Understanding the Role of MMPs in Basement Membrane Breaching In vivo

描述（由申请人提供）：基底膜是一种致密的片状细胞外基质，可包裹和分隔组织隔室。细胞通过基底膜屏障侵入的能力在正常和疾病过程中是重要的。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。研究脊椎动物细胞如何突破基底膜一直受到成像和实验研究体内细胞-基底膜相互作用的困难的阻碍。因此，细胞如何穿过基底膜仍然知之甚少。我们的实验室已经开发了将联合收割机高分辨率活细胞成像与秀丽隐杆线虫中锚细胞侵袭的高度定型和遗传上易处理的模型相结合的方法，以揭示体内调节基底膜破裂的分子机制。我们已经确定C.线虫c-fos癌基因同源物，fos-1a在锚细胞中起作用以特异性介导基底膜穿透。在fos-1a突变的动物中，锚细胞延长了在完整基底膜处变平的细胞突起。FOS-1A蛋白转录靶介导基底膜破坏的完整库尚不清楚。最近，我们发现FOS-1A在侵袭过程中调节锚细胞中三种MMPs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表达，并且被假设为酶促促进BM去除。基质金属蛋白酶定位于侵袭性机器，并且是体外转移性癌细胞系中细胞外基质降解所必需的。由于在脊椎动物中大量表达的基质金属蛋白酶和直接检测细胞的困难， 尽管基质金属蛋白酶在细胞侵袭中的作用与其在体内侵袭中的作用有关，但基质金属蛋白酶在通过基底膜的细胞侵袭中的相关性和潜在功能尚不清楚。本研究的目的是利用锚细胞侵袭模型在C. elegans -遗传分析，活细胞成像，分子微扰-以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，以确定新的基因和途径，功能下游的FOS-1A与基质金属蛋白酶，以促进入侵。本提案目标的完成将增加我们对调节细胞侵袭的遗传途径以及FOS-1A指导的基质金属蛋白酶驱动的基底膜破裂的功能意义的了解。本提案中概述的这项工作将通过确定可以限制侵入性行为的特定途径直接影响人类健康。

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.