Role of MT-MMPs in Renal Development

描述（由申请人提供）：基质金属蛋白酶（MMP）是参与细胞外基质（ECM）组分降解的20多种蛋白水解酶的多基因家族。大多数MMPs作为可溶性酶分泌到细胞外环境中;然而，一些是膜结合的，称为膜靶向金属蛋白酶-MMPs。尽管有体外证据表明可溶性MMPs在肾脏发育中起重要作用，但在缺乏此类MMPs的小鼠中没有描述显著的肾脏表型。相反，我们发现MT 1-MMP缺失的小鼠由于ECM组分，特别是层粘连蛋白5（Ln-5）的切割减少而具有发育不良的发育不良肾。最近，我们观察到，MT4-MMP裸小鼠具有发育不良的肾，由于一个不确定的机制。基于这些数据，我们假设MT-MMPs，即MT 1-和MT4-MMPs在正常肾脏发育中起关键作用。这些酶在正常肾脏发育中的作用将在以下3个目标中确定。1)a）将Ln-5缺失小鼠与MT 1-MMP缺失小鼠杂交，并将双缺失突变体的表型与单Ln-5缺失和MT 1-MMP缺失小鼠进行比较，以观察是否可以挽救MT 1-MMP缺失小鼠的肾表型; B）从Ln- 5、MT 1-MMP和Ln-5/MT 1-MMP缺失小鼠中分离完整的胚胎肾和集合管细胞，并测定它们经历分支运动发生的能力。在微管发生实验中，将用Ln-5的不同裂解产物重构凝胶，以确定Ln-5的哪些特定结构域对分支形态发生至关重要。2)我们将通过a）分析来自野生型和MT4-MMP-缺失小鼠的胚胎和成年肾脏，和B）分离来自野生型和MT4-MMP-缺失小鼠的集合管细胞，以确定缺乏这种酶对ECM依赖性细胞功能如迁移、粘附和小管生成的影响，来确定MT4- MMP的缺乏如何导致发育不良和发育不良的肾脏。在目的3中，我们将通过以下方式确定MT 1-和MT 4-MMP的相关ECM底物：a）在体外通过单独的纯化的MT 1-和MT 4-MMP或与可溶性MMP组合的纯化的肾基底膜组分的切割产物，和B）在体内确定MT 1-和MT 4-MMP的肾基底膜组成是否存在差异。MMP无效小鼠与其野生型对应物相比。

DESCRIPTION (provided by applicant): Matrix metalloproteinases (MMP) are a multigenic family of more than 20 proteolytic enzymes involved in the degradation of extracellular matrix (ECM) components. Most MMPs are secreted as soluble enzymes into the extracellular milieu; however some are membrane-bound and called the membrane-targeted metalloproteinases-MMPs. Despite in vitro evidence that soluble MMPs play an important role in renal development, no significant renal phenotypes have been described in mice lacking this class of MMPs. In contrast, we have found that mice null for MT1-MMP have dysplastic dysgenic kidneys due to decreased cleavage of ECM components, particularly laminin 5 (Ln-5). More recently we observed that MT4-MMP null mice have hypoplastic dysgenic kidneys, due to an undetermined mechanism. Based on these data we hypothesize that MT-MMPs, namely MT1- and MT4-MMPs play a critical role in normal renal development. The role of these enzymes in normal renal development will be determined in the following 3 Aims. 1) We will determine whether Ln-5 cleavage is required for normal kidney development by a) crossing the Ln-5-null mouse with the MT1-MMP-null mouse and comparing the phenotype of the double null mutants with the single Ln-5-null and MT1-MMP null mice to see if the renal phenotype of the MT1-MMP-null mouse can be rescued; b) isolating whole embryonic kidneys and collecting duct cells from the Ln- 5, MT1-MMP and Ln-5/MT1-MMP null mice and determine their ability to undergo branching movphogenesis. In the tubulogenesis experiments the gels will be reconstituted with different cleavage products of Ln-5 to determine which specific domains of Ln-5 are critical for branching morphogenesis. 2) We will determine how lack of MT4- MMP results in hypoplastic and dysgenic kidneys by a) analyzing embryonic and adult kidneys from wild type and MT4-MMP-null mice and b) isolating collecting duct cells from wild type and MT4-MMP-null mice to determine the effects of lack of this enzyme on ECM-dependent cellular functions such as migration, adhesion and tubulogeneisis. In Aim 3 we will determine the relevant ECM substrates for MT1- and MT4-MMP by determining a) in vitro the cleavage products of purified renal basement membrane components by purified MT1- and MT4-MMPs alone or in combination with soluble MMPs and b) in vivo whether there are differences in the composition of the renal basement membranes of the MT 1- and MT4-MMP null mice compared to their wild type counterparts.