Matrix metalloproteinases in Drosophila wound healing

描述(申请人提供)：修复表皮伤口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)是一类在体外、培养和发育过程中裂解细胞外基质并调节信号通路的酶，但它们在创伤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的模型来阐明MMPs的功能，因为它的复杂性降低了--例如，小鼠有24个MMPs，而果蝇只有两个MMPs，MMP1和MMP2。这种有机体还提供了强大的遗传工具，可以对几乎每个基因进行时空可控的功能获得和功能丧失的表型分析。我们推测，果蝇MMPs形成一个复合体，作为基底膜重塑的全球调节因子。这一假说是基于对损伤和发育环境中的MMP1、MMP2和MMP2结合蛋白TIMP的遗传分析。所有这些都是愈合穿刺伤、蛹形态发生的特定方面以及正常生长过程中扩张基底膜所必需的。虽然这些基因有其他独立的表型，但共有的表型都表明这三个基因在基底膜重塑中发挥了基础性作用。由于已在哺乳动物细胞培养中描述了MMP1、MMP2和TIMP的三分子复合体，我们假设MMP1、MMP2和TIMP形成一个类似的复合体，指导基底膜的扩张和修复。支持这一假说的是，在MMP2和TIMP突变体中，MMP1‘S在基底膜和伤口边缘的正常定位丢失。这一假设是新颖而令人兴奋的：尽管MMP被认为可以切割ECM，但它们被认为是降解ECM，而不是促进其扩张和修复。MMPs似乎还调节伤口边缘的ERK信号，其中MMP1促进信号转导，MMP2将信号限制在边缘。这些相反的表型暗示了相同的基质金属蛋白酶复合体的另一种功能。目的1确定MMP1、MMP2和TIMP之间的分子相互作用，验证基质金属蛋白酶复合体假说。我们将确定结合伙伴，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伙伴。目的2通过生物化学方法和一种新的蛋白质组学方法(iTRAQ-tails)确定MMPs如何单独和共同修饰基底膜；利用条件遗传学确定控制重构的组织；以及检测突变基底膜的精细结构。目的3确定两种基质金属蛋白酶在体内对创面ERK信号的反向调控，确定受体和配体，确定ERK信号在创面愈合中的作用，以及确定负责改变该通路的特异性基质金属蛋白酶底物(S)。为了实现这些目标，我们获得了广泛的专家团队的支持，以告知和指导我们的实验。

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.