Role of MT-Matrix Metalloproteins (MMPs) in Renal Development

描述(申请人提供)：基质金属蛋白酶(MMP)是一个多基因家族，由20多种蛋白水解酶组成，参与细胞外基质(ECM)成分的降解。大多数MMPs以可溶性酶的形式分泌到细胞外环境中，但也有一些是膜结合的，称为膜靶向金属蛋白酶-MMPs。尽管体外证据表明，可溶性MMPs在肾脏发育中发挥重要作用，但在缺乏这类MMPs的小鼠中，尚未描述显著的肾脏表型。相反，我们发现MT1-MMP零的小鼠有发育不良的肾脏，这是由于ECM成分，特别是层粘连蛋白5(Ln-5)的切割减少。最近，我们观察到MT4-MMP零基因小鼠的肾脏发育不良，原因不明。根据这些数据，我们推测MT-MMPs，即MT1-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)我们将通过a)将Ln-5缺失小鼠与MT1-MMP零小鼠杂交，并将双零突变体的表型与单个Ln-5缺失和MT1-MMP零小鼠进行比较，以确定是否可以挽救MT1-MMP零小鼠的肾脏表型；b)分离整个胚胎肾脏，收集Ln-5、MT1-MMP零和Ln-5/MT1-MMP零小鼠的导管细胞，并确定它们进行分支运动的能力。在小管发生实验中，凝胶将与Ln-5的不同切割产物重组，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)分析野生型和MT4-MMP零缺失型小鼠的胚胎和成年肾脏，以及b)分离野生型和MT4-MMP零缺失型小鼠的收集导管细胞，以确定缺乏MT4-MMPs对细胞外基质依赖的细胞功能(如迁移、黏附和小管生成)的影响，从而确定MT4-MMPs缺失是如何导致发育不全和发育不全的肾脏的。在目标3中，我们将通过以下方法确定MT1-和MT4-MMPECM的相关底物：a)在体外，纯化的MT1-和MT4-MMPs单独或与可溶性MMPs一起切割纯化的肾基膜成分；b)在体内，MT1-和MT4-MMP零小鼠的肾基底膜的组成与野生型小鼠相比是否存在差异。

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.