RESUMEN
The integrin αvß8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-ß. Through its association with LAP, TGF-ß is maintained in a latent form that must be activated to function. Binding to the integrin αvß8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-ß activation in vivo. Altered expression of the integrin ß8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1ß (IL-1ß) increases ITGB8 expression on lung fibroblasts, which increases αvß8-mediated TGF-ß activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1ß. 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, αvß8 surface expression, and αvß8-mediated TGFß activation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Cadenas beta de Integrinas/biosíntesis , Interleucina-1beta/biosíntesis , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta/metabolismo , Acetilación , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , ADN/genética , ADN/metabolismo , Células HeLa , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Integrina alfa5/biosíntesis , Integrina alfa5/genética , Cadenas beta de Integrinas/genética , Interleucina-1beta/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
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.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Cadenas beta de Integrinas/metabolismo , Factor de Transcripción Sp3/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Islas de CpG , Células HeLa , Humanos , Inflamación , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Células Epiteliales/metabolismo , Epitelio , Metaplasia/patología , Enfermedad Pulmonar Obstructiva Crónica , Mucosa Respiratoria , Animales , Comunicación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Células Epiteliales/patología , Epitelio/metabolismo , Epitelio/patología , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Integrinas/genética , Integrinas/metabolismo , Interleucina-1/genética , Interleucina-1/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Mesodermo , Metaplasia/metabolismo , Ratones , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/citología , Mucosa Respiratoria/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
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.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Factor de Transcripción E2F2/metabolismo , Enfermedades Gastrointestinales/virología , Hiperplasia/virología , Virus 40 de los Simios/patogenicidad , Animales , Factor de Transcripción E2F2/deficiencia , Factor de Transcripción E2F4/metabolismo , Enterocitos/química , Enterocitos/virología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína p130 Similar a la del Retinoblastoma/metabolismoRESUMEN
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.