Dean Sheppard

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

Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix

Mechanisms leading to fibroblast accumulation during pulmonary fibrogenesis remain unclear. Although there is in vitro evidence of lung alveolar epithelial-to-mesenchymal transition (EMT), whether EMT occurs within the lung is currently unknown. Biopsies from fibrotic human lungs demonstrate epithelial cells with mesenchymal features, suggesting EMT. To more definitively test the capacity of alveolar epithelial cells for EMT, mice expressing β-galactosidase (β-gal) exclusively in lung epithelial cells were generated, and their fates were followed in an established model of pulmonary fibrosis, overexpression of active TGF-β1. β-gal-positive cells expressing mesenchymal markers accumulated within 3 weeks of in vivo TGF-β1 expression. The increase in vimentin-positive cells within injured lungs was nearly all β-gal-positive, indicating epithelial cells as the main source of mesenchymal expansion in this model. Ex vivo, primary alveolar epithelial cells cultured on provisional matrix components, fibronectin or fibrin, undergo robust EMT via integrin-dependent activation of endogenous latent TGF-β1. In contrast, primary cells cultured on laminin/collagen mixtures do not activate the TGF-β1 pathway and, if exposed to active TGF-β1, undergo apoptosis rather than EMT. These data reveal alveolar epithelial cells as progenitors for fibroblasts in vivo and implicate the provisional extracellular matrix as a key regulator of epithelial transdifferentiation during fibrogenesis.

Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma

Asthma is an increasingly common disease that remains poorly understood and difficult to manage. This disease is characterized by airway hyperreactivity (AHR, defined by exaggerated airflow obstruction in response to bronchoconstrictors), mucus overproduction and chronic eosinophilic inflammation. AHR and mucus overproduction are consistently linked to asthma symptoms and morbidity. Asthma is mediated by Th2 lymphocytes, which produce a limited repertoire of cytokines, including interleukin-4 (IL-4), IL-5, IL-9 and IL-13. Although each of these cytokines has been implicated in asthma, IL-13 is now thought to be especially critical. In animal models of allergic asthma, blockade of IL-13 markedly inhibits allergen-induced AHR, mucus production and eosinophilia. Furthermore, IL-13 delivery to the airway causes all of these effects. IL-13 is thus both necessary and sufficient for experimental models of asthma. However, the IL-13-responsive cells causing these effects have not been identified. Here we show that mice lacking signal transducer and activator of transcription 6 (STAT6) were protected from all pulmonary effects of IL-13. Reconstitution of STAT6 only in epithelial cells was sufficient for IL-13-induced AHR and mucus production in the absence of inflammation, fibrosis or other lung pathology. These results demonstrate the importance of direct effects of IL-13 on epithelial cells in causing two central features of asthma.

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.

Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans

Pulmonary fibrosis is a progressive and largely untreatable group of disorders that affects up to 100,000 people on any given day in the United States. To elucidate the molecular mechanisms that lead to end-stage human pulmonary fibrosis we analyzed samples from patients with histologically proven pulmonary fibrosis (usual interstitial pneumonia) by using oligonucleotide microarrays. Gene expression patterns clearly distinguished normal from fibrotic lungs. Many of the genes that were significantly increased in fibrotic lungs encoded proteins associated with extracellular matrix formation and degradation and proteins expressed in smooth muscle. Using a combined set of scoring systems we determined that matrilysin (matrix metalloproteinase 7), a metalloprotease not previously associated with pulmonary fibrosis, was the most informative increased gene in our data set. Immunohistochemisry demonstrated increased expression of matrilysin protein in fibrotic lungs. Furthermore, matrilysin knockout mice were dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin. Our results identify matrilysin as a mediator of pulmonary fibrosis and a potential therapeutic target. They also illustrate the power of global gene expression analysis of human tissue samples to identify molecular pathways involved in clinical disease.

Loss of integrin |[alpha]|v|[beta]|6-mediated TGF-|[beta]| activation causes Mmp12-dependent emphysema

Integrins are heterodimeric cell-surface proteins that regulate cell growth, migration and survival. We have shown previously that the epithelial-restricted integrin αvβ6 has another critical function; that is, it binds and activates latent transforming growth factor-β (TGF-β). Through a global analysis of pulmonary gene expression in the lungs of mice lacking this integrin (Itgb6 null mice) we have identified a marked induction of macrophage metalloelastase (Mmp12)—a metalloproteinase that preferentially degrades elastin and has been implicated in the chronic lung disease emphysema. Here we report that Itgb6-null mice develop age-related emphysema that is completely abrogated either by transgenic expression of versions of the β6 integrin subunit that support TGF-β activation, or by the loss of Mmp12. Furthermore, we show that the effects of Itgb6 deletion are overcome by simultaneous transgenic expression of active TGF-β1. We have uncovered a pathway in which the loss of integrin-mediated activation of latent TGF-β causes age-dependent pulmonary emphysema through alterations of macrophage Mmp12 expression. Furthermore, we show that a functional alteration in the TGF-β activation pathway affects susceptibility to this disease.

TGF-β is a critical mediator of acute lung injury

We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding αv integrin subunit because various αv-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride–induced hepatic fibrosis, whereas global loss of β3, β5 or β6 integrins or conditional loss of β8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the αv integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of αv-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Src-mediated coupling of focal adhesion kinase to integrin αvβ5 in vascular endothelial growth factor signaling

Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin αvβ5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with β1 and β3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/αvβ5 signaling complex. Moreover, formation of this FAK/αvβ5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin β5, but not integrin β3, have a reduced VP response to VEGF. This FAK/αvβ5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin αvβ5 into a FAK-containing signaling complex during growth factor–mediated biological responses.

Therapy for Fibrotic Diseases: Nearing the Starting Line

An emerging consensus indicates that fibrotic diseases in lung, liver, and kidney exhibit common underlying mechanisms, which can be targeted therapeutically. Fibrosis, the accumulation of excess extracellular matrix in injured tissue, is the final common pathway for numerous diseases, including many of the lung, liver, kidney, and skin. This State of the Art Review outlines an emerging consensus that these diseases have many underlying features in common. The authors further describe ongoing efforts to develop effective therapies, including a discussion of obstacles to progress. Fibrosis, or the accumulation of extracellular matrix molecules that make up scar tissue, is a common feature of chronic tissue injury. Pulmonary fibrosis, renal fibrosis, and hepatic cirrhosis are among the more common fibrotic diseases, which in aggregate represent a huge unmet clinical need. New appreciation of the common features of fibrosis that are conserved among tissues has led to a clearer understanding of how epithelial injury provokes dysregulation of cell differentiation, signaling, and protein secretion. At the same time, discovery of tissue-specific features of fibrogenesis, combined with insights about genetic regulation of fibrosis, has laid the groundwork for biomarker discovery and validation, and the rational identification of mechanism-based antifibrotic drugs. Together, these advances herald an era of sustained focus on translating the biology of fibrosis into meaningful improvements in quality and length of life in patients with chronic fibrosing diseases.