Regulation of TGF-Beta Activity in the Lung by LTBP-4

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DESCRIPTION (provided by applicant): Transforming growth factor-beta (TGF-¿) is released from cells as part of a tripartite latent complex that includes, in addition to TGF-¿, the latency associated protein (LAP) and latent TGF-¿ binding protein (LTBP), which is disulfide bonded to LAP. We have reversed the impaired terminal alveolar development phenotype observed in mice deficient in LTBP-4 by generating Ltbp4-/-;Tgfb2-/- mice and thereby lowering TGF-¿ levels. This result suggests that the defect in lung septation in Ltbp4-/- animals is related to increased TGF-¿2 levels. We propose that LTBP-4 acts primarily as an organizer of elastic microfibrils, multi-protein assemblies, which contain fibrillins, fibulins, elastin, and LTBPs, and not as a binder of latent TGF-¿. In our view, the TGF-¿-mediated effects are secondary to abnormal matrix. We will test this hypothesis in two aims. In Aim 1, we will generate mice in which the two cysteine residues in LTBP-4 that bind to LAP are mutated to serines so that Ltbp-4 cannot bind to TGF-¿. These mice will produce Ltbp-4 and TGF-¿, but no Ltbp-4-TGF-¿ complexes. If the lung alveolarization abnormality in Ltbp4-/- mice is due to the absence of the structural activity of LTBP-4, these new mutant animals should have a normal phenotype. Conversely, if the lung defect in Ltbp-4-/- mice relates to the loss of TGF-¿ bound to Ltbp-4, the mutant animals will display abnormal air sac septation. We will also validate our hypothesis in vitro using Ltbp4-/- cells and measuring matrix organization and active TGF-¿ levels under conditions in which either LTBP-4's structural function or TGF-¿ levels are normalized. We will normalize the LTBP-4 structural function by adding either cells that express WT LTBP-4 or purified LTBP-4 protein. TGF-¿ levels will be normalized by adding a pan-neutralizing antibody to TGF-¿. In Aim 2, we will examine the role and source of TGF- ¿ in the lung pathology. We will characterize the contribution of TGF-¿ to the lung defect by producing Ltbp4-/-;Tgfb1-/- mice and examining their phenotypes. The results of this experiment will establish whether normalization of the lung phenotype in Ltbp4-/-;Tgfb2-/- animals is due to a decrease in total TGF-¿; i.e. the sum of TGF-¿1 and TGF-¿2, or is specific for TGF-¿2. We will also identify the nature of the activator of latent TGF-¿ in cultured cells and/or animals deficient in LTBP-4 by using specific inhibitors of, or mice with null mutations for, latent TGF-¿ activators. Finally, we will determine whether the excess active TGF-¿ formed in the absence of LTBP- 4 derives from complexes of LTBP-1 or LTBP-3 with TGF-¿, or from latent TGF-¿ not bound to an LTBP. These experiments will yield important insights as to how latent TGF-¿ is controlled in the lung and by cultured lung cells using novel genetic and cellular approaches. The results may suggest mechanisms for normalizing TGF-¿ in certain pathological states, such as lung fibrosis.