Establishment and validation of computational model for MT1-MMP dependent ECM degradation and intervention strategies.
Establishment and validation of computational model for MT1-MMP dependent ECM degradation and intervention strategies.
复制标题
DOI：
10.1371/journal.pcbi.1002479
复制
发表时间：
2012
影响因子：
4.3
通讯作者：
Ichikawa K
中科院分区：
文献类型：
--
作者：
Hoshino D;Koshikawa N;Suzuki T;Quaranta V;Weaver AM;Seiki M;Ichikawa K
MT 1-MMP是一种有效的促进侵袭的膜蛋白酶，被侵袭性癌细胞所利用。MT 1-MMP优先定位在称为侵袭伪足的膜突起处，在那里它在周围细胞外基质（ECM）的降解中起中心作用。以前的报道表明，在ECM降解中持续供应MT 1-MMP的作用。然而，MT 1-MMP的周转率和周转在多大程度上有助于ECM降解的侵袭伪足尚未澄清。为了解决这个问题，我们首先进行FRAP（荧光恢复后光漂白）实验与荧光标记的MT 1-MMP集中在一个单一的invadopodium，并发现FRAP信号非常快速的恢复，近似的双指数图与时间常数为26秒和259秒。恢复主要取决于囊泡运输，但可以忽略不计的横向扩散。接下来，我们构建了一个计算模型，采用所观察到的FRAP实验的动力学。模拟成功地再现了我们的FRAP实验。接下来，我们在实验和模拟中抑制囊泡运输。添加抑制囊泡运输的药物实验上阻断ECM降解，并且模拟显示在抑制囊泡运输的条件下没有明显的ECM降解。此外，ECM降解的减少程度取决于MT 1-MMP周转的减少程度。因此，我们的实验和模拟已经确立了MT 1-MMP在侵袭伪足ECM降解中的快速周转的作用。此外，我们的模拟表明协同作用的蛋白水解活性和MT 1-MMP营业额ECM降解，因为有一个非线性和显着减少ECM降解，如果这两个因素同时减少。因此，我们的计算模型提供了一个新的计算机工具，设计和评估干预策略，在癌细胞的侵袭。在癌症治疗中，预防侵袭是重要的。MT 1-MMP是参与ECM（细胞外基质）降解的膜蛋白，其在侵袭伪足（invadopodia）处高度表达，侵袭伪足是癌细胞的小突起。ECM降解MT 1-MMP在侵袭伪足被假设为癌细胞侵袭的初始步骤。然而，MT 1-MMP被内源性抑制剂TIMP-2抑制，因此需要在侵袭伪足表面持续更新MT 1-MMP。与此一致，据报道，囊泡运输的阻断，这是一种参与周转的机制，阻止ECM降解。然而，MT 1-MMP在侵袭伪足的周转率和周转对ECM降解的关键程度尚未阐明。在这份报告中，我们测量了MT 1-MMP的周转率在一个单一的invadopodium和发现快速周转率的时间常数为26秒和259秒，这主要取决于囊泡运输。基于所观察到的动力学构建了计算模型。如果我们阻止快速周转，ECM降解在实验和模拟中都被阻止。这些结果确立了MT 1-MMP的快速周转在侵袭伪足ECM降解中的作用。
MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion. Prevention of invasion is important in cancer therapy. MT1-MMP is a membrane protein involved in degradation of ECM (extracellular matrix) that is highly expressed at invadopodia, which are small protrusions of cancer cells. ECM degradation by MT1-MMP at invadopodia is hypothesized as the initial step of cancer cell invasion. However, MT1-MMP is inhibited by the endogenous inhibitor TIMP-2, so continuous turnover of MT1-MMP at the surface of invadopodia would be required. In agreement, it has been reported that the blockade of vesicle transport, which is one mechanism involved in the turnover, blocked the ECM degradation. However, the turnover rate of MT1-MMP at invadopodia and the extent to which the turnover is critical for the degradation of ECM have not been clarified. In this report we measured the turnover rate of MT1-MMP at a single invadopodium and found rapid turnover rates with time constants of 26 s and 259 s, which primarily depended on the vesicle transport. A computational model was constructed based on the observed kinetics. If we blocked the rapid turnover, the ECM degradation was blocked both experimentally and in simulations. These results established the role of the rapid turnover of MT1-MMP in the ECM degradation at invadopodia.
登录
查看更多内容
DOI：
10.1083/jcb.200709076
发表时间：
2008-06-16
期刊：
The Journal of cell biology
影响因子：
--
作者：
Sakurai-Yageta M;Recchi C;Le Dez G;Sibarita JB;Daviet L;Camonis J;D'Souza-Schorey C;Chavrier P
通讯作者：
Chavrier P
影响因子：
11.4
作者：
Bravo-Cordero, Jose J.;Marrero-Diaz, Raquel;Montoya, Maria C.
通讯作者：
Montoya, Maria C.
影响因子：
4.8
作者：
Hoshino, Daisuke;Tomari, Taizo;Seiki, Motoharu
通讯作者：
Seiki, Motoharu
影响因子：
8
作者：
Maquoi, E.;Assent, D.;Noel, A.
通讯作者：
Noel, A.
影响因子：
5.7
作者：
Nonaka, T;Nishibashi, K;Seiki, M
通讯作者：
Seiki, M