Regulation of matrix metalloproteinase activity during tissue remodelling

组织由细胞和细胞分泌并附着的复杂蛋白质基质组成。这种细胞外基质(ECM)不断重塑，最明显的是在胚胎发育的戏剧性生长和细胞重组期间，也包括在伤口愈合期间和作为老化过程的一部分。细胞使用一种名为基质金属蛋白酶(MMPs)的复杂酶家族来重塑ECM。由于MMPs消化所有的ECM蛋白，它们的活性必须受到仔细的调控，否则组织将会解体。不足为奇的是，基质金属蛋白酶调节缺陷是多种疾病的原因和/或影响，包括关节炎、心脏病和癌症。但令人惊讶的是，人们对活组织中基质金属蛋白酶活性的调节知之甚少；我们所了解的大部分知识来自于在培养皿中对分离细胞进行的实验，以及对纯化蛋白质的生化实验。 斑马鱼是生物医学研究的优秀模式生物，我们已经在斑马鱼基因组中确定了所有MMPs及其已知的调节因子。由于斑马鱼胚胎是透明的，并且外部发育迅速，我们可以相对容易地观察到操纵基质金属蛋白酶活性的效果。此外，我们已经开发了利用鱼胚胎的透明度来可视化基质金属蛋白酶活性的技术，并检测在完整的胚胎中调节这些ECM重塑蛋白的生化事件。我们已经开发了一种新型的斑马鱼转基因品系，它表达一种工程蛋白，报告了其中一种MMPs的激活。使用这些胚胎，我们将产生有史以来第一张地图，显示这种基质金属蛋白酶在发育过程中何时何地被激活。此外，使用这些胚胎，我们可以发现被认为调节这种基质金属蛋白酶的生化机制是否真的发生在活着的动物身上。 侵袭性细胞使用基质金属蛋白酶降解组织之间的边界，允许它们在体内迁移。这对于正常的胚胎发育、伤口愈合和免疫反应是必不可少的，但也是肿瘤细胞转移的方式。因此，我们想更好地了解MMPs是如何在正常和病理侵袭细胞类型之间的交界处被调控的。我们可以将人类肿瘤细胞移植到斑马鱼胚胎中，并观察它们在胚胎中增殖和扩散的过程。使用我们的转基因品系和其他工具，我们可以测试有关侵袭性细胞如何使用自己的MMPs和/或周围组织中的MMPs来降解ECM的假设，从而使它们能够入侵并穿过胚胎。这不仅有助于我们了解胚胎是如何自我构建的，以及组织在受伤后如何维持和再生其结构，而且还将有助于了解MMPs如何在对组织造成损害的病理条件下被激活，并允许肿瘤扩散到全身。

Tissues are comprised of cells and a complex matrix of proteins cells secrete and adhere to. This extracellular matrix (ECM) is constantly remodeled, most obviously during the dramatic growth and cellular rearrangements of embryonic development, but also during wound healing and as part of the process of ageing. Cells remodel the ECM using a complex family of enzymes called matrix metalloproteinases (MMPs). Because MMPs digest all of the ECM proteins, their activity must be carefully regulated or tissues would disintegrate. Not surprisingly, defects in MMP regulation are causes and/or effects of a variety of diseases, including arthritis, heart disease and cancer. But surprisingly little is known about the regulation of MMP activity in living tissues; most of what we have learned comes from experiments done on isolated cells in petri dishes, and biochemical experiments with purified proteins. Zebrafish are an excellent model organism for biomedical research and we have identified all of the MMPs and their known regulators in the zebrafish genome. Because the zebrafish embryo is transparent, and develops externally and rapidly, we can observe the effects of manipulating MMP activity relatively easily. Furthermore, we have developed techniques that take advantage of the transparency of the fish embryo to visualize MMP activity, and to detect the biochemical events that regulate these ECM-remodeling proteases in the intact embryo. We have developed a novel transgenic line of zebrafish that expresses an engineered protein, which reports on the activation of one of these MMPs. Using these embryos we will generate the first ever map' of when and where this MMP becomes activated during development. Furthermore, using these embryos we can find out if the biochemical mechanisms that are thought to regulate this MMP are actually occurring in the living animal. Invasive cells use MMPs to degrade the boundaries between tissues, allowing them to migrate through the body. This is essential for normal embryonic development, wound healing, and the immune response, but it is also how tumour cells metastasize. We would therefore like to better understand how MMPs are regulated at the interface between normally and pathologically invasive cell types. We can transplant human tumour cells into zebrafish embryos and observe them as they proliferate and spread through the embryo. Using our transgenic line and other tools, we can test hypotheses about how invasive cells use their own MMPs and/or the MMPs present in the surrounding tissues to degrade the ECM, allowing them to invade and move through the embryo. This will not only help us understand how embryos build themselves and how tissues maintain and regenerate their structure after injury, but also how MMPs become activated under pathological conditions that cause damage to tissues, and allow tumors to spread throughout the body.