SQUAMOUS EPITHELIAL CELL INVASION IN ORGAN CULTURE

拟议的研究的总体目标是阐明细胞和分子调控机制，在人类皮肤器官培养模型的入侵入侵表型收购的基础。 在该模型中，将器官培养的皮肤暴露于某些外源性生长因子（特别是表皮生长因子或肝细胞生长因子）导致真皮-表皮基底膜的侵蚀和上皮对下层基质的侵入。 侵袭性表型的获得伴随着基质金属蛋白酶-9（MMP-9）（92-kD明胶酶B/IV型胶原酶）量的增加和上皮细胞群运动性的诱导，以及相邻基质中胶原溶解活性的上调。 假设靶组织暴露于外源性生长因子通过表皮中的两个丝裂原活化蛋白激酶（MAPK）级联启动信号转导，并且这些途径的激活导致MMP-9的产生和角质形成细胞运动的诱导。 进一步假设刺激的上皮细胞精心制作诱导邻近成纤维细胞产生MMP-1（间质胶原酶）的因子。这些事件一起导致了入侵。 在拟议研究的具体目标I中，目标将是确定上调MMP-9产生和刺激表皮细胞运动所需的细胞内信号传导事件。 将使用分析实验和干预方法的组合来定义相关的信号事件并干扰其表达。 在特定目标II中，将进行研究以证明真皮成纤维细胞的侵袭相关MMP-1的产生是真皮成纤维细胞与生长因子刺激的上皮细胞产物（而不是与外源性生长因子本身）相互作用的结果;并评估成纤维细胞上调MMP-1所需的细胞内信号传导事件。

The overall goal of the proposed studies is to elucidate cellular and molecular regulatory mechanisms that underlie acquisition of the invasive phenotype in a human skin organ culture model of invasion. In this model, exposure of organ-cultured skin to certain exogenous growth factors (notably epidermal growth factor or hepatocyte growth factor) results in erosion of the dermal-epidermal basement membrane and invasion of the underlying stroma by the epithelium. Acquisition of the invasive phenotype is accompanied by elaboration of increased amounts of matrix metalloproteinase-9 (MMP-9) (92-kD gelatinase B/type IV collagenase) and induction of motility in the epithelial cell population, and by up-regulation of collagenolytic activity in the adjacent stroma. It is hypothesized that exposure of the target tissue to the exogenous growth factors initiates signal-transduction through two mitogen-activated protein kinase (MAPK) cascades in the epidermis, and that the activation of these pathways results in MMP-9 elaboration and in the induction of keratinocyte motility. It is hypothesized further that stimulated epithelial cells elaborate factors that induce production of MMP-1 (interstitial collagenase) by adjacent fibroblasts. Together, these events bring about invasion. In Specific Aim I of the proposed studies, the goal will be to define the intracellular signaling events that are required for up-regulation of MMP-9 production and for stimulation of cell motility in the epidermis. A combination of analytical experiments and interventional approaches will be used to define the relevant signaling events and to interfere with their expression. In Specific Aim II, studies will be carried out to demonstrate that invasion-associated MMP-1 elaboration by dermal fibroblasts is a result of interaction of dermal fibroblasts with products of growth factor-stimulated epithelial cells (rather than with the exogenous growth factors themselves); and to assess intracellular signaling events that are required for fibroblast up-regulation of MMP-1.