Matrix metalloproteinases in Drosophila wound healing

果蝇伤口愈合中的基质金属蛋白酶

• 批准号: 8518365
• 负责人: Andrea W Page-McCaw
• 金额: $ 30.08万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-25 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8518365
• 关键词: Affect; Architecture; Basement membrane; Binding; Binding Proteins; Biochemical; Biochemistry; Cancer Patient; Cell Culture Techniques; Cells; Chronic; Cicatrix; Cleaved cell; Complex; Data; Dermis; Development; Dissection; Drosophila genus; Epithelium; Extracellular Matrix; Family; Genes; Genetic; Growth; Healed; Health; Human; In Vitro; Inflammation; Knowledge; Ligands; Link; Mammalian Cell; Mammals; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Mediating; Methods; Microscopy; Modeling; Molecular Genetics; Morphogenesis; Mus; Organism; Pathway interactions; Peptide Hydrolases; Phenotype; Process; Property; Proteins; Proteolysis; Proteomics; Puncture wound; RNA Splicing; Receptor Protein-Tyrosine Kinases; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Structure; Testing; Tissues; Transmission Electron Microscopy; Ursidae Family; Wound Healing; base; cell motility; extracellular; genetic analysis; healing; in vivo; innovation; loss of function; member; migration; mutant; novel; receptor; repaired; research study; tool; tumor; tumor progression; wound

DESCRIPTION (provided by applicant): Healing an epidermal wound requires the coordination of tissues and extracellular matrix (ECM), and this coordination is mediated by signaling pathways. Matrix metalloproteinases (MMPs) are proteases that cleave ECM and regulate signaling pathways in vitro, in culture, and during development, but their roles in wound healing are unclear. MMPs are medically important: they are highly upregulated during tumor progression, inflammation, and wound healing. We use a Drosophila model of epidermal wound healing to elucidate MMP function because of its reduced complexity - for example, there are 24 MMPs in mouse, whereas there are only two MMPs in Drosophila, Mmp1 and Mmp2. This organism also offers powerful genetic tools that allow spatio- temporally controlled gain- and loss-of-function phenotypic analyses for nearly every gene. We hypothesize that the Drosophila MMPs form a complex that acts as a global regulator of basement membrane remodeling. This hypothesis is based on genetic analyses of Mmp1, Mmp2, and the MMP binding-protein Timp in wounding and developmental contexts. All are required for healing puncture wounds, for specific aspects of pupal morphogenesis, and for expanding basement membrane during normal growth. Although these genes have other independent phenotypes, the shared phenotypes all point to a fundamental role for the three genes in basement membrane remodeling. As an MMP-Timp trimolecular complex has been described in mammalian cell culture, we hypothesize that Mmp1, Mmp2, and Timp form a similar complex that directs expansion and repair of basement membranes. Supporting this hypothesis, Mmp1's normal localization to the basement membrane and wound margins is lost in Mmp2 and Timp mutants. This hypothesis is novel and exciting: although MMPs are known to cleave ECM, they are considered to degrade ECM, rather than promote its expansion and repair. The MMPs also appear to regulate ERK signaling at the wound margin, with Mmp1 promoting signaling and Mmp2 confining signaling to the margin. These opposing phenotypes suggest another function of the same MMP complex. Aim 1 determines the molecular interactions among Mmp1, Mmp2 and Timp, testing the MMP-complex hypothesis. We will identify binding partners, determine if they co-localize in tissues, and if different Mmp1 splice forms have different partners. Aim 2 determines how MMPs individually and together modify basement membrane, identifying substrates by biochemical methods and an innovative proteomics approach (iTRAQ- TAILS); identifying the tissues controlling remodeling with conditional genetics; and examining the fine structure of mutant basement membrane. Aim 3 determines how the two MMPs oppositely regulate ERK signaling at wounds in vivo, identifying the receptor and ligand, determining the function of ERK signaling in wound healing; and identifying the specific MMP substrate(s) responsible for altering the pathway. To accomplish these aims we have garnered support from a broad team of experts to inform and guide our experiments.

描述（由申请人提供）：表皮伤口愈合需要组织和细胞外基质（ECM）的协调，这种协调是由信号通路介导的。基质金属蛋白酶（MMPs）是一种在体外、培养和发育过程中切割ECM并调节信号通路的蛋白酶，但它们在伤口愈合中的作用尚不清楚。MMPs在医学上很重要：它们在肿瘤进展、炎症和伤口愈合过程中高度上调。我们使用果蝇表皮伤口愈合模型来阐明MMP的功能，因为它降低了复杂性-例如，小鼠中有24个MMPs，而果蝇中只有两个MMPs， Mmp1和Mmp2。这种生物还提供了强大的遗传工具，允许对几乎每个基因进行时空控制的功能增益和功能丧失表型分析。我们假设果蝇MMPs形成一种复合物，作为基底膜重塑的全球调节剂。这一假设是基于Mmp1、Mmp2和MMP结合蛋白Timp在损伤和发育背景下的遗传分析。所有这些都是愈合穿刺伤口、蛹形态发生的特定方面以及在正常生长过程中扩大基底膜所必需的。尽管这些基因具有其他独立的表型，但共同的表型都指向这三个基因在基底膜重塑中的基本作用。由于在哺乳动物细胞培养中已经发现了一种MMP-Timp三分子复合物，我们假设Mmp1、Mmp2和Timp形成了一种类似的复合物，指导基底膜的扩张和修复。支持这一假设的是，Mmp1在Mmp2和Timp突变体中失去了在基底膜和伤口边缘的正常定位。这一假设新颖而令人兴奋：尽管已知MMPs可以切割ECM，但它们被认为是降解ECM，而不是促进其扩张和修复。MMPs似乎也调节创伤边缘的ERK信号，其中Mmp1促进信号传导，而Mmp2将信号传导限制在边缘。这些相反的表型表明相同的MMP复合物具有另一种功能。Aim 1确定了Mmp1、Mmp2和Timp之间的分子相互作用，验证了mmp复合物假说。我们将识别结合伙伴，确定它们是否在组织中共定位，以及不同的Mmp1剪接形式是否有不同的合作伙伴。目标2确定MMPs如何单独和共同修饰基底膜，通过生化方法和创新的蛋白质组学方法识别底物（iTRAQ- TAILS）；用条件遗传学鉴定控制重塑的组织；并对突变基膜的精细结构进行了检测。目的3确定两种MMPs如何在体内反向调节伤口中的ERK信号，识别受体和配体，确定ERK信号在伤口愈合中的作用；并确定负责改变该途径的特定MMP底物。为了实现这些目标，我们获得了一个广泛的专家团队的支持，为我们的实验提供信息和指导。