Role of MT-MMPs in Renal Development

MT-MMP 在肾脏发育中的作用

• 批准号: 6862399
• 负责人: ROY ZENT
• 金额: $ 33.35万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6862399
• 关键词: cell adhesion; cell migration; collagenase; enzyme activity; extracellular matrix; genetically modified animals; laboratory mouse; laminin; metalloendopeptidases; nephrogenesis; phenotype; protein structure function; renal tubule

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.

描述（由申请人提供）：基质金属蛋白酶（MMP）是一个由 20 多种蛋白水解酶组成的多基因家族，参与细胞外基质（ECM）成分的降解。大多数 MMP 作为可溶性酶分泌到细胞外环境中；然而，有些是膜结合的，称为膜靶向金属蛋白酶-MMP。尽管体外证据表明可溶性 MMP 在肾脏发育中发挥重要作用，但在缺乏此类 MMP 的小鼠中尚未描述出显着的肾脏表型。相比之下，我们发现 MT1-MMP 缺失的小鼠由于 ECM 成分（尤其是层粘连蛋白 5 (Ln-5)）裂解减少而具有发育不良的发育不良肾脏。最近，我们观察到 MT4-MMP 缺失小鼠由于未确定的机制而具有发育不良的发育不良肾脏。基于这些数据，我们假设 MT-MMP，即 MT1-和 MT4-MMP 在正常肾脏发育中发挥关键作用。这些酶在正常肾脏发育中的作用将由以下 3 个目标决定。 1) 我们将确定正常肾脏发育是否需要 Ln-5 裂解 a) 将 Ln-5 缺失小鼠与 MT1-MMP 缺失小鼠杂交，并比较双缺失突变体与单 Ln-5 的表型- MT1-MMP缺失小鼠和MT1-MMP缺失小鼠，看看MT1-MMP缺失小鼠的肾脏表型是否可以被挽救； b)从Ln-5、MT1-MMP和Ln-5/MT1-MMP无效小鼠中分离整个胚胎肾和集合管细胞，并确定它们进行分支运动发生的能力。在管发生实验中，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于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，以及 b) 从野生型和 MT4-MMP- 中分离集合管细胞，确定 MT4-MMP 的缺乏如何导致肾脏发育不全和发育不良。小鼠，以确定缺乏这种酶对 ECM 依赖性细胞功能（例如迁移、粘附和管生成）的影响。在目标 3 中，我们将通过确定 a) 体外纯化的 MT1- 和 MT4-MMP 单独或与可溶性 MMP 组合对纯化的肾基底膜成分的裂解产物来确定 MT1- 和 MT4-MMP 的相关 ECM 底物，以及 b)体内 MT 1-和 MT4-MMP 缺失小鼠的肾基底膜组成与其野生型对应物相比是否存在差异。