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

New York University School Of Medicine, New York NY

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Abstract

DESCRIPTION (provided by applicant): Transforming growth factor-beta (TGF-ß) is released from cells as part of a tripartite latent complex that includes, in addition to TGF-ß, the latency associated protein (LAP) and latent TGF-ß 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-ß levels. This result suggests that the defect in lung septation in Ltbp4-/- animals is related to increased TGF-ß2 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-ß. In our view, the TGF-ß-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-ß. These mice will produce Ltbp-4 and TGF-ß, but no Ltbp-4-TGF-ß 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-ß 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-ß levels under conditions in which either LTBP-4's structural function or TGF-ß 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-ß levels will be normalized by adding a pan-neutralizing antibody to TGF-ß. In Aim 2, we will examine the role and source of TGF-ß in the lung pathology. We will characterize the contribution of TGF-ß 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-ß; i.e. the sum of TGF-ß1 and TGF-ß2, or is specific for TGF-ß2. We will also identify the nature of the activator of latent TGF-ß in cultured cells and/or animals deficient in LTBP-4 by using specific inhibitors of, or mice with null mutations for, latent TGF-ß activators. Finally, we will determine whether the excess active TGF-ß formed in the absence of LTBP- 4 derives from complexes of LTBP-1 or LTBP-3 with TGF-ß, or from latent TGF-ß not bound to an LTBP. These experiments will yield important insights as to how latent TGF-ß is controlled in the lung and by cultured lung cells using novel genetic and cellular approaches. The results may suggest mechanisms for normalizing TGF-ß in certain pathological states, such as lung fibrosis.