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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.

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.

The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.

Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. 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.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -β (TGF-β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-β family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Squamous metaplasia (SM) is common in smokers and is associated with airway obstruction in chronic obstructive pulmonary disease (COPD). A major mechanism of airway obstruction in COPD is thickening of the small airway walls. We asked whether SM actively contributes to airway wall thickening through alteration of epithelial-mesenchymal interactions in COPD. Using immunohistochemical staining, airway morphometry, and fibroblast culture of lung samples from COPD patients; genome-wide analysis of an in vitro model of SM; and in vitro modeling of human airway epithelial-mesenchymal interactions, we provide evidence that SM, through the increased secretion of IL-1beta, induces a fibrotic response in adjacent airway fibroblasts. We identify a pivotal role for integrin-mediated TGF-beta activation in amplifying SM and driving IL-1beta-dependent profibrotic mesenchymal responses. Finally, we show that SM correlates with increased severity of COPD and that fibroblast expression of the integrin alpha(v)beta(8), which is the major mediator of airway fibroblast TGF-beta activation, correlated with disease severity and small airway wall thickening in COPD. Our findings have identified TGF-beta as a potential therapeutic target for COPD.

Erythropoiesis, the process of cell proliferation and differentiation that produces erythrocytes, is a tightly regulated process, but apart from early progenitor development and the EPO-dependent response, very little is known about other molecular signals which control cellular fate during RBC production. Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept, an ActRIIA ligand trap, binds to and inhibits activin and other members of the TGFβ superfamily to induce a rapid increase in red cell number and hemoglobin in healthy volunteers. Pharmacological findings demonstrate that RAP-011, a murine ortholog of sotatercept, stimulates RBC parameters in mice through a mechanism distinct from EPO. We conducted the current study to evaluate if RAP-011 may stimulate expansion of a late-stage erythroblast population that is not normally expanded and/or may induce faster differentiation of erythroid precursors. In order to determine if RAP-011 promotes proliferation or differentiation during erythropoiesis, the number of cell divisions was quantified by CFSE staining. During in vitro erythroid differentiation, RAP-011 did not appear to alter the number of cell divisions; however, the percentage of cells that underwent the last division was higher in cultures treated with RAP-011, suggesting that the drug induced faster cellular maturation/differentiation. We also analyzed cell viability of GPA+ cells at the end of the differentiation process and observed that the percentage of apoptotic death was higher in control vs. RAP-011-treated cells. This suggests that RAP-011 may promote survival of late-stage precursors. To assess potential candidates which may mediate the erythropoietic effects of RAP-011, we selected three high affinity RAP-011 ligands, Activin A, Activin B and GDF-11, and proceeded to evaluate their effects on Smad signaling and on erythroid differentiation of human bone marrow progenitors. First, we observed that RAP-011 blocked ligand-induced Smad2/3 phosphorylation in the bone marrow-derived cells. Secondly, RAP-011 rescued activin A-induced inhibition of BFU-E colony formation. Finally, when mature CD36+ cells were differentiated in liquid media containing each of the three ligands, RAP-011 was able to reverse GDF-11- and Activin A-induced inhibition of of erythroid cell proliferation. GDF-11 and Activin A also significantly decreased the percentage of GPA-positive cells in culture, while significantly increasing the percentage of CD45-positive cells. Consistent with proliferation results, RAP-011 blocked these ligand effects. Treatment of CD36+ cells with Activin B did not alter growth or differentiation. These data suggest that GDF-11 and Activin A may contribute, in part, to the erythropoietic stimulatory effects of RAP-011. Several members of the TGFβ superfamily of ligands have been implicated as negative growth regulators, or “chalones”, functioning in homeostasis to maintain specific, mature tissue size. The results from our studies using the ActRIIA-Fc ligand trap, RAP-011, suggest that GDF-11 and Activin A, as well as other sotatercept ligands, may also be “chalones” for the blood, specifically regulating homeostasis of mature RBCs. We suggest that sotatercept increases red blood cell maturation and survival by blocking the negative growth regulation by TGFβ members. In pathologic states such as ineffective erythropoiesis, sotatercept may have an even greater impact than in the healthy, homeostatically-balanced environment. Disclosures: Carrancio: Celgene Corp.: Employment. Markovics:Celgene Corp.: Employment. Wong:Celgene Corp.: Employment. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene Corp.: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Sung:Celgene Corp.: Employment.

Abstract 372 The regulation of erythropoiesis requires stem cell factor and erythropoietin (EPO) for the proliferation and survival of erythroid progenitor and early precursor cells. While recombinant EPO is widely used for treating various types of anemia, it often lacks efficacy in cases of anemia due to ineffective erythropoiesis in which immature erythroid precursors undergo apoptosis. Thus, there is an need for new therapies to treat the later stages of erythropoiesis. Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept (ACE-011), a recombinant fusion protein consisting of the extracellular domain of the human activin receptor IIA (ActRIIA) linked to the human immunoglobulin G1 (IgG1) Fc domain, is a ligand trap which binds a number of TGFβ superfamily ligands including activin A, activin B, growth differentiation factor-11 (GDF-11) and bone morphogenetic protein-10 (BMP-10). Administration of sotatercept led to substantial increases in red cell number and hemoglobin in human subjects, but the mechanism is not fully understood. We utilized both mouse in vivo and human in vitro models to investigate the mechanism of sotatercept in promoting erythropoiesis. In order to compare the effects of RAP-011 (the murine version of sotatercept) to EPO on red blood cell (RBC) parameters, C57/Bl mice were dosed with RAP-011, EPO or control vehicle. RAP-011-treated mice had a rapid and statistically significant increase in hematocrit, hemoglobin, and RBC number in less than 72-hours. As rapidly as 24 hours after treatment, RAP-011 induced a significant increase in RNA-negative, enucleated cells in the bone marrow (BM). RAP-011 also rapidly increased BM BFU-e and CFU-e erythroid progenitors, while EPO was more effective on spleen-derived progenitors. These data suggest that RAP-011 acts primarily on both bone marrow progenitor cells and late erythroblasts to promote erythropoiesis. In order to investigate the cellular mechanism by which RAP-011 increases red blood cell parameters, we conducted a series of in vitro experiments and found no evidence to support direct effects of RAP-011 on human CD34+ cells assessed in colony formation assays and in erythroid differentiation in liquid culture. As both clinical and pharmacological findings point to a clear role for RAP-011 in stimulating RBC parameters, we hypothesized that RAP-011 effects may be mediated by accessory cells in the BM microenvironment. Human CD36+ cells, which are highly enriched for erythroid progenitors, were co-cultured with long-term BM cultures and erythroid differentiation was assessed following 6 days of culture in EPO (2U/mL)-supplemented media. At day 6 the output of these cultures was predominantly characterized as EryA (∼basophilic erythroblast) but with the addition of RAP-011 (50μM), a significant fraction of CD36+ cells matured into EryB/C cells (polychromatic/orthochromatic erythroblasts), suggesting that factors produced by BM accessory cells mediate RAP-011 erythropoietic effects and that, in contrast to EPO, RAP-011 may play a role in the latter stages of erythroblast maturation. To identify cytokines that may mediate RAP-011 effects, CD36+ cells were treated with several activin receptor IIA ligands. GDF-11 treatment significantly decreased proliferation of GPA+ cells during the differentiation process and RAP-011 effectively reversed this effect, but had no consequence on untreated cells. These data suggest that GDF-11 may mediate the erythroipoietic stimulatory effects of RAP-011. In summary, RAP-011 induced a rapid increase in RBC parameters in mice (reflected in the number of enucleated cells found in the bone marrow), likely mediated by BM accessory cells. Our data also suggest that effects of sotatercept may be mediated at least partly by GDF-11, acting as a potential negative regulator of the terminal stages of erythropoiesis. The ability of sotatercept to reverse this inhibition would lead to a rapid release of terminal erythroid cells into the circulation. These data support the rationale to develop sotatercept for the treatment of anemia and ineffective erythropoiesis, especially in patients who may not respond to EPO. Disclosures: Carrancio: Celgene Corporation: Employment. Markovics:Celgene Corporation: Employment. Wong:Celgene Corporation: Employment. Leisten:Celgene Corporation: Employment. Groza:Celgene Corporation: Employment. Raymon:Celgene Corporation: Employment. Heise:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Sung:Celgene Corporation: Employment.