MECHANISM OF ACTIVATION OF LATENT TGF BETA BY VASCULAR E

细胞因子转化因子-β（TGF-β）从细胞中释放， 一种由活性分子组成的非活性复合物， 通过静电相互作用裂解的前肽。 此外，潜在的 TGF-β蛋白（LTBP）是一种单独的基因产物，与 前肽通过二硫键连接。 虽然细胞产生潜伏的TGF-β TGF-β血管细胞的形成通常需要 两种不同细胞类型如内皮细胞和 平滑肌细胞 激活也需要尿激酶的作用 型纤溶酶原激活剂（uPA）、纤溶酶、组织II型转谷氨酰胺酶 （TGase），复合物与细胞表面甘露糖6- 磷酸盐/IGF-II受体（M6 P/IGF-IIr）和活性LTBP。 每个 异型对中的细胞似乎提供特异性反应物， 活化反应。 这些分子以协调一致的方式作用于 细胞表面或基质从潜伏的TGF-β形成TGF-β。 我们 我认为这种反应将潜在的复合物集中在表面上，或者 通过将潜在复合物与M6 P/IGF-IIr结合，然后通过交叉- 通过TGase将LTBP连接至未知基质或表面分子。 由尿激酶形成的纤溶酶然后从细胞中释放活性细胞因子。 通过切割前肽并使固定化结构不稳定， 离子相互作用 在本申请中，我们提出阐明 在分子水平上形成TGF-β所需的特定成分 并建立该机制的体内意义。 一是 将表征TGase与LTBP的相互作用。 使用生化 和分子技术，我们将建立TGase反应 残基在LTBP中，LTBP与什么分子交联，以及是否 这种交联对于潜在的TGF-β活化是至关重要的。 后一 实验将测试LTBP的功能改变，使反应性 残留物不见了。 我们还将研究纤溶酶对 前肽，并确定哪个键被纤溶酶切割，以及这是否 从潜在的复合物中释放TGF-β。 第二，我们会设立 我们已经确定的TGF-β所必需的各个步骤的顺序 阵 我们将利用命令的添加和撤回 对反应中的每个步骤具有特异性的抑制剂，然后测量 测试细胞产生TGF-β的顺序， 个人反应。 第三，我们将研究在体内的意义， 我们的体外结果是小鼠肺损伤模型， TGF-β形成。 在该模型中，肺中的出血性休克可以是 通过施用TGF-β 1的中和抗体来预防。 我们将监测药物抑制TGase或 LTBP、uPA和M6 P/IGF-IIr的抗体， 损伤，以防止肺病理学的出现， 组织学和生物化学方法。

The cytokine transforming factor-beta (TGF-beta) is released from cells as an inactive complex consisting of the active molecule still bound to its cleaved propeptide by electrostatic interactions. In addition, the latent TGF-beta protein (LTBP), a separate gene product, is linked to the propeptide by a disulfide bond. Although cells produce latent TGF-beta constitutively, formation of TGF-beta vascular cells normally requires the interaction of two different cell types such as endothelial cells and smooth muscle cells. Activation also requires the action of urokinase type plasminogen activator (uPA), plasmin, tissue type II transglutaminase (TGase), interaction of the complex with the cell surface mannose 6- phosphate/IGF-II receptor (M6P/IGF-IIr), and active LTBP. Each of the cells in a heterotypic pair appears to contribute specific reactants to the activation reaction. These molecules act in a concerted fashion on the cell surface or matrix to form TGF-beta from latent TGF-beta. We propose that this reaction focuses the latent complex on the surface or matrix by binding the latent complex to the M6P/IGF-IIr followed by cross- linking of the LTBP to an unknown matrix or surface molecule by TGase. Plasmin formed by urokinase then releases the active cytokine from the immobilized structure by cleaving the propeptide and destabilizing the ionic interactions. In this application we propose to elucidate the interactions of the specific components required for TGF-beta formation at a molecular level and to establish the in vivo significance of this mechanism. First, we will characterize the interaction of TGase with LTBP. Using biochemical and molecular techniques, we will establish where the TGase reactive residues are in LTBP, to what molecule LTBP is cross-linked, and whether this cross-linking is crucial for latent TGF-beta activation. This latter experiment will test the function of LTBP altered so that the reactive residue is missing. We will also examine the action of plasmin on the propeptide and establish which bond is cleaved by plasmin and whether this releases TGF-beta from the latent complex. Second, we will establish the order of the various steps we have identified as essential for TGF-beta formation. We will utilize the ordered addition and withdrawal of inhibitors specific for each step in the reaction followed by measurement of TGF-beta production by the test cells to place in sequence the individual reactions. Third, we will examine the in vivo significance of our in vitro results in a mouse lung injury model that is dependent upon TGF-beta formation. In this model hemorrhagic shock in the lung can be prevented by the administration of neutralizing antibodies to TGF-beta1. We will monitor the ability of agents that either inhibit TGase or antibodies to LTBP, uPA, and M6P/IGF-IIr, administered to mice at the time of injury, to prevent the appearance of lung pathology as measured by histological and biochemical approaches.