Proliferation of Pulmonary Interstitial Fibroblasts Is Mediated by Transforming Growth Factor-β1-induced Release of Extracellular Fibroblast Growth Factor-2 and Phosphorylation of p38 MAPK and JNK

Idiopathic pulmonary fibrosis (IPF; a progressive lung disease) is characterized by parenchymal remodeling with enlarged air spaces called honeycomb cysts and palisades of fibroblasts called fibroblast foci. In IPF, lung epithelial cells covering honeycomb cysts and fibroblast foci aberrantly express the active conformation of the potent fibrogenic cytokine transforming growth factor-β1 (TGF-β1). Using explanted rat lung slices, we transfected alveolar epithelial cells with the retrovirus pMX containing a site-directed mutation in which Cys223 and Cys225 were substituted with serines, resulting in release of biologically active TGF-β1 and fibroblast proliferation and remodeling that resembled IPF. Fibroblasts obtained from transfected explants and in culture for 6 weeks incorporated 6.59 ± 1.55-fold more [3H]thymidine compared with control fibroblasts without transfection or fibroblasts obtained from transfected explants cultured with antibody to fibroblast growth factor-2 (FGF-2). Primary lung fibroblasts obtained from normal rat lungs cultured with TGF-β1 expressed increased levels of phosphorylated p38 MAPK and JNK, but not ERK1/2. The presence of TGF-β1 caused an immediate release of extracellular FGF-2 from primary pulmonary fibroblasts; and in the presence of anti-FGF-2 antibody, phosphorylated p38 MAPK and JNK were abrogated. TGF-β inhibits cell proliferation by suppression of c-Myc and induction of p15INK46, p21CIP1, or p27KIP. Fibroblasts cultured with TGF-β1 showed no regulation of c-Myc or induction of p15INK46, p21CIP1,or p27KIP. These findings suggest that pulmonary fibroblasts may not respond to the anti-proliferative effects of TGF-β1, but proliferate in response to TGF-β1 indirectly by the release of FGF-2, which induces phosphorylation of p38 MAPK and JNK. Idiopathic pulmonary fibrosis (IPF; a progressive lung disease) is characterized by parenchymal remodeling with enlarged air spaces called honeycomb cysts and palisades of fibroblasts called fibroblast foci. In IPF, lung epithelial cells covering honeycomb cysts and fibroblast foci aberrantly express the active conformation of the potent fibrogenic cytokine transforming growth factor-β1 (TGF-β1). Using explanted rat lung slices, we transfected alveolar epithelial cells with the retrovirus pMX containing a site-directed mutation in which Cys223 and Cys225 were substituted with serines, resulting in release of biologically active TGF-β1 and fibroblast proliferation and remodeling that resembled IPF. Fibroblasts obtained from transfected explants and in culture for 6 weeks incorporated 6.59 ± 1.55-fold more [3H]thymidine compared with control fibroblasts without transfection or fibroblasts obtained from transfected explants cultured with antibody to fibroblast growth factor-2 (FGF-2). Primary lung fibroblasts obtained from normal rat lungs cultured with TGF-β1 expressed increased levels of phosphorylated p38 MAPK and JNK, but not ERK1/2. The presence of TGF-β1 caused an immediate release of extracellular FGF-2 from primary pulmonary fibroblasts; and in the presence of anti-FGF-2 antibody, phosphorylated p38 MAPK and JNK were abrogated. TGF-β inhibits cell proliferation by suppression of c-Myc and induction of p15INK46, p21CIP1, or p27KIP. Fibroblasts cultured with TGF-β1 showed no regulation of c-Myc or induction of p15INK46, p21CIP1,or p27KIP. These findings suggest that pulmonary fibroblasts may not respond to the anti-proliferative effects of TGF-β1, but proliferate in response to TGF-β1 indirectly by the release of FGF-2, which induces phosphorylation of p38 MAPK and JNK. The most common pulmonary fibrotic disorder is idiopathic pulmonary fibrosis (IPF), 2The abbreviations used are: IPFidiopathic pulmonary fibrosisECMextracellular matrixTGF-β1transforming growth factor-β1L-TGF-β1latent transforming growth factor-β1AECsalveolar epithelial cellsKGFkeratinocyte growth factorFGF-2fibroblast growth factor-2MAPKmitogen-activated protein kinaseJNKc-Jun N-terminal kinaseCKIscyclin-dependent kinase inhibitorsDMEMDulbecco's modified Eagle's mediumELISAenzyme-linked immunosorbent assayPDGFplatelet-derived growth factorIGF-1insulin-like growth factor-1SAPKstress-activated protein kinaseERK1/2extracellular signal-regulated kinase-1/2TCID50tissue culture infectious doseTβRtransforming growth factor-β1 receptorTBSTris-buffered saline. a progressive and lethal disease of unknown etiology and uncertain pathogenesis (1American Thoracic Society and European Respiratory Society Am. J. Respir. Crit. Care Med. 2000; 161: 646-664Crossref PubMed Scopus (997) Google Scholar). The incidence of IPF is ∼15–30 cases/100,000 persons/year (1American Thoracic Society and European Respiratory Society Am. J. Respir. Crit. Care Med. 2000; 161: 646-664Crossref PubMed Scopus (997) Google Scholar, 2Coultas D.B. Zumwalt R.E. Black W.C. Sobonya R.E. Am. J. Respir. Crit. Care Med. 1994; 150: 967-972Crossref PubMed Scopus (793) Google Scholar, 3Hansell A. Hollowell J. Nichols T. McNiece R. Strachan D. Thorax. 1999; 54: 413-419Crossref PubMed Scopus (142) Google Scholar). The histological findings in IPF are called usual interstitial pneumonia and are characterized by temporal heterogeneity (1American Thoracic Society and European Respiratory Society Am. J. Respir. Crit. Care Med. 2000; 161: 646-664Crossref PubMed Scopus (997) Google Scholar, 2Coultas D.B. Zumwalt R.E. Black W.C. Sobonya R.E. Am. J. Respir. Crit. Care Med. 1994; 150: 967-972Crossref PubMed Scopus (793) Google Scholar, 3Hansell A. Hollowell J. Nichols T. McNiece R. Strachan D. Thorax. 1999; 54: 413-419Crossref PubMed Scopus (142) Google Scholar, 4Katzenstein A.L. Zisman D.A. Litzky L.A. Nguyen B.T. Kotloff R.M. Am. J. Surg. Pathol. 2002; 26: 1567-1577Crossref PubMed Scopus (188) Google Scholar) in which the normal appearing lung is seen adjacent to interstitial fibrosis, honeycomb cysts that are distorted, and enlarged airspaces and fibroblast foci (1American Thoracic Society and European Respiratory Society Am. J. Respir. Crit. Care Med. 2000; 161: 646-664Crossref PubMed Scopus (997) Google Scholar, 4Katzenstein A.L. Zisman D.A. Litzky L.A. Nguyen B.T. Kotloff R.M. Am. J. Surg. Pathol. 2002; 26: 1567-1577Crossref PubMed Scopus (188) Google Scholar). Fibroblast foci are small aggregates of actively proliferating fibroblasts and myofibroblasts that are surrounded by the extracellular matrix (ECM) (1American Thoracic Society and European Respiratory Society Am. J. Respir. Crit. Care Med. 2000; 161: 646-664Crossref PubMed Scopus (997) Google Scholar, 4Katzenstein A.L. Zisman D.A. Litzky L.A. Nguyen B.T. Kotloff R.M. Am. J. Surg. Pathol. 2002; 26: 1567-1577Crossref PubMed Scopus (188) Google Scholar). In IPF, fibroblast foci are widely dispersed, and their numbers correlate with worsening lung function, progression of disease, and poor prognosis (5Flaherty K.R. Colby T.V. Travis W.D. Toews G.B. Mumford J. Murray S. Thannickal V.J. Kazerooni E.A. Gross B.H. Lynch J.P. Martinez F.J. Am. J. Respir. Crit. Care Med. 2003; 167: 1410-1415Crossref PubMed Scopus (225) Google Scholar, 6Flaherty K.R. Thwaite E.L. Kazerooni E.A. Gross B.H. Toews G.B. Colby T.V. Travis W.D. Mumford J.A. Murray S. Flint A. Lynch J.P. Martinez F.J. Thorax. 2003; 58: 143-148Crossref PubMed Scopus (459) Google Scholar). idiopathic pulmonary fibrosis extracellular matrix transforming growth factor-β1 latent transforming growth factor-β1 alveolar epithelial cells keratinocyte growth factor fibroblast growth factor-2 mitogen-activated protein kinase c-Jun N-terminal kinase cyclin-dependent kinase inhibitors Dulbecco's modified Eagle's medium enzyme-linked immunosorbent assay platelet-derived growth factor insulin-like growth factor-1 stress-activated protein kinase extracellular signal-regulated kinase-1/2 tissue culture infectious dose transforming growth factor-β1 receptor Tris-buffered saline. Using lung sections from patients with IPF, we previously demonstrated by immunohistochemistry that the biologically active conformation of transforming growth factor-β1 (TGF-β1), a potent fibrogenic cytokine, is expressed in epithelial cells lining honeycomb cysts in areas of advanced disease and remodeling (7Khalil N. O'Connor R.N. Flanders K.C. Unruh H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 131-138Crossref PubMed Scopus (283) Google Scholar, 8Khalil N. O'Connor R.N. Unruh H.W. Warren P.W. Flanders K.C. Kemp A. Bereznay O.H. Greenberg A.H. Am. J. Respir. Cell Mol. Biol. 1991; 5: 155-162Crossref PubMed Scopus (448) Google Scholar, 9Khalil N. Parekh T.V. O'Connor R. Antman N. Kepron W. Yehaulaeshet T. Xu Y.D. Gold L.I. Thorax. 2001; 56: 907-915Crossref PubMed Scopus (142) Google Scholar). In regions of the lung where fibroblast foci are present, the biologically active conformation of TGF-β1 is observed in the hyperplastic pneumocytes overlying the fibroblast foci (7Khalil N. O'Connor R.N. Flanders K.C. Unruh H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 131-138Crossref PubMed Scopus (283) Google Scholar, 8Khalil N. O'Connor R.N. Unruh H.W. Warren P.W. Flanders K.C. Kemp A. Bereznay O.H. Greenberg A.H. Am. J. Respir. Cell Mol. Biol. 1991; 5: 155-162Crossref PubMed Scopus (448) Google Scholar). Alveolar epithelial cells (AECs) in lung sections with no evidence of inflammation or fibrosis do not express the active conformation of TGF-β1. Based on our observations in IPF lungs demonstrating that AECs that line areas of fibrosis and fibroblast foci aberrantly express biologically active TGF-β1, we designed an in vitro model to determine the role of AEC-derived TGF-β1 in interstitial pulmonary fibrosis and parenchymal remodeling (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). In this model, we used slices of normal rat lungs, which were free of inflammatory cells. The AECs in the explanted lung slices were successfully transfected with the retrovirus pMX carrying an insert of TGF-β1 designated as L-s223,225-TGF-β1 (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). The L-s223,225-TGF-β1 cDNA contains a mutation in the TGF-β1 gene in which Cys223 and Cys225 have been substituted with serines (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). This mutation results in constitutive release of biologically active TGF-β1 (23Sakamoto R. Nitta T. Kamikawa Y. Kono S. Sugihara K. Tsuyama S. Murata F. Med. Electron Microsc. 2002; 35: 248-254Crossref PubMed Scopus (23) Google Scholar). Retroviruses such as pMX enter only actively proliferating cells (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). To selectively transfect the AECs of the explant, the explants were treated with keratinocyte growth factor (KGF), a potent AEC mitogen (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). In situ hybridization using a digoxigenin-labeled probe of the puromycin resistance gene contained in the pMX retrovirus confirmed that, in the presence of KGF, only AECs of the explant were transfected with the vector (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). In lung explants in which the AECs were successfully transfected with the L-s223,225-TGF-β1 gene, there was release of active TGF-β1 and extensive fibrosis with lesions that resembled fibroblast foci and honeycomb cysts (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). The main sources of connective tissue proteins are fibroblasts and myofibroblasts (11Eckes B. Zigrino P. Kessler D. Holtkotter O. Shephard P. Mauch C. Krieg T. Matrix Biol. 2000; 19: 325-332Crossref PubMed Scopus (201) Google Scholar), which are increased in pMX-L-s223,225-TGF-β1-transfected explants (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). These findings suggest that the release of active TGF-β1 by AECs in this model directly or indirectly regulates the proliferation of interstitial pulmonary fibroblasts. Because the appearance of fibroblasts (especially in fibroblast foci) is associated with the pathogenesis of IPF and correlates with progression of fibrosis in IPF (5Flaherty K.R. Colby T.V. Travis W.D. Toews G.B. Mumford J. Murray S. Thannickal V.J. Kazerooni E.A. Gross B.H. Lynch J.P. Martinez F.J. Am. J. Respir. Crit. Care Med. 2003; 167: 1410-1415Crossref PubMed Scopus (225) Google Scholar, 6Flaherty K.R. Thwaite E.L. Kazerooni E.A. Gross B.H. Toews G.B. Colby T.V. Travis W.D. Mumford J.A. Murray S. Flint A. Lynch J.P. Martinez F.J. Thorax. 2003; 58: 143-148Crossref PubMed Scopus (459) Google Scholar), the current model provides an opportunity to determine the mechanism by which interstitial fibroblasts increase in number in response to AEC-derived TGF-β1 (7Khalil N. O'Connor R.N. Flanders K.C. Unruh H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 131-138Crossref PubMed Scopus (283) Google Scholar, 8Khalil N. O'Connor R.N. Unruh H.W. Warren P.W. Flanders K.C. Kemp A. Bereznay O.H. Greenberg A.H. Am. J. Respir. Cell Mol. Biol. 1991; 5: 155-162Crossref PubMed Scopus (448) Google Scholar, 9Khalil N. Parekh T.V. O'Connor R. Antman N. Kepron W. Yehaulaeshet T. Xu Y.D. Gold L.I. Thorax. 2001; 56: 907-915Crossref PubMed Scopus (142) Google Scholar, 10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). In this study, we demonstrate that fibroblasts obtained from lung tissue of explants with successful transfection of AECs with pMX-L-s223,225-TGF-β1 compared with control fibroblasts proliferated actively. However, when fibroblasts were obtained from transfected explants that were cultured with antibody to TGF-β1 or fibroblast growth factor-2 (FGF-2), DNA synthesis was decreased. Primary lung fibroblasts obtained from normal rat lungs cultured with TGF-β1 caused an almost immediate release of extracellular FGF-2 and expression of increased quantities of phosphorylated p38 MAPK and JNK; but in the presence of antibody to FGF-2, phosphorylation of p38 MAPK and JNK was totally abrogated. TGF-β inhibited proliferation of epithelial and hematopoietic cells by suppression of c-Myc and induction of cyclin-dependent kinase inhibitors (CKIs) p15INK46, p21CIP1, and p27KIP. Fibroblasts cultured with TGF-β1 showed no regulation of c-Myc, p15INK46, p21CIP1, or p27KIP. These findings suggest that TGF-β1 regulates the proliferation of pulmonary fibroblasts indirectly by the release of FGF-2, which, by induction of phosphorylation of p38 MAPK and JNK, leads to fibroblast proliferation. Furthermore, fibroblasts do not respond to the anti-proliferative effects of TGF-β1 by suppression of c-Myc and induction of CKIs p15INK46, p21CIP1, and p27KIP. Materials—Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum were purchased from Invitrogen (Burlington, Ontario, Canada). Agarose, hydrocortisone, retinol acetate, insulin-transferrin-sodium selenium, and fetuin were purchased from Sigma. Recombinant KGF/FGF-7, the TGF-β1 enzyme-linked immunosorbent assay (ELISA) kit, porcine TGF-β1, anti-FGF-2 antibody, anti-human platelet-derived growth factor (PDGF) antibody, anti-human insulin-like growth factor-1 (IGF-1) antibody, and neutralizing anti-TGF-β1 antibody were purchased from R&D Systems (Minneapolis, MN). Antibodies to detect total p38 MAPK, SAPK/JNK, and ERK1/2 were purchased from Stressgen (Victoria, British Columbia, Canada), New England Biolabs Inc. (Pickering, Ontario), and Upstate Cell Signaling Solutions (Charlottesville, VA), respectively. Anti-phospho-Thr183/Tyr185 SAPK/JNK, anti-phospho-Thr180/Tyr182 p38 MAPK, anti-phospho-Thr202/Tyr204 p44/42 MAPK, and anti-phospho-ERK1/2 (E10) antibodies were purchased from Cell Signaling (Beverly, MA). Anti-c-Myc antibody 9E10 and anti-p15INK46 antibody K-18 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p21CIP1 and anti-p27KIP antibodies were purchased from Pharmingen (Mississauga, Ontario). Anti-vimentin and biotinylated, rat-absorbed rabbit anti-mouse antibodies were purchased from Dako Corp. (Carpinteria, CA). The L2, C3H/10T1/2, and NIH/3T3 cell lines were obtained from American Type Culture Collection (Manassas, VA). Preparation of the Retroviral Vector—pPK9A (a gift from Dr. Lalage Wakefield, Laboratory of Chemoprevention, National Institutes of Health, Bethesda, MD) is a plasmid that contains the entire length of the cDNA of TGF-β1 with Cys223 and Cys225 substituted with serines (12Brunner A.M. Marquardt H. Malacko A.R. Lioubin M.N. Purchio A.F. J. Biol. Chem. 1989; 264: 13660-13664Abstract Full Text PDF PubMed Google Scholar). The substitution of cysteines with serines at these positions results in secretion of TGF-β1 in its biologically active form (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar, 12Brunner A.M. Marquardt H. Malacko A.R. Lioubin M.N. Purchio A.F. J. Biol. Chem. 1989; 264: 13660-13664Abstract Full Text PDF PubMed Google Scholar), and this cDNA was named L-s223,225-TGF-β1 (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). Using BglII digestion, the 1.2-kb TGF-β1 was isolated and purified with a DNA gel extraction kit (Qiagen Inc., Mississauga) (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). L-s223,225-TGF-β1 was subcloned into the retroviral vector pMX (a gift from Dr. Alice Mui, Department of Surgery, University of British Columbia) (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). The pMX retrovirus with no TGF-β1 cDNA was designated as pMX, whereas the retrovirus containing the TGF-β1 gene was designated as pMX-L-s223,225-TGF-β1 (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). The retroviruses were produced using the packaging cell line Plat-E, followed by quantitation based on the number of infected NIH/3T3 cells (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). Preparation of Normal Rat Lung Slices—Female Sprague-Dawley rats that were free of respiratory disease and that weighed 200–250 g were purchased from the University of British Columbia Vivarium. Intraperitoneal administration of 0.4 ml of ketamine (Biomedia-MTC, Cambridge, Ontario) and 0.2 ml of Rompun (Bayer, Etobicoke, Ontario) was used for euthanization. The trachea was exposed, and an 18-gauge catheter was inserted through the cartilaginous rings. After exposure of the thoracic and abdominal cavities, the inferior vena cava and abdominal aorta were severed. The peripheral blood was removed with 10 ml of normal saline injected into the right ventricle until the lungs turned white. The trachea and lungs were taken out. To remove alveolar cells, the lungs were lavaged with 50–60 ml of warm normal saline through the trachea and then infused with 5 ml of 0.4% agarose/DMEM (2× solution of 1:1 serum-free DMEM and 0.8% agarose (Invitrogen)) at 40 °C. The medium was supplemented with hydrocortisone (0.2 μg/ml), retinol acetate (0.2 μg/ml), and 0.02% insulin-transferrin-sodium selenium. A thread was used to tie the tracheas, and lungs were placed on culture dishes on ice overnight to further solidify the lungs. The lungs were sliced manually at 1–2-mm thickness from each lobe with a sterilized scalpel (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). In some instances, alveolar macrophages were isolated from the lavage fluid, cultured as described previously (7Khalil N. O'Connor R.N. Flanders K.C. Unruh H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 131-138Crossref PubMed Scopus (283) Google Scholar), and stained with vimentin. Culture of Rat Lung Slices—1.5 ml of warm 0.4% agarose/DMEM was added to each 6-well plate. After the agarose/DMEM solidified, four to six lung slices were placed on top of the agarose, and 1.5 ml of serum-free DMEM supplemented with hydrocortisone (0.2 μg/ml), retinol acetate (0.2 μg/ml), and 0.02% insulin-transferrin-sodium selenium was added. The lung slices were incubated at 37 °C under 5% CO2. The medium was changed twice each week, and lung slices were turned every other day and collected after culture. Treatment of the lung slices consisted of medium, KGF (25 ng/ml), pMX (106 TCID50), or pMX-L-s223,225-TGF-β1 (106 TCID50) + KGF (25 ng/ml) in the absence or presence of neutralizing anti-TGF-β1 antibody (0.1 μg/ml), fetuin (10 μm), anti-FGF-2 antibody (4.5 μg/ml), anti-IGF-1 antibody (5 μg/ml), or anti-PDGF antibody (5 μg/ml). Fetuin was used under some conditions because, as a glycoprotein, it can associate with TGF-β1 and prevent TGF-β1 from binding to TGF-β1 receptor (TβR) II and mediating a signal (13Demetriou M. Binkert C. Sukhu B. Tenenbaum H.C. Dennis J.W. J. Biol. Chem. 1996; 271: 12755-12761Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). KGF was used because retroviruses transfect only proliferating cells and because it is an AEC mitogen that induces only AEC proliferation in lung explants (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar, 14Wang G. Slepushkin V.A. Bodner M. Zabner J. van Es H.H.G Thomas P. Jolly D.J. Davidson B.L. McCray P.B. J. Gene Med. 1999; 1: 22-30Crossref PubMed Scopus (27) Google Scholar). Quantitation of TGF-β1 by ELISA—The TGF-β1 present in the conditioned medium was quantitated using an ELISA kit designed to detect only biologically active TGF-β1 that is not associated with lamin-associated polypeptide-1 (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar, 15Chen G. Grotendorst G. Eichholtz T. Khalil N. Am. J. Physiol. 2003; 284: L548-L556PubMed Google Scholar, 16Chen G. Khalil N. Respir. Physiol. Neurobiol. 2002; 132: 341-346Crossref PubMed Scopus (23) Google Scholar). To determine the quantity of the total TGF-β1 in the sample, an aliquot of the conditioned media was acidified to remove latency-associated polypeptide-1 from any latent TGF-β1 (L-TGF-β1) present in the conditioned medium. The same conditioned medium was neutralized and used in the ELISA to determine the total TGF-β1 present in the sample. Preparation of Fibroblast Cultures—In the explant model, the release of TGF-β1 in the conditioned medium overlying the explants was detected in maximal quantities 7 days after transfection, but was barely detectable 14 days after transfection and was not detected in the conditioned medium 21 and 28 days after transfection (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). After 14 days, the explant lung tissue demonstrated maximal fibroblast and myofibroblast numbers and connective tissue synthesis, suggesting that, 14 days after the release of TGF-β1, there is a maximal expansion of fibroblasts (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). For this reason, it was expected that the isolation of fibroblasts from the explant 14 days after transfection of AECs was likely to be the ideal time interval from the release of TGF-β1 to obtain fibroblasts. 14 days after transfection, the explanted lung slices were cut manually into small pieces (∼0.5 mm3), placed on culture dishes, and cultured with DMEM in the presence of 10% fetal calf serum, 100 unit/ml penicillin, and 100 μg/ml streptomycin. Fibroblasts were observed to grow out from the explant 7 days later. At this time, the pieces of lung explant were discarded, and the fibroblasts were obtained by trypsinization and plated in equal numbers on culture dishes for 6 weeks. The control fibroblasts were obtained from lung explants cultured in medium, pMX with no TGF-β1 insert, or KGF. In some instances, fibroblasts were obtained from explants transfected with pMX-L-s223,225-TGF-β1 but also cultured with fetuin (10 μm) or antibody to TGF-β1 (0.1 μg/ml), FGF-2 (4.5 μg/ml), IGF-1 (5 μg/ml), or PDGF (5 μg/ml). For some experiments, fibroblasts from untreated explants were cultured with TGF-β1 (10 ng/ml) in the absence or presence of anti-FGF-2 (4.5 μg/ml), anti-IGF-1 (5 μg/ml), or anti-PDGF (5 μg/ml) antibody or an inhibitor of MAPK activation (SB203580 (10 μm), PD98059 (40 μm), or SP60025 (20 μm)). The fibroblasts were then used for [3H]thymidine incorporation or Western analysis. Vimentin Staining of Rat Lung Tissue and Fibroblasts in Culture—Rat lung tissue pieces and/or fibroblasts were fixed in -10 °C methanol. After air-drying, the cells and/or lung sections were incubated with 3% H2O2 in methanol and then washed twice with Tris-buffered saline (TBS)/Tween. The slides were incubated with universal blocking solution and incubated with anti-vimentin antibody (1:80 dilution) for 30 min. The slides were washed twice with TBS/Tween, incubated with biotinylated, rat-absorbed rabbit anti-mouse antibody (1:300 dilution) for 20 min, and rinsed in TBS/Tween. Horseradish peroxidase-conjugated streptavidin was applied to lung tissue pieces and/or fibroblasts for 20 min, washed with TBS/Tween, and incubated with 3,3′-diaminobenzidine chromogen for 5 min. The slides were rinsed in distilled water, counterstained with hematoxylin or stained with hematoxylin and eosin, and mounted in aqueous permanent mounting medium. Extraction and Quantitation of FGF-2—To release and quantitate extracellular FGF-2 in fibroblast cultures, the fibroblasts were washed with DMEM and then incubated with 0.25 ml of 20 mm Tris-HCl (pH 7.2) and 2 m NaCl for 2 min (17Murakami-Mori K. Mori S. Nakamura S. J. Immunol. 1998; 161: 1694-1704PubMed Google Scholar). The FGF-2 in this medium was quantitated using an FGF-2 detection ELISA kit (R&D Systems) according to the manufacturer's instructions. The fibroblasts remaining were washed with DMEM, lysed with triple-detergent lysis buffer (50 mm Tris-HCl (pH 8.0), 0.15 m NaCl, 1% Triton X-100, 0.1% SDS, and 5 mg/ml sodium deoxycholate) in the presence of 1 μg/ml aprotinin, 1 μg/ml leupeptin, 5 μg/ml pepstatin A, 1 mm PMSF, and phosphatase inhibitors (1 mm NaF and 1 mm Na3VO4) (all from Sigma). Cell lysates were centrifuged at 12,000 rpm for 20 min, and the supernatant was used to quantitate FGF-2 in the same ELISA as described above. [3H]Thymidine Incorporation—To determine fibroblast DNA synthesis, [3H]thymidine incorporation was used. 6 weeks after culture, the fibroblasts were cultured in serum-free DMEM cell concentration of 2 × 104/ml for 24 h. [3H]Thymidine was added to the cell cultures at 0.5 μCi/ml for 24 h, and the fibroblasts were harvested. The degradations/min were measured and used as an index of DNA synthesis and fibroblast proliferation. Expression of c-Myc, p15INK46, p21CIP1, or p27KIP by Fibroblast Cell Lines and L2 Cells—To determine whether fibroblasts other than primary interstitial fibroblasts respond to TGF-β1 by regulation of c-Myc, p15INK46, p21CIP1, or p27KIP, C3H/10T1/2 and NIH/3T3 fibroblasts were cultured with TGF-β1 in the absence or presence of neutralizing anti-FGF-2 antibody. Because the regulation of c-Myc, p15INK46, p21CIP1, or p27KIP has classically been described for epithelial cells, L2 cells, an AEC line (18Hoffmann G. Grote J. Friedrich F. Mutz N. Schobersberger W. Biochem. Biophys. Res. Commun. 1995; 217: 575-583Crossref PubMed Scopus (24) Google Scholar), were cultured with TGF-β1 in the absence or presence of neutralizing anti-FGF-2 antibody in the same manner and used to determine the expression of c-Myc, p15INK46, p21CIP1,orp27KIP, and the results were compared with those obtained with the fibroblast cell lines. Western Analysis to Detect and Quantitate Total and Phosphorylated p38 MAPK, ERK1/2, and JNK and c-Myc, p15INK46, p21CIP1, and p27KIP — The fibroblasts remaining after collection of the conditioned medium were washed with phosphate-buffered saline and detached by trypsinization (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar). Whole cell protein was extracted with triple-detergent lysis buffer. The protein levels of each sample were determined using a Bio-Rad protein assay. Samples of protein (25 μg) were electrophoresed on a 10% SDS-polyacrylamide gel in a Mini-Protean II electrophoresis cell (Bio-Rad). Protein molecular weight markers (Amersham Biosciences) were run parallel to each blot as an indicator of the molecular weight. Equality of protein loading was evaluated as described (10Xu Y.D. Hua J. Mui A. O'Connor R. Grotendorst G. Khalil N. Am. J. Physiol. 2003; 285: L527-L539Crossref PubMed Scopus (143) Google Scholar) using silver stain (data not shown), Ponceau S staining solution (Sigma) (data not shown), or Coomassie Brilliant Blue (Sigma) (data not shown). In some instances, equality of protein loading, cell viability, a