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.
| Mazzieri, R; Munger, J S; Rifkin, D B (2000) Measurement of active TGF-beta generated by cultured cells. Methods Mol Biol 142:13-27 |
| Munger, J S; Harpel, J G; Giancotti, F G et al. (1998) Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1. Mol Biol Cell 9:2627-38 |
