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Understanding the Role of MMPs in Basement Membrane Breaching In vivo

了解 MMP 在体内基底膜破裂中的作用

基本信息

批准号：
8398457
负责人：
Laura Catherine Kelley
金额：
$ 4.92万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-12-01 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8398457
关键词：
Animals Basement membrane Behavior Binding Biological Caenorhabditis elegans Cancer cell line Cell Culture Techniques Cell physiology Cells Cessation of life Complex Data Detection Development Developmental Process Disease Disease Progression Disseminated Malignant Neoplasm Drug Delivery Systems Encapsulated Enzymes Event Excision Extracellular Matrix Extracellular Matrix Degradation FOS Protein FOS gene Gene Expression Gene Expression Regulation Genes Genetic Genetic Screening Goals Health Homologous Gene Human Human Development Image Immune System Diseases Immunologic Surveillance In Vitro Infiltration Invaded Knowledge Laboratories Lead Leukocyte Trafficking Life Localized Malignant Neoplasm Malignant Neoplasms Matrix Metalloproteinases Mediating Membrane Proteins Metalloproteases Metalloproteinase Gene Methods Modeling Molecular Molecular Genetics Movement Neoplasm Metastasis Neural Crest Normal Cell Oncogenes Pathway interactions Patients Penetration Process Proteins RNA Interference Research Resolution Role Site Site-Directed Mutagenesis Specificity Stereotyping Time Tissues Transgenes Vertebrates Work Wound Healing Zinc cancer cell cardiogenesis cellular imaging crosslink gene function genetic analysis immune function improved in vivo matrix metalloproteinase 25 migration mutant natural Blastocyst Implantation novel outcome forecast overexpression programs promoter transcription factor tumor progression

项目摘要

DESCRIPTION (provided by applicant): Basement membrane is a dense sheet-like extracellular matrix that encapsulates and separates tissue compartments. The ability of cells to invade through basement membrane barriers is important during normal and disease processes. For example, basement membrane breaching is required for embryo implantation, neural crest migration, heart development, leukocyte trafficking and cancer cell metastasis-a critical step in tumor progression and the leading cause of patient death. Studying how cells breach basement membrane in vertebrates has been hindered by the difficulty of imaging and experimentally examining cell-basement membrane interactions in vivo. As a result, how cells cross basement membrane remains poorly understood. Our laboratory has developed methods that combine high-resolution live-cell imaging with the highly- stereotyped and genetically tractable model of anchor cell invasion in Caenorhabditis elegans to uncover the molecular mechanisms regulating basement membrane breaching in vivo. We have determined that the C. elegans c-fos oncogene homologue, fos-1a functions in the anchor cell to specifically mediate basement membrane penetration. In fos-1a mutant animals, the anchor cell extends cellular processes that flatten at an intact basement membrane. The complete repertoire of FOS-1A protein transcriptional targets mediating basement membrane breaching is not known. Recently, we have found that FOS-1A regulates the expression of three MMPs in the anchor cell during the time of invasion. Matrix metalloproteinases are overexpressed in cells responsible for tissue remodeling, wound healing, and cancer and are hypothesized to enzymatically facilitate BM removal. Matrix metalloproteinases localize to invasive machinery and are required for extracellular matrix degradation in metastatic cancer cell lines in vitro. Due to the high number of matrix metalloproteinases expressed in vertebrates and the difficulty of directly examining cell invasion in vivo, the relevance, and potential function of matrix metalloproteinases in cell invasion through basement membrane is unclear. The goal of the proposed research is to use the strengths of the model of anchor cell invasion in C. elegans - genetic analysis, live cell-imaging, molecular perturbation - to determine the function of matrix metalloproteinases during basement membrane breaching. In addition, I will perform a sensitized genetic screen to identify novel genes and pathways that function downstream of FOS-1A with matrix metalloproteinases to promote invasion. Completion of the aims in this proposal will increase our knowledge of the genetic pathways regulating cell invasion and the functional significance of FOS-1A-directed matrix metalloproteinase-driven basement membrane breaching. This work outlined in this proposal will directly impact human health by identifying specific pathways that could be targeted to limit invasive behavior. PUBLIC HEALTH RELEVANCE: Cell invasion through basement membrane is an important and fundamental process in human development, immune functioning and is a defining step in the lethal progression of most cancers. The proposed research will increase our understanding of the way in which invasive cells penetrate basement membrane barriers and invade into tissues. Completion of this work will lead to better strategies to therapeutically target invasive cellular behavior and directly improve patient prognosis in diseases such as cancer.

描述（由申请人提供）：基底膜是致密的片状细胞外基质，其封装并分隔组织区室。细胞侵入基底膜屏障的能力在正常和疾病过程中很重要。例如，基底膜破裂是胚胎植入、神经嵴迁移、心脏发育、白细胞运输和癌细胞转移所必需的——这是肿瘤进展的关键步骤，也是患者死亡的主要原因。由于体内成像和实验检查细胞与基底膜相互作用的难度，研究脊椎动物细胞如何突破基底膜受到阻碍。因此，人们对细胞如何穿过基底膜仍知之甚少。我们的实验室开发了将高分辨率活细胞成像与秀丽隐杆线虫锚细胞侵袭的高度定型和遗传易处理模型相结合的方法，以揭示体内调节基底膜破裂的分子机制。我们已经确定，线虫 c-fos 癌基因同源物 fos-1a 在锚细胞中发挥作用，特异性介导基底膜渗透。在 fos-1a 突变动物中，锚细胞延伸了在完整基底膜上变平的细胞过程。介导基底膜破裂的 FOS-1A 蛋白转录靶点的完整功能尚不清楚。最近，我们发现FOS-1A在侵袭过程中调节锚细胞中三种MMP的表达。基质金属蛋白酶在负责组织重塑、伤口愈合和癌症的细胞中过度表达，并被假设可以通过酶促方式促进 BM 去除。基质金属蛋白酶定位于侵袭性机器，是体外转移性癌细胞系中细胞外基质降解所必需的。由于脊椎动物中表达的基质金属蛋白酶数量较多且直接检查细胞的困难体内侵袭、基质金属蛋白酶在细胞基底膜侵袭中的相关性和潜在功能尚不清楚。本研究的目标是利用线虫锚细胞入侵模型的优势——遗传分析、活细胞成像、分子扰动——来确定基质金属蛋白酶在基底膜破裂过程中的功能。此外，我将进行敏化遗传筛选，以确定在 FOS-1A 下游与基质金属蛋白酶一起发挥作用以促进侵袭的新基因和途径。完成本提案中的目标将增加我们对调节细胞侵袭的遗传途径以及 FOS-1A 导向的基质金属蛋白酶驱动的基底膜破裂的功能意义的了解。该提案中概述的这项工作将通过确定可限制入侵行为的具体途径来直接影响人类健康。公共健康相关性：细胞通过基底膜的侵袭是人类发育、免疫功能中一个重要且基本的过程，也是大多数癌症致命进展的决定性步骤。拟议的研究将增加我们对侵袭性细胞穿透基底膜屏障并侵入组织的方式的理解。这项工作的完成将带来更好的策略来治疗靶向侵袭性细胞行为，并直接改善癌症等疾病的患者预后。