Role of MT-MMPs in Renal Development

描述（由申请人提供）：基质金属蛋白酶（MMP）是一个多基因家族，包括20多种参与细胞外基质（ECM）成分降解的蛋白水解酶。大多数MMPs作为可溶性酶分泌到细胞外环境；然而，有些是膜结合的，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，但在缺乏这类MMPs的小鼠中没有发现明显的肾脏表型。相反，我们发现MT1-MMP缺失的小鼠由于ECM成分，特别是层粘连蛋白5 （Ln-5）的切割减少而出现发育不良的肾脏。最近，我们观察到MT4-MMP缺失小鼠由于一种不确定的机制而具有发育不良的肾脏。基于这些数据，我们假设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缺失的小鼠与MT1-MMP缺失的小鼠杂交，并将双缺失突变体与单个Ln-5缺失和MT1-MMP缺失的小鼠进行表型比较，以确定正常肾脏发育是否需要Ln-5的切割，看看是否可以挽救MT1-MMP缺失小鼠的肾脏表型；b)从Ln-5、MT1-MMP和Ln-5/MT1-MMP缺失小鼠中分离整个胚胎肾，收集肾管细胞，测定其分支运动发生能力。在微管形成实验中，将用不同的Ln-5裂解产物重建凝胶，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)分析野生型和MT4-MMP缺失小鼠的胚胎和成年肾脏，b)分离野生型和MT4-MMP缺失小鼠的收集管细胞，以确定缺乏这种酶对ecm依赖的细胞功能（如迁移、粘附和小管形成）的影响，来确定缺乏MT4-MMP是如何导致肾脏发育不良和发育不良的。在Aim 3中，我们将确定MT1-和MT4-MMP的相关ECM底物，通过测定a)纯化的MT1-和MT4-MMPs单独或与可溶性MMPs联合纯化的肾基底膜成分的体外裂解产物，b)体内MT1-和MT4-MMP缺失小鼠的肾基底膜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.