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CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the increased recruitment of dendritic cells observed in fibroinflammatory diseases such as chronic obstructive pulmonary disease (COPD). CCL20 expression is increased by the proinflammatory cytokine IL-1β. We have determined that IL-1β-dependent CCL20 expression is also dependent on the multifunctional cytokine TGF-β. TGF-β is expressed in a latent form that must be activated to function, and activation is achieved through binding to the integrin αvβ8 (itgb8). Here we confirm correlative increases in αvβ8 and IL-1β with CCL20 protein in lung parenchymal lysates of a large cohort of COPD patients. How IL-1β- and αvβ8-mediated TGF-β activation conspire to increase fibroblast CCL20 expression remains unknown, because these pathways have not been shown to directly interact. We evaluate the 5'-flanking region of CCL20 to determine that IL-1β-driven CCL20 expression is dependent on αvβ8-mediated activation of TGF-β. We identify a TGF-β-responsive element (i.e. SMAD) located on an upstream enhancer of the human CCL20 promoter required for efficient IL-1β-dependent CCL20 expression. By chromatin immunoprecipitation, this upstream enhancer complexes with the p50 subunit of NF-κB on a NF-κB-binding element close to the transcriptional start site of CCL20. These interactions are confirmed by electromobility shift assays in nuclear extracts from human lung fibroblasts. These data define a mechanism by which αvβ8-dependent activation of TGF-β regulates IL-1β-dependent CCL20 expression in COPD., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor-β (TGF-β) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-β is expressed in a latent form that requires activation. The integrin αvβ8 (encoded by the itgb8 gene) is a receptor for latent TGF-β and is essential for its activation. Expression of integrin αvβ8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvβ8 (B5) inhibited TGF-β activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-β activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvβ8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvβ8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-β pathway to treat fibroinflammatory airway diseases., (Copyright © 2014, American Association for the Advancement of Science.)

The integrin αvβ8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-β. Through its association with LAP, TGF-β is maintained in a latent form that must be activated to function. Binding to the integrin αvβ8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-β activation in vivo. Altered expression of the integrin β8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1β (IL-1β) increases ITGB8 expression on lung fibroblasts, which increases αvβ8-mediated TGF-β activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1β. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing "opening" of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvβ8 surface expression, and αvβ8-mediated TGFβ activation.

The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models - conditional deletion in mouse fibroblasts of the TGF-β-activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts - we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.

Integrin alphavbeta8 is a critical regulator of transforming growth factor beta activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin beta8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551-3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018-1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480-7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084-7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin beta8. Additionally, reduction of SP3 or inhibition of p38 blocks alphavbeta8-mediated transforming growth factor beta activation. These results place integrin beta8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway.

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.

Transforming growth factor (TGF)-beta is a potent multifunctional cytokine that is an essential regulator of epithelial proliferation. Because TGF-beta is expressed almost entirely in a latent state in vivo, a major source of regulation of TGF-beta function is its activation. A subset of integrins, alphavbeta8 and alphavbeta6, which are expressed in the human airway, has recently been shown to activate latent TGF-beta in vitro, suggesting a regulatory role for integrins in TGF-beta function in vivo. Here we have developed a novel, biologically relevant experimental model of human airway epithelium using intact human bronchial tissue. We have used this model to determine the function of integrin-mediated activation of TGF-beta in the airway. In human bronchial fragments cultured in vitro, authentic epithelial-stromal interactions were maintained and integrin and TGF-beta expression profiles correlated with profiles found in normal lung. In addition, in this model, we found that either the integrin alphavbeta8 or TGF-beta could inhibit airway epithelial cell proliferation. Furthermore, we found that one mechanism of integrin-alphavbeta8-dependent inhibition of cell proliferation was through activation of TGF-beta because anti-beta8 antibody blocked the majority (76%) of active TGF-beta released from bronchial fragments. These data provide compelling evidence for a functional role for integrin-mediated activation of TGF-beta in control of human airway epithelial proliferation in vivo.

Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, 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-beta, 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.