COLLAGENOLYSIS-DRIVEN MOLECULAR MOTORS IN CELL MIGRATION AND MATRIX REMODELING

描述（由申请人提供）：金属蛋白酶（MMP）在形态发生、伤口愈合、血管生成、子宫复旧和骨吸收过程中的组织重塑中发挥关键作用。基质金属蛋白酶催化的细胞周蛋白水解是确定正常和肿瘤组织驻留细胞微环境的重要因素。恶性细胞利用MMPs促进肿瘤侵袭和转移。自20世纪90年代本实验室首次发现细胞表面MMP-2活化复合物（（MT 1-MMP）2/TIMP-2/MMP-2）以来，我们一直致力于研究该复合物催化的空间调控的分子机制，以阐明MMP在细胞- ECM相互作用中的作用。我们最近发现了MMPs的一些显著特征。我们证明了我。基质金属蛋白酶通过基质表面扩散机制与胶原蛋白相互作用。跨膜MT 1-MMP和分泌的MMP-1、MMP-2和MMP 9都可以在天然胶原纤维的表面上扩散; ii. MMP-1充当由胶原蛋白水解驱动的独特的、基于扩散的、ATP非依赖性运动酶; iii. MT 1-MMP的细胞外部分通过类似的偏置扩散机制与胶原相互作用; iv. MMP-2 C-末端结构域与抑制剂TIMP-2的复合物形成不影响扩散速率。因此，整个膜栓系的胶原溶解复合物（MT 1-MMP）2/TIMP-2/MMP-2相对于下面的胶原基质是移动的。这些发现对于理解基质金属蛋白酶在富含胶原的微环境中细胞运动中的作用具有深远的意义。因此，我们提出了一个移动的细胞表面胶原溶解界面的模型。在该模型中，我们假设膜束缚的胶原溶解复合物通过其沿下面的胶原原纤维沿着定向滑动的能力来辅助细胞运动。这种机制可能有助于协调细胞运动过程中的细胞粘附-解吸事件与细胞粘附素-整合素粘附装置。为了支持这一观点，我们现在提出的证据表明，细胞表面胶原溶解复合物的活性有助于细胞在3D胶原组织构建中发挥的力的产生。这促使我们进一步研究细胞周围胶原溶解是否有助于在富含胶原的微环境中细胞产生力。因此，我们在这里建议I。完成MT 1-MMP作为蛋白水解驱动的布朗棘轮的研究; ii.确定MMP-1和/或MT 1-MMP中与基质表面扩散机制相关的结构-功能关系，以及iii.使用几种实验方法确定细胞周围胶原溶解对富含胶原微环境中细胞产生力的贡献，包括2D培养物中的牵引力显微镜，3D组织构建体中的力测量，最后使用具有消融酶基因的细胞和具有突变胶原酶切割位点的胶原测量细胞可以施加在单个胶原原纤维上的力。这些研究将为进一步研究细胞如何利用细胞周围蛋白水解来协调细胞运动和组织重塑中的粘附-脱离事件提供必要的背景，这是转移性肿瘤侵袭的关键过程。 公共卫生相关性：基质金属蛋白酶（Matrix Metalloproteins，MMPs）是一类具有独特催化胶原等细胞外基质成分转化能力的酶。这些酶在形态发生、伤口愈合、血管生成、子宫复旧和骨吸收的正常过程中的组织重塑中起关键作用。恶性细胞利用MMPs促进肿瘤侵袭和转移。该提案的目标是提供更好地理解由MMPs催化的空间调节的细胞周围蛋白水解的分子机制及其在许多正常和病理条件下的功能作用。

DESCRIPTION (provided by applicant): Metalloproteases (MMPs) play a pivotal role in tissue remodeling during morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Pericellular proteolysis catalyzed by MMPs is an important factor in defining the microenvironment of the resident cells of normal and neoplastic tissues. Malignant cells exploit MMPs to promote tumor invasion and metastasis. Since the cell surface MMP-2 activation complex ((MT1-MMP)2/TIMP-2/MMP-2) was described in our lab in the 1990s, we have studied molecular mechanisms of the spatial regulation catalyzed by this complex to elucidate the role of MMPs in cell - ECM interactions. We have recently identified some remarkable features of MMPs. We showed that i. MMPs interact with collagen via a substrate surface diffusion mechanism. Both trans-membrane MT1-MMP and secreted MMP-1, -2 and 9 can diffuse on the surface of native collagen fibrils; ii. MMP-1 acts as a unique, diffusion-based, ATP- independent motor enzyme driven by collagen proteolysis; iii. The extra-cellular portion of MT1-MMP interacts with collagen through a similar biased-diffusion mechanism; iv. Complex formation of MMP-2 C-terminal domain with the inhibitor TIMP-2 does not affect the rate of diffusion. Thus the entire membrane tethered collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 is mobile relative to the underlying collagen substratum. These findings have profound implications for a mechanistic understanding of the role of MMPs in cell locomotion in a collagen rich microenvironment. We thus propose a model for a Mobile Cell Surface Collagenolytic Interface. In this model we hypothesize that the membrane tethered Collagenolytic Complex assists cell locomotion by virtue of its ability to slide directionally along the underlying collagen fibril. This mechanism is likely to be instrumental in orchestration of cell adhesion-desorption events during cell locomotion in collaboration with the cytoskeleton-integrin adhesion apparatus. To support this notion we now present evidence that the activity of the cell surface collagenolytic complex aids in force generation that cells exert in 3D collagen tissue constructs. This motivates us to further examine whether pericellular collagenolysis contributes to force generation by cells in a collagen- rich microenvironment. Thus here we propose i. To complete the investigation of the MT1-MMP as a proteolysis driven Brownian ratchet; ii. To define the structure-function relationship in MMP-1 and/or MT1-MMP relevant to the mechanism of substrate surface diffusion and iii. To determine the contribution of pericellular collagenolysis to force generation by cells in a collagen rich microenvironment using several experimental approaches including traction force microscopy in 2D cultures, the force measurements in 3D tissue constructs, and finally to measure the force that cells can exert on an individual collagen fibril utilizing cells with ablated enzyme gene(s) and collagen with mutated collagenase cleavage site. These studies will provide the necessary background for further investigation of how cells utilize pericellular proteolysis to orchestrate the adhesion-detachment events in cell locomotion and tissue remodeling that is a key process in metastatic tumor invasion. Public Health Relevance: Matrix Metalloproteases (MMPs) are the specialized group of enzyme with unique ability to catalyze turnover of extracellular matrix components such as collagen. These enzymes play a pivotal role in tissue remodeling during normal processes of morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Malignant cells exploit MMPs to promote tumor invasion and metastasis. The goal of this proposal is to provide for a better understanding of the molecular mechanisms of spatially regulated peri-cellular proteolysis catalyzed by MMPs and its functional role in numerous normal and pathological conditions.