β 1的反应增加，不受肿瘤的影响， 促进佛波醇酯，并显示在结肠直肠肿瘤中的水平升高 与邻近的正常粘膜组织相比。 我们有 确定了与细胞表面相关的细胞活化机制， 并且对72 kDa IV型胶原酶具有特异性，并且其可以 用伴刀豆球蛋白A的佛波醇酯预处理诱导。 这 细胞激活机制不影响其他成员的 胶原酶基因家族 进一步表征和纯化 这一启动机制的组成部分正在进行之中。 我们已经研究了潜在酶TIMP-2复合物的结构， 酶缺失突变体和酶抑制剂交联的产生 问题研究 这些研究表明，72 kDa IV型胶原酶具有 至少两个TIMP-2结合结构域。 主要结合域是 位于C-末端，血红素样结构域的酶。 这 结合位点以潜在酶的形式存在。 第二结合 该位点位于酶活性位点，并且仅在以下情况下变得可用： 有机汞介导的酶激活。 最后，针对92 kDa IV型胶原酶的抗肽抗体， 间质胶原酶、溶基质素-1和溶基质素-2已经被 制备和表征。

In order to investigate the role of type IV collagenase in tumor invasion and metastases, we have focused on the multilevel regulation of this enzyme. These studies have shown that in contrast with other members of the collagenase enzyme family, the 72 kDa type IV collagenase mRNA levels are increased in response to TGFbeta1, are unaffected by the tumor promoting phorbol esters, and show elevated levels in colorectal tumor tissues when compared with adjacent normal mucosa tissues. We have identified a cellular activation mechanism which is cell surface associated and specific for the 72 kDa type IV collagenase enzyme, and which can be induced by pretreatment with phorbol esters of concanavalin A. This cellular activation mechanism does not affect other members of the collagenase gene family. Further characterization and purification of components of this activation mechanism are ongoing. We have studied the structure of the latent enzyme TIMP-2 complex through production of enzyme deletion mutants and enzyme inhibitor cross linking studies. These studies demonstrate that the 72 kDa type IV collagenase has at least two TIMP-2 binding domains. The principal binding domain is located in the C-terminal, hemopexin-like domain of the enzyme. This binding site is available in the latent enzyme form. The second binding site is at the enzyme active site and only becomes available following organomercurial mediated enzyme activation. Finally, antipeptide antibodies against the 92 kDa type IV collagenase, interstitial collagenase, stromelysin-1 and stromelysin-2 have been prepared and characterized.