TGFB INDUCED DECREASE OF ENDOTHELIAL MONOLAYER INTEGRITY

内皮细胞（EC）的形状和间隙形成是由信号转导途径控制的，这些信号转导途径改变了对立的粘附力和收缩力的平衡。我们最近的研究表明，转化生长因子β (tgfβ)降低肺内皮单层完整性，这种降低与细胞间隙形成和肌球蛋白轻链（MLC）磷酸化的增加暂时相关。这种反应的时间过程和蛋白质合成抑制剂的初步数据表明，转录激活的变化调节了这种反应。我们假设，钙粘蛋白介导的细胞-细胞连接的粘附强度下降，加上MLC磷酸化的增加和随后的细胞收缩，是tgf β诱导的单层完整性下降的原因。我们进一步假设TGFbeta诱导的基因表达的不同信号通路调节TGFbeta诱导的EC表型变化的不同方面，这些变化与单层完整性降低有关。在Specific Aim 1中，我们将使用来自两种激活转录的tgf β途径的显性阴性和构成活性重组蛋白的表达来确定这些途径对tgf β诱导EC单层完整性降低的贡献。在特异性目标2中，我们将通过直接评估钙粘蛋白依赖的粘附强度变化和使用钙粘蛋白-5 α -钙粘蛋白融合蛋白，通过降低钙粘蛋白与肌动蛋白细胞骨架的关联，来验证tgf β降低钙粘蛋白介导的细胞-细胞粘附的假设。在具体目标3中，我们将检测MLC磷酸酶和激酶活性的变化，以确定这些酶中的一种或两种的变化是否有助于tgf β诱导的MLC磷酸化增加和随后的细胞收缩。这些目标的完成将是我长期研究目标的第一步，我的长期研究目标是：1)确定细胞因子，包括tgf β，如何通过改变肌动蛋白细胞骨架与细胞-细胞和细胞-基质粘附位点的相互作用来调节内皮屏障功能；2)确定这些途径在体内导致肺循环血管完整性丧失的重要性。

Endothelial cell (EC) shape and gap formation are controlled by signal transduction pathways that alter the balance of opposing adhesive and contractile forces. We have recently shown that transforming growth factor beta (TGFbeta) decreases pulmonary endothelial monolayer integrity and that this decrease was temporally associated with intercellular gap formation and an increase in myosin light chain (MLC) phosphorylation. The time course of this response and preliminary data with protein synthesis inhibitors suggest that changes in transcriptional activation regulate this response. We hypothesize that decreases in the adhesion strength of cadherin mediated cell-cell junctions in conjunction with increased MLC phosphorylation and subsequent cell contraction are responsible for TGFbeta induced decrease in monolayer integrity. We further hypothesize that different signaling pathways of TGFbeta induced gene expression regulate different aspects of the TGFbeta induced changes in EC phenotype that are associated with decreased monolayer integrity. In Specific Aim 1 we will use expression of dominant negative and constitutively active recombinant proteins from two TGFbeta pathways that activate transcription to determine the contribution of theses pathways to TGFbeta induced decrease of EC monolayer integrity. In Specific Aim 2 we will test the hypothesis that TGFbeta decreases cadherin mediated cell-cell adhesion by decreasing cadherin association with the actin cytoskeleton by directly assessing changes in cadherin dependent adhesion strength and by using a cadherin-5 alpha-catenin fusion protein. In specific aim 3 we will assay for changes in MLC phosphatase and kinase activity to determine if changes in one or both of these enzymes contribute to the TGFbeta induced increase of MLC phosphorylation and the subsequent cell contraction. Completion of these aims will be a first step toward my long term research goals that are 1) to deterine how cytokines, including TGFbeta, modulate endothelial barrier function by altering the interaction of the actin cytoskeleton with cell-cell and cell- matrix adhesion sites and 2) to determine the significance of these pathways in vivo as contributing to the loss of vascular integrity in the pulmonary circulation.