Understanding the Role of MMPs in Basement Membrane Breaching In vivo

描述（由申请人提供）：基膜是一种致密的片状细胞外基质，包裹和分离组织隔室。细胞侵入基底膜屏障的能力在正常和疾病过程中都很重要。例如，基底膜的破裂是胚胎植入、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。脊椎动物细胞如何突破基底膜的研究一直受到成像和实验检测细胞-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。我们的实验室开发了一种方法，将高分辨率活细胞成像与秀丽隐杆线虫锚细胞入侵的高度定型和遗传易学模型相结合，揭示体内调节基底膜破裂的分子机制。我们已经确定了线虫c-fos癌基因同源物fos-1a在锚定细胞中特异性介导基底膜渗透。在fos-1a突变动物中，锚定细胞扩展细胞过程，在完整的基底膜上变平。介导基底膜破裂的FOS-1A蛋白转录靶点的完整库尚不清楚。最近，我们发现FOS-1A在入侵期间调控锚细胞中三种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度表达，并被认为可以通过酶促基质金属蛋白酶去除。基质金属蛋白酶定位于侵袭性机器，并且是转移性癌细胞细胞系细胞外基质降解所必需的。由于基质金属蛋白酶在脊椎动物中大量表达，且难以直接检测细胞

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.