Integrin-Mediated Transforming Growth Factor-β Activation Regulates Homeostasis of the Pulmonary Epithelial-Mesenchymal Trophic Unit
2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.060049
ISSN1525-2191
AutoresJun Araya, Stephanie Cambier, Alanna Morris, Walter E. Finkbeiner, Stephen L. Nishimura,
Tópico(s)Pleural and Pulmonary Diseases
ResumoTrophic 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)-β 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, αvβ6 and αvβ8, are responsible for almost all of the TGF-β activation in the EMTU. Both αvβ8 and αvβ6 contribute to fetal tracheal epithelial activation of TGF-β, whereas only αvβ8 contributes to fetal tracheal fibroblast activation of TGF-β. Interestingly, fetal tracheal epithelial αvβ8-mediated TGF-β activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in αvβ8-mediated activation of TGF-β. Autocrine αvβ8-mediated TGF-β 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-β within the EMTU. 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)-β 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, αvβ6 and αvβ8, are responsible for almost all of the TGF-β activation in the EMTU. Both αvβ8 and αvβ6 contribute to fetal tracheal epithelial activation of TGF-β, whereas only αvβ8 contributes to fetal tracheal fibroblast activation of TGF-β. Interestingly, fetal tracheal epithelial αvβ8-mediated TGF-β activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in αvβ8-mediated activation of TGF-β. Autocrine αvβ8-mediated TGF-β 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-β within the EMTU. The lung develops from tightly orchestrated interactions between opposing layers of epithelial and mesenchymal cells separated by a basement membrane, the components of the epithelial-mesenchymal trophic unit (EMTU).1Holgate ST Davies DE Lackie PM Wilson SJ Puddicombe SM Lordan JL Epithelial-mesenchymal interactions in the pathogenesis of asthma.J Allergy Clin Immunol. 2000; 105: 193-204Abstract Full Text Full Text PDF PubMed Google Scholar During development these reciprocal interactions are driven by paracrine factors that determine the differentiation state of airway epithelial cells and fibroblasts.2Shannon JM Nielsen LD Gebb SA Randell SH Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea.Dev Dyn. 1998; 212: 482-494Crossref PubMed Scopus (136) Google Scholar, 3Demayo F Minoo P Plopper CG Schuger L Shannon J Torday JS Mesenchymal-epithelial interactions in lung development and repair: are modeling and remodeling the same process?.Am J Physiol. 2002; 283: L510-L517Google Scholar In the adult, reactivation of the EMTU has been proposed to occur in response to injury contributing either to the maintenance of homeostasis or to lung pathology, depending on the nature and duration of the insult.1Holgate ST Davies DE Lackie PM Wilson SJ Puddicombe SM Lordan JL Epithelial-mesenchymal interactions in the pathogenesis of asthma.J Allergy Clin Immunol. 2000; 105: 193-204Abstract Full Text Full Text PDF PubMed Google Scholar, 4Knight DA Lane CL Stick SM Does aberrant activation of the epithelial-mesenchymal trophic unit play a key role in asthma or is it an unimportant sideshow?.Curr Opin Pharmacol. 2004; 4: 251-256Crossref PubMed Scopus (30) Google Scholar, 5Holgate ST Epithelial damage and response.Clin Exp Allergy. 2000; 30: 37-41Crossref PubMed Google Scholar The multifunctional cytokine transforming growth factor (TGF)-β has been widely implicated as a master regulatory cytokine, acting as both an autocrine and paracrine factor in lung development and in the regulation of homeostasis of the EMTU.5Holgate ST Epithelial damage and response.Clin Exp Allergy. 2000; 30: 37-41Crossref PubMed Google Scholar, 6Kaartinen V Voncken JW Shuler C Warburton D Bu D Heisterkamp N Groffen J Abnormal lung development and cleft palate in mice lacking TGF-β3 indicates defects of epithelial-mesenchymal interaction.Nat Genet. 1995; 11: 415-421Crossref PubMed Scopus (864) Google Scholar Thus, mice genetically engineered to be deficient in TGF-β1 develop pulmonary inflammation, airway epithelial hyperplasia, and dysplasia.7Crawford SE Stellmach V Murphy-Ullrich JE Ribeiro SM Lawler J Hynes RO Boivin GP Bouck N Thrombospondin-1 is a major activator of TGF-β1 in vivo.Cell. 1998; 93: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar, 8Bottinger EP Letterio JJ Roberts AB Biology of TGF-β in knockout and transgenic mouse models.Kidney Int. 1997; 51: 1355-1360Crossref PubMed Scopus (150) Google Scholar, 9Diebold RJ Eis MJ Yin M Ormsby I Boivin GP Darrow BJ Saffitz JE Doetschman T Early-onset multifocal inflammation in the transforming growth factor β1-null mouse is lymphocyte mediated.Proc Natl Acad Sci USA. 1995; 92: 12215-12219Crossref PubMed Scopus (230) Google Scholar In addition, mice that are haploinsufficient in TGF-β1 are more susceptible to chemically induced pulmonary carcinoma, whereas overexpression of TGF-β in the mouse lung results in pulmonary fibrosis.10Sime PJ Xing Z Graham FL Csaky KG Gauldie J Adenovector-mediated gene transfer of active transforming growth factor-β1 induces prolonged severe fibrosis in rat lung.J Clin Invest. 1997; 100: 768-776Crossref PubMed Scopus (877) Google Scholar, 11Wang A Yokosaki Y Ferrando R Balmes J Sheppard D Differential regulation of airway epithelial integrins by growth factors.Am J Respir Cell Mol Biol. 1996; 15: 664-672Crossref PubMed Scopus (94) Google Scholar Mice deficient in TGF-β3 show defective airway branching.6Kaartinen V Voncken JW Shuler C Warburton D Bu D Heisterkamp N Groffen J Abnormal lung development and cleft palate in mice lacking TGF-β3 indicates defects of epithelial-mesenchymal interaction.Nat Genet. 1995; 11: 415-421Crossref PubMed Scopus (864) Google Scholar These results highlight the importance of TGF-β in the homeostatic regulation of pulmonary immunity, epithelial growth, and extracellular matrix deposition as well as in lung development. TGF-β is ubiquitously expressed in multiple cell types in the lung in three isoforms (TGF-β1 to TGF-β3), but almost entirely in an inactive (latent) form.12Annes JP Munger JS Rifkin DB Making sense of latent TGF-β activation.J Cell Sci. 2003; 116: 217-224Crossref PubMed Scopus (1286) Google Scholar Latent-TGF-β is further covalently linked to the extracellular matrix by binding to latent TGF-β-binding proteins.13Rifkin DB Latent transforming growth factor-β (TGF-β) binding proteins: orchestrators of TGF-β availability.J Biol Chem. 2005; 280: 7409-7412Crossref PubMed Scopus (341) Google Scholar Therefore, the activation of TGF-β from these latent stores is a major point of regulation of TGF-β function. TGF-β is activated by diverse mechanisms in the lung, ultimately involving either proteolysis or conformational alteration of the latency-associated peptide (LAP) of TGF-β.12Annes JP Munger JS Rifkin DB Making sense of latent TGF-β activation.J Cell Sci. 2003; 116: 217-224Crossref PubMed Scopus (1286) Google Scholar Recently, it has become evident that certain integrins are able to mediate activation of TGF-β and may be essential components of the TGF-β activation apparatus.14Munger JS Huang X Kawakatsu H Griffiths MJ Dalton SL Wu J Pittet JF Kaminski N Garat C Matthay MA Rifkin DB Sheppard D The integrin αvβ6 binds and activates latent TGF β1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 15Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar Thus, integrins have the potential to sequester latent TGF-β to the cell surface, where activation can be tightly coupled to cellular responses to environmental stress to maintain homeostasis. The LAP of TGF-β1 and −3 contains the RGD tri-peptide motif, which binds to a subset of integrins.14Munger JS Huang X Kawakatsu H Griffiths MJ Dalton SL Wu J Pittet JF Kaminski N Garat C Matthay MA Rifkin DB Sheppard D The integrin αvβ6 binds and activates latent TGF β1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 15Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar, 16Ludbrook SB Barry ST Delves CJ Horgan CM The integrin αvβ3 is a receptor for the latency-associated peptides of transforming growth factors β1 and β3.Biochem J. 2003; 369: 311-318Crossref PubMed Scopus (94) Google Scholar, 17Lu M Munger JS Steadele M Busald C Tellier M Schnapp LM Integrin α8β1 mediates adhesion to LAP-TGF-β1.J Cell Sci. 2002; 115: 4641-4648Crossref PubMed Scopus (66) Google Scholar, 18Munger JS Harpel JG Giancotti FG Rifkin DB Interactions between growth factors and integrins: latent forms of transforming growth factor-β are ligands for the integrin αvβ1.Mol Biol Cell. 1998; 9: 2627-2638Crossref PubMed Scopus (194) Google Scholar Of the six different integrins that bind to LAP-β1, only two appear to be able to activate TGF-β, αvβ6, and αvβ8 efficiently.14Munger JS Huang X Kawakatsu H Griffiths MJ Dalton SL Wu J Pittet JF Kaminski N Garat C Matthay MA Rifkin DB Sheppard D The integrin αvβ6 binds and activates latent TGF β1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 15Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar The αvβ6 integrin mediates activation of TGF-β through a mechanism involving the conformational alteration of TGF-β; the αvβ8 integrin mediates activation of TGF-β through the transmembrane matrix-metalloprotease-1 (MT1-MMP)-dependent cleavage of LAP.14Munger JS Huang X Kawakatsu H Griffiths MJ Dalton SL Wu J Pittet JF Kaminski N Garat C Matthay MA Rifkin DB Sheppard D The integrin αvβ6 binds and activates latent TGF β1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 15Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar These different mechanisms of integrin-mediated activation may have evolved to serve cell-type-specific roles in particular physiological settings. In the airway and alveolar epithelium, the integrin αvβ6 is normally expressed at low levels but can be induced by inflammatory stimuli; the integrin αvβ8 is normally expressed at high levels by normal airway epithelium.11Wang A Yokosaki Y Ferrando R Balmes J Sheppard D Differential regulation of airway epithelial integrins by growth factors.Am J Respir Cell Mol Biol. 1996; 15: 664-672Crossref PubMed Scopus (94) Google Scholar, 19Cambier S Mu D O'Connell D Boylen K Travis W Liu W Broaddus VC Nishimura SL The integrin αvβ8 negatively regulates epithelial cell growth.Cancer Res. 2000; 60: 7084-7093PubMed Google Scholar The implications of these patterns of integrin expression are that the αvβ6-dependent conformational mechanism of TGF-β activation would be biased toward TGF-β activation at injured sites after the local induction of αvβ6 expression; the αvβ8-dependent mechanism of TGF-β activation involving the metalloprotease-dependent liberation of active TGF-β would support homeostatic paracrine interactions between normal cells. We have recently developed a system based on the use of intact human bronchial tissue to evaluate the role of integrin-mediated activation of TGF-β in the wounded adult human airway. Thus, we found that the integrin αvβ8 is expressed at high levels in fragments of intact bronchial tissues and that it mediates activation of TGF-β, ultimately inhibiting epithelial cell growth.20Fjellbirkeland L Cambier S Broaddus VC Hill A Brunetta P Dolganov G Jablons D Nishimura SL Integrin αvβ8-mediated activation of transforming growth factor-β inhibits human airway epithelial proliferation in intact bronchial tissue.Am J Pathol. 2003; 163: 533-542Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar These findings suggest a possible role for TGF-β during the reactivation of the EMTU in the injured adult airway. However, the EMTU has only been rigorously experimentally defined in nonhuman lung systems, which have significant differences in anatomy and cell-type distribution from human lungs.3Demayo F Minoo P Plopper CG Schuger L Shannon J Torday JS Mesenchymal-epithelial interactions in lung development and repair: are modeling and remodeling the same process?.Am J Physiol. 2002; 283: L510-L517Google Scholar, 21Harkema JR Mariassy A George JS Hyde DM Plopper CG Epithelial cells of the conducting airways: a species comparison.in: Farmer SG Hay DWP The Airway Epithelium. Marcel Decker, New York1991: 3-39Google Scholar For instance, rodent airways are relatively lacking in basal cells, the cell type in which αvβ8 and αvβ6 are expressed in human airway.11Wang A Yokosaki Y Ferrando R Balmes J Sheppard D Differential regulation of airway epithelial integrins by growth factors.Am J Respir Cell Mol Biol. 1996; 15: 664-672Crossref PubMed Scopus (94) Google Scholar, 19Cambier S Mu D O'Connell D Boylen K Travis W Liu W Broaddus VC Nishimura SL The integrin αvβ8 negatively regulates epithelial cell growth.Cancer Res. 2000; 60: 7084-7093PubMed Google Scholar Therefore, we sought to develop a model of the human EMTU to define the role that integrin-mediated activation of TGF-β plays in epithelial-mesenchymal interactions because these same interactions are likely to contribute to airway disease in the adult. Here, we describe the individual roles that fetal tracheal epithelial cells and fetal tracheal fibroblasts play in integrin-mediated activation of TGF-β and then demonstrate the cell-type-dependent role of TGF-β activation in autocrine and paracrine interactions within the human EMTU. Surprisingly, we found that despite expressing robust levels of αvβ8, fetal tracheal epithelial cells fail to efficiently activate TGF-β unless stimulated with phorbol esters, an effect most likely attributable to the increased activity of MT1-MMP. In contrast, we find that fetal tracheal fibroblasts express relatively low levels of αvβ8 but efficiently support αvβ8-dependent activation of TGF-β. Furthermore, autocrine activation of TGF-β by fetal tracheal fibroblasts leads to the suppression of transcription and secretion of hepatocyte growth factor (HGF), a potent airway epithelial cell mitogen. This suppression of HGF is sufficient to negatively impact both phosphorylation of the HGF receptor c-Met, expressed by fetal tracheal epithelial cells, as well as proliferation of fetal tracheal epithelial cells. Taken together, our results suggest that integrins, in particular the integrin αvβ8, are central regulators of homeostatic interactions within the human pulmonary EMTU. Fetal tracheas were obtained at the time of elective termination of intrauterine pregnancy (18 to 22 weeks gestation) from otherwise healthy females. Informed consent was obtained from all participants as part of an approved ongoing research protocol by the University of California, San Francisco, Committee on Human Research. Tracheal fragments were microdissected into 0.2- to 0.5-mm fragments and cultured in an individual well of an agar-coated 96-well plate using the liquid overlay culture technique as described.20Fjellbirkeland L Cambier S Broaddus VC Hill A Brunetta P Dolganov G Jablons D Nishimura SL Integrin αvβ8-mediated activation of transforming growth factor-β inhibits human airway epithelial proliferation in intact bronchial tissue.Am J Pathol. 2003; 163: 533-542Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Fetal tracheal epithelial cells were isolated as previously described.22Finkbeiner WE Respiratory cell culture.in: Crystal RG The Lung: Scientific Foundations. Lippincott-Raven Publishers, Philadelphia1997: 415-433Google Scholar Freshly isolated fetal tracheal epithelial cells were plated onto rat-tail collagen type I (10 μg/ml)-coated dishes and incubated overnight, and then the medium was changed to bronchial epithelial growth medium (Clonetics, San Diego, CA). Fetal tracheal fibroblasts were cultured from the tracheal tissues remaining after epithelial cell isolation by the explant technique.22Finkbeiner WE Respiratory cell culture.in: Crystal RG The Lung: Scientific Foundations. Lippincott-Raven Publishers, Philadelphia1997: 415-433Google Scholar Briefly, fibroblasts outgrown from tracheal fragments were cultured in fibroblast growth media (Dulbecco's modified Eagle's medium with 10% fetal calf serum and penicillin-streptomycin; UCSF Cell Culture Facility). Confluent cells were passaged by trypsin treatment (0.025%; UCSF Cell Culture Facility) and used for the experiments at the initial passage (P1). Wild-type SW480 (American Type Culture Collection, Rockville, MD) and SW480 cells stably transduced with integrin β8 were prepared and passaged as described.19Cambier S Mu D O'Connell D Boylen K Travis W Liu W Broaddus VC Nishimura SL The integrin αvβ8 negatively regulates epithelial cell growth.Cancer Res. 2000; 60: 7084-7093PubMed Google Scholar All cells were incubated at 37°C in a humidified incubator in 7.5% CO2. Cultures were characterized immunohistochemically using anti-prolyl-4-hydroxylase (DAKO, Carpinteria, CA), anti-vimentin (Sigma, St. Louis, MO), and anti-cyto-keratin antibodies (Lu-5; BioCare Medical, Concord, CA). Fetal tracheal epithelial cells showed >95% positive staining with anti-cytokeratin and 95% positive staining with anti-prolyl-4-hydroxylase and anti-vimentin antibodies and <5% positive staining with the anti-cytokeratin antibody (data not shown). Additional antibodies used were mouse anti-β8 (clones 14E5 and 37e115Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar); rabbit-anti-β823McCarty JH Lacy-Hulbert A Charest A Bronson RT Crowley D Housman D Savill J Roes J Hynes RO Selective ablation of αv integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death.Development. 2005; 132: 165-176Crossref PubMed Scopus (182) Google Scholar (generous gift of Joseph McCarty, Massachusetts Institute of Technology, Cambridge, MA); anti-αv (8B815Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar, clone L230; American Tissue Type Collection, Manassas, VA); anti-β1 (clone P5D2; Developmental Studies Hybridoma Bank, Iowa City, IA); anti-β3 [clone AP3 (American Type Culture Collection) and LM609 (Chemicon, Temecula, CA)]; anti-β5 (clone P1F6, Chemicon); anti-β6 (10D524Weinacker A Chen A Agrez M Cone RI Nishimura S Wayner E Pytela R Sheppard D Role of the integrin αvβ6 in cell attachment to fibronectin. Heterologous expression of intact and secreted forms of the receptor.J Biol Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar; gift of Dean Sheppard, University of California at San Francisco, San Francisco, CA); anti-pan-TGF-β, anti-TGF-β1, anti-TGF-β3, anti-HGF (R&D Systems, Minneapolis, MN); anti-phosphorylated SMAD-225Yamagata H Matsuzaki K Mori S Yoshida K Tahashi Y Furukawa F Sekimoto G Watanabe T Uemura Y Sakaida N Yoshioka K Kamiyama Y Seki T Okazaki K Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH(2)-terminal kinase during human colorectal carcinogenesis.Cancer Res. 2005; 65: 157-165PubMed Google Scholar (gift of Dr. Matsuzaki, Kansai Medical University, Osaka, Japan); anti-phosphorylated c-Met and anti-c-Met (Cell Signaling Technology, Beverly, MA); and anti-smooth muscle actin (SMA, Sigma). Recombinant active TGF-β1 and HGF (R&D Systems), gelatin (Sigma), pro-MMP-2 (Chemicon), and the pan-metalloprotease inhibitor GM6001 (Ryss Lab, Union City, CA) were purchased. Rat-tail collagen was isolated as previously described.26Montesano R Mouron P Amherdt M Orci L Collagen matrix promotes reorganization of pancreatic endocrine cell monolayers into islet-like organoids.J Cell Biol. 1983; 97: 935-939Crossref PubMed Scopus (149) Google Scholar The GST-β8 cytoplasmic domain fusion protein was prepared as previously described.27Nishimura SL Sheppard D Pytela R Integrin αvβ8. Interaction with vitronectin and functional divergence of the β8 cytoplasmic domain.J Biol Chem. 1994; 269: 28708-28715Abstract Full Text PDF PubMed Google Scholar Immunohistochemistry was performed as previously described with minor modification.19Cambier S Mu D O'Connell D Boylen K Travis W Liu W Broaddus VC Nishimura SL The integrin αvβ8 negatively regulates epithelial cell growth.Cancer Res. 2000; 60: 7084-7093PubMed Google Scholar Briefly, the fetal tracheal tissue was formalin-fixed and paraffin-embedded or flash-frozen in embedding medium (VWR, Westchester, PA). The anti-β8 primary antibody was applied after preabsorption onto frozen sections of fetal liver. Flow cytometry was performed as previously described.19Cambier S Mu D O'Connell D Boylen K Travis W Liu W Broaddus VC Nishimura SL The integrin αvβ8 negatively regulates epithelial cell growth.Cancer Res. 2000; 60: 7084-7093PubMed Google Scholar RNA isolation, reverse transcription, and polymerase chain reaction were performed as previously described.15Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar The primers used were β8 integrin sense primer, 5′-CATCTGAAAAACAACGTCTACG-3′; β8 integrin anti-sense primer, 5′-ATCTGGACAGATGGCGGTAAT-3′; HGF sense primer 5′-CAGAGGGACAAAGGAAAAGAA-3′, HGF anti-sense primer 5′-GCAAGTGAATGGAAGTCCTTTA-3′; β-actin sense primer 5′-TGACGGGGTCACCCACACTGTGCC-3′, β-actin anti-sense primer 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′. These primer sets yielded PCR products of 306, 167, and 662 bp for β8 integrin, HGF,28Plantier L Marchand-Adam S Marchal-Somme J Leseche G Fournier M Dehoux M Aubier M Crestani B Defect of hepatocyte growth factor production by fibroblasts in human pulmonary emphysema.Am J Physiol. 2005; 288: L641-L647Google Scholar and β-actin, respectively. Confluent dishes of cells were surface-labeled with 0.1 mg/ml NHS-LC-biotin (Pierce Corp., Rockford, IL) and lysed in Tris-buffered saline with 1% Triton X-100, 1 mmol/L phenylmethyl sulfonyl fluoride, and protease inhibitor cocktail (Calbiochem, San Diego, CA). Immunoprecipitations and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as previously described.27Nishimura SL Sheppard D Pytela R Integrin αvβ8. Interaction with vitronectin and functional divergence of the β8 cytoplasmic domain.J Biol Chem. 1994; 269: 28708-28715Abstract Full Text PDF PubMed Google Scholar After transfer to polyvinylidene difluoride membrane (Immobilon-P; Amersham, Arlington Heights, IL), biotinylated proteins were detected by horseradish peroxidase-streptavidin conjugate (Amersham) followed by enhanced chemiluminescence (Amersham). Western blotting was performed as described with minor modification.19Cambier S Mu D O'Connell D Boylen K Travis W Liu W Broaddus VC Nishimura SL The integrin αvβ8 negatively regulates epithelial cell growth.Cancer Res. 2000; 60: 7084-7093PubMed Google Scholar Briefly, fibroblast-conditioned medium was used to treat fetal tracheal epithelial cells for 1 hour, followed by lysis in Tris-buffered saline with 1% Triton X-100, 1 mmol/L phenylmethyl sulfonyl fluoride, 5 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L sodium orthovanadate (Sigma), and protease inhibitor cocktail (Calbiochem). Zymography was performed as previously described.15Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar The TGF-β bioassays were performed as previously described.15Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar, 20Fjellbirkeland L Cambier S Broaddus VC Hill A Brunetta P Dolganov G Jablons D Nishimura SL Integrin αvβ8-mediated activation of transforming growth factor-β inhibits human airway epithelial proliferation in intact bronchial tissue.Am J Pathol. 2003; 163: 533-542Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar TMLC cells (the generous gift of John Munger and Daniel Rifkin, New York University, New York, NY)29Abe M Harpel JG Metz CN Nunes I Loskutoff DJ Rifkin DB An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.Anal Biochem. 1994; 216: 276-284Crossref PubMed Scopus (672) Google Scholar were cultured with fetal tracheal fragments, epithelial cells, or fibroblasts pretreated (3 hours) or not with 1 or 10 nmol/L phorbol 12-myristate-13-acetate (PMA, Sigma), anti-pan-TGF-β-blocking antibody, or isoform-specific TGF-β antibodies, anti-β8-specific antibody (clone 37E1), or pan-metalloprotease inhibitor GM6001 (Ryss Lab, Inc., Union City, CA). TGF-β standard curves were generated with a 1:2 dilution series of recombinant TGF-β added to the TMLC bioassay. Fetal tracheal fibroblasts were grown to confluence in 12-well tissue culture plates, washed two times with PBS, and then incubated in serum-free Dulbecco's modified Eagle's medium in the presence or absence of anti-pan-TGF-β-blocking antibody or anti-β8-specific antibody with or without recombinant active TGF-β1 for 24 hours. HGF was measured in conditioned media with an HGF Quantikine ELISA kit (R&D Systems). Fetal tracheal fibroblasts (10 × 104) were cultured for 12 to 16 hours on culture inserts (1 μm pore size; Fisher Scientific, Pittsburgh, PA) in a 24-well plate in fibroblast growth media. Fetal tracheal epithelial cells (5 × 104) were cultured for 12 to 16 hours on rat-tail collagen (10 μg/ml coating concentration)-coated wells of a 12-well tissue culture plate (Fisher Scientific, Pittsburg, PA) in 1 ml of bronchial epithelial growth medium (Clonetics). The culture inserts containing the fetal tracheal fibroblasts were placed into wells containing fetal tracheal epithelial cells, and the media in the upper and lower chambers was changed to Dulbecco's modified Eagle's medium with 0.1% fetal calf serum and penicillin and streptomycin in the presence or absence of neutralizing anti-pan-TGF-β, anti-β8, anti-HGF, anti-β8 plus anti-TGF-β, or a control nonfunction-blocking anti-αv integrin subunit antibody (8b815Mu D Cambier S Fjellbirkeland L Baron JL Munger JS Kawakatsu H Sheppard D Broaddus VC Nishimura SL The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (572) Google Scholar). Cell proliferation was determined by cell counting, as described.19Cambier S Mu D O'Connell D Boylen K Travis W Liu W Broaddus VC Nishimura SL The integrin αvβ8 negatively regulates epithelial cell growth.Cancer Res. 2000; 60: 7084-7093PubMed Google Scholar Cell adhesion assays were perfor
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