John S. Munger

A Mechanism for Regulating Pulmonary Inflammation and Fibrosis: The Integrin αvβ6 Binds and Activates Latent TGF β1

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.ated, and in this configuration TGFb is unable to bind University of California, San Francisco to its receptors; that is, TGFb is latent. In most cases, San Francisco, California 94143-0854 the complex of LAP and TGFb (the small latent complex 5 Department of Medicine SLC) is joined by latent TGFb binding protein 1 (LTBP1), 6 Cell Biology and Kaplan Cancer Center a matrix protein with sequence similarity to the fibrillins, New York University School of Medicine and the complex of all three proteins is called the large New York, New York 10016-6402 latent complex (LLC). Latent TGFb can be linked by

The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP–dependent activation of TGF-β1

Întegrins, matrix metalloproteases (MMPs), and the cytokine TGF-β have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-β exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-β. Because the latent domain of TGF-β1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-β (SLC) to the cell surface where TGF-β activation could be locally controlled. Here, we show that SLC binds to αvβ8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP–dependent release of active TGF-β, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell–matrix interactions.

Integrin αVβ6-mediated activation of latent TGF-β requires the latent TGF-β binding protein-1

Transforming growth factor-βs (TGF-β) are secreted as inactive complexes containing the TGF-β, the TGF-β propeptide, also called the latency-associated protein (LAP), and the latent TGF-β binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-β regulation. We have investigated the role of LTBP in modulating TGF-β generation by the integrin αVβ6. We show that even though αvβ6 recognizes an RGD on LAP, LTBP-1 is required for αVβ6-mediated latent TGF-β activation. The domains of LTBP-1 necessary for activation include the TGF-β propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in αVβ6-mediated latent TGF-β activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-β activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.

TGF‐β Latency: Biological Significance and Mechanisms of Activation

Transforming growth factor (TGF‐)β is secreted as a latent complex in which the mature growth factor remains associated with its propeptide. In order to elicit a biological response, the cytokine must be released from the latent complex, a process termed latent TGF‐β activation or TGF‐β formation. Although latent TGF‐β activation is a critical step in the regulation of its activity, little is known about the molecular mechanisms that lead to the production of active TGF‐β. In this article, we present an overview of the data available on this topic, and we propose a tentative model for the mechanism of TGF‐β formation based upon the observations with different cell systems and on recent findings on the structure of the latent TGF‐β complex.

Absence of integrin-mediated TGFβ1 activation in vivo recapitulates the phenotype of TGFβ1-null mice

The multifunctional cytokine transforming growth factor (TGF) β1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFβ1 must be functionally released from this complex before it can engage TGFβ receptors. One mechanism of latent TGFβ1 activation involves interaction of the integrins αvβ6 and αvβ8 with an RGD sequence in LAP; other putative latent TGFβ1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFβ1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1RGE/RGE) display the major features of Tgfb1−/− mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFβ1. These findings indicate that RGD-binding integrins are requisite latent TGFβ1 activators during development and in the immune system.

Cross Talk among TGF-β Signaling Pathways, Integrins, and the Extracellular Matrix

The growth factor TGF-β is secreted in a latent complex consisting of three proteins: TGF-β, an inhibitor (latency-associated protein, LAP, which is derived from the TGF-β propeptide) and an ECM-binding protein (one of the latent TGF-β binding proteins, or LTBPs). LTBPs interact with fibrillins and other ECM components and thus function to localize latent TGF-β in the ECM. LAP contains an integrin-binding site (RGD), and several RGD-binding integrins are able to activate latent TGF-β through binding this site. Mutant mice defective in integrin-mediated activators, and humans and mice with fibrillin gene mutations, show the critical role of ECM and integrins in regulating TGF-β signaling.

Proteolytic control of growth factor availability

Most growth factors are released from cells in a form that does not permit immediate interaction with their high affinity receptors. An important mechanism for presentation of these released latent growth factors is activation by the plasminogen activator‐plasmin system. The involvement of this system in the biology of Transforming Growth Factor‐β (TGF‐β) is reviewed.

Mice that lack activity of αvβ6- and αvβ8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice

The arginine-glycine-aspartate (RGD)-binding integrins αvβ6 and αvβ8 activate latent TGFβ1 and TGFβ3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins αvβ5 and αvβ3 activate these TGFβ isoforms. To define the combined role of αvβ6- and αvβ8-integrin in TGFβ activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6–/–;Itgb8–/– embryos die at mid-gestation; those that survive develop cleft palate–as observed in Tgfb3–/– mice. Itgb8–/– mice treated with an anti-αvβ6-integrin antibody develop severe autoimmunity and lack Langerhans cells–similar to Tgfb1-null mice. These results support a model in which TGFβ3-mediated palate fusion and TGFβ1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins αvβ6 and αvβ8 but not other RGD-binding integrins as TGFβ activators.

The integrin αVβ6 binds and activates latent TGFβ3

Transforming growth factors‐β (TGFβ1, 2 and 3) are secreted in a complex with their propeptides (latency‐associated peptide 1 (LAP1), 2 and 3). TGFβ signaling requires the dissociation of LAP and TGFβ, a process termed latent TGFβ activation. This process is a critical but incompletely understood step in the regulation of TGFβ function. In particular, the extent to which activation mechanisms differ among the three TGFβ isoforms is relatively unexplored. We show here that αVβ6 binds and activates latent TGFβ3.

Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1.

The development of fibrosis is a common response to a variety of injuries and results in the net accumulation of matrix proteins and impairment of normal organ function. We previously reported that the integrinα 8β1 is expressed by alveolar interstitial cells in normal lung and is upregulated during the development of fibrosis. TGFβ1 is an important mediator of the inflammatory response in pulmonary fibrosis. TGFβ1 is secreted as a latent protein that is non-covalently associated with latency-associated peptide (LAP) and requires activation to exert its effects. LAP-TGFβ1 and LAP-TGFβ3 contain the tripeptide sequence, arginine-glycine-aspartic acid (RGD), a known integrin recognition motif. The integrin α8β1 binds to several ligands such as fibronectin and vitronectin through the RGD sequence. Recent reports demonstrate that the integrins αvβ1, αvβ6 and αvβ8 adhere to LAP-TGFβ1 through the RGD site. Therefore, we asked whether LAP-TGFβ1 might be a ligand for α8β1 and whether this may be important in the development of fibrosis. We found that cell lines transfected with α8 subunit were able to spread on and adhere to recombinant LAP-TGFβ1 significantly better than mock transfected cell lines.α 8-transfected cells were also able to adhere to LAP-TGFβ3 significantly better than mock transfected cells. Adhesion to LAP-TGFβ1 was enhanced by activation of α8β1 by Mn2+, or 8A2, an integrin β1 activating antibody. Furthermore, cell adhesion was abolished when we used a recombinant LAP-TGFβ1 protein in which the RGD site was mutated to RGE. α8β1 binding to LAP-TGFβ1 increased cell proliferation and phosphorylation of FAK and ERK, but did not activate of TGFβ1. These data strongly suggest that LAP-TGFβ1 is a ligand ofα 8β1 and interaction of α8β1 with LAP-TGFβ1 may influence cell behavior.