Pathological Alteration of FoxO3a Activity Promotes Idiopathic Pulmonary Fibrosis Fibroblast Proliferation on Type I Collagen Matrix
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.07.020
ISSN1525-2191
AutoresRichard Nho, Polla Hergert, J Kahm, José Jessurun, Craig A. Henke,
Tópico(s)Biomarkers in Disease Mechanisms
ResumoIdiopathic pulmonary fibrosis (IPF) is a prevalent, progressive, and incurable fibroproliferative lung disease. The phenotype of IPF fibroblasts is characterized by their ability to elude the proliferation-suppressive properties of polymerized type I collagen. The mechanism underlying this pathological response is incompletely understood but involves aberrant activation of the phosphatidylinositol 3-kinase–Akt signaling pathway owing to inappropriately low phosphatase and tensin homolog phosphatase activity. Akt can phosphorylate and inactivate the forkhead box O3a (FoxO3a) transcriptional factor, which, when transcriptionally active, increases the expression of the CDK inhibitor p27 and promotes cell cycle arrest. Herein, we demonstrate that IPF fibroblasts display high levels of inactive FoxO3a compared with nonfibrotic control fibroblasts because of their high Akt activity. We found that p27 levels are decreased in IPF compared with control fibroblasts cultured on polymerized collagen. Furthermore, overexpression of FoxO3a in IPF fibroblasts increases p27 levels and suppresses the ability of IPF fibroblasts to proliferate on polymerized collagen. In contrast, the expression of dominant-negative FoxO3a augmented control fibroblast proliferation. IHC examination of fibroblastic foci in IPF lung tissue demonstrates the presence of inactive FoxO3a in cells within fibroblastic foci. These data indicate that the ability of IPF fibroblasts to circumvent the proliferation-suppressive properties of polymerized collagen involves inactivation of FoxO3a by high Akt activity, resulting in down-regulation of p27. Idiopathic pulmonary fibrosis (IPF) is a prevalent, progressive, and incurable fibroproliferative lung disease. The phenotype of IPF fibroblasts is characterized by their ability to elude the proliferation-suppressive properties of polymerized type I collagen. The mechanism underlying this pathological response is incompletely understood but involves aberrant activation of the phosphatidylinositol 3-kinase–Akt signaling pathway owing to inappropriately low phosphatase and tensin homolog phosphatase activity. Akt can phosphorylate and inactivate the forkhead box O3a (FoxO3a) transcriptional factor, which, when transcriptionally active, increases the expression of the CDK inhibitor p27 and promotes cell cycle arrest. Herein, we demonstrate that IPF fibroblasts display high levels of inactive FoxO3a compared with nonfibrotic control fibroblasts because of their high Akt activity. We found that p27 levels are decreased in IPF compared with control fibroblasts cultured on polymerized collagen. Furthermore, overexpression of FoxO3a in IPF fibroblasts increases p27 levels and suppresses the ability of IPF fibroblasts to proliferate on polymerized collagen. In contrast, the expression of dominant-negative FoxO3a augmented control fibroblast proliferation. IHC examination of fibroblastic foci in IPF lung tissue demonstrates the presence of inactive FoxO3a in cells within fibroblastic foci. These data indicate that the ability of IPF fibroblasts to circumvent the proliferation-suppressive properties of polymerized collagen involves inactivation of FoxO3a by high Akt activity, resulting in down-regulation of p27. Idiopathic pulmonary fibrosis (IPF) is a condition in which the lung parenchyma becomes progressively scarred over time. Consequently, this pathological condition, this distorted pulmonary architecture, severely disrupts lung function, often with fatal consequences.1Ryu J.H. Colby T.V. Hartman T.E. Idiopathic pulmonary fibrosis: current concepts.Mayo Clin Proc. 1998; 73: 1085-1101Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 2Gharaee-Kermani M. Gyetko M.R. Hu B. Phan S.H. Pharm: new insights into the pathogenesis and treatment of idiopathic pulmonary fibrosis: a potential role for stem cells in the lung parenchyma and implications for therapy.Pharm Res. 2007; 24: 819-841Crossref PubMed Scopus (95) Google Scholar However, the pathogenesis of this deadly disease is not well understood and treatment to cure IPF is not available. During tissue repair, normal lung fibroblasts sense type I collagen matrix via integrins and the induction of key integrin-mediated signaling pathways suppress fibroblast proliferation, thus limiting the fibroblast response.3Kuzuya M. Satake S. Ramos M.A. Kanda S. Koike T. Yoshino K. Ikeda S. Iguchi A. Induction of apoptotic cell death in vascular endothelial cells cultured in three-dimensional collagen lattice.Exp Cell Res. 1999; 248: 498-508Crossref PubMed Scopus (57) Google Scholar, 4Xia H. Nho R.S. Kahm J. Kleidon J. Henke C.A. Fibroblast survival in response to contraction of type I collagen matrices via a beta 1 integrin viability signaling pathway.J Biol Chem. 2004; 279: 33024-33034Crossref PubMed Scopus (133) Google Scholar, 5Nho R.S. Xia H. Kahm J. Kleidon J. Diebold D. Henke C.A. Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway.J Biol Chem. 2005; 280: 26630-26639Crossref PubMed Scopus (81) Google Scholar Our prior studies6Nho R.S. Xia H. Diebold D. Kahm J. Kleidon J. White E. Henke C.A. PTEN regulates fibroblast elimination during collagen matrix contraction.J Biol Chem. 2006; 281: 33291-33301Crossref PubMed Scopus (42) Google Scholar, 7Xia H. Nho R.S. Kahm J. Kleidon J. Henke C.A. Focal adhesion kinase is upstream of phosphatidylinositol 3-kinase/Akt in regulating fibroblast survival in response to contraction of type I collagen matrices via a beta 1 integrin viability signaling pathway.J Biol Chem. 2004; 279: 33024-33034Crossref PubMed Scopus (278) Google Scholar demonstrate that, when normal lung fibroblasts attach to polymerized collagen via β1 integrin, phosphatase and tensin homolog (PTEN) activity increases, inhibiting phosphatidylinositol 3-kinase (PI3K)–Akt function. In contrast, IPF fibroblasts have low PTEN activity, rendering inappropriately high PI3K-Akt activity on collagen.8Xia H. Diebold D. Nho R. Perlman D. Kleidon J. Kahm J. Avdulov S. Peterson M. Nerva J. Bitterman P. Henke C. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (175) Google Scholar, 9Xia H. Khalil W. Kahm J. Jessurun J. Kleidon J. Henke C.A. Pathologic caveolin-1 regulation of PTEN in idiopathic pulmonary fibrosis.Am J Pathol. 2010; 176: 2626-2637Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar Thus, the IPF fibroblast phenotype is characterized by an aberrant integrin-mediated signaling pathway, enabling these cells to elude the proliferation-suppressing properties of polymerized collagen. Forkhead box O3a (FoxO3a) is a transcription factor that activates the CDK inhibitor protein p27. Up-regulation of FoxO3a activity increases p27 expression, which promotes cell cycle inhibition.10Nakao T. Geddis A.E. Fox N.E. Kaushansky K. PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27(Kip1).Cell Cycle. 2008; 7: 257-266Crossref PubMed Scopus (67) Google Scholar, 11Zhao Y. Fei M. Wang Y. Lu M. Cheng C. Shen A. Expression of Foxo3a in non-Hodgkin's lymphomas is correlated with cell cycle inhibitor p27.Eur J Haematol. 2008; 81: 83-93Crossref PubMed Scopus (14) Google Scholar, 12Li C.J. Chang J.K. Chou C.H. Wang G.J. Ho M.L. The PI3K/Akt/FOXO3a/p27Kip1 signaling contributes to anti-inflammatory drug-suppressed proliferation of human osteoblasts.Biochem Pharmacol. 2010; 79: 926-937Crossref PubMed Scopus (34) Google Scholar Seminal studies13Castrillon D.H. Miao L. Kollipara R. Horner J.W. DePinho R.A. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a.Science. 2003; 301: 215-218Crossref PubMed Scopus (724) Google Scholar, 14Mikse O.R. Blake Jr, D.C. Jones N.R. Sun Y.W. Amin S. Gallagher C.J. Lazarus P. Weisz J. Herzog C.R. FOXO3 encodes a carcinogen-activated transcription factor frequently deleted in early-stage lung adenocarcinoma.Cancer Res. 2010; 70: 6205-6215Crossref PubMed Scopus (33) Google Scholar, 15Sunters A. Fernández de Mattos S. Stahl M. Brosens J.J. Zoumpoulidou G. Saunders C.A. Coffer P.J. Medema R.H. Coombes R.C. Lam E.W. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines.J Biol Chem. 2003; 278: 49795-49805Crossref PubMed Scopus (433) Google Scholar have shown that FoxO3a activity is abnormally suppressed in several cancers. These observations link FoxO3a function to human disease. Several upstream proteins regulate FoxO3a activity by phosphorylating many of its Ser-Thr residues.16Chapuis N. Park S. Leotoing L. Tamburini J. Verdier F. Bardet V. Green A.S. Willems L. Agou F. Ifrah N. Dreyfus F. Bismuth G. Baud V. Lacombe C. Mayeux P. Bouscary D. IκB kinase overcomes PI3K/Akt and ERK/MAPK to control FOXO3a activity in acute myeloid leukemia.Blood. 2010; 116: 4240-4250Crossref PubMed Scopus (66) Google Scholar, 17Brunet A. Park J. Tran H. Hu L.S. Hemmings B.A. Greenberg M.E. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a).Mol Cell Biol. 2001; 21: 952-965Crossref PubMed Scopus (711) Google Scholar, 18Nho R.S. Kahm J. β1-Integrin-collagen interaction suppresses FoxO3a by the coordination of Akt and PP2A.J Biol Chem. 2010; 285: 14195-14209Crossref PubMed Scopus (20) Google Scholar Among them, Akt is an important protein kinase in regulating its activity by phosphorylating a crucial Ser253 residue.19Zheng W.H. Kar S. Quirion R. Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells.J Biol Chem. 2000; 275: 39152-39158Crossref PubMed Scopus (132) Google Scholar, 20Yusuf L. Zhu X. Kharas M.G. Chen J. Fruman D.A. Optimal B-cell proliferation requires phosphoinositide 3-kinase-dependent inactivation of FOXO transcription factors.Blood. 2004; 104: 784-787Crossref PubMed Scopus (114) Google Scholar Phosphorylation of FoxO3a by Akt retains FoxO3a in the cytoplasm, thereby inhibiting its activity.21Chen Y.R. Liu M.T. Chang Y.T. Wu C.C. Hu C.Y. Chen J.Y. Epstein-Barr virus latent membrane protein 1 represses DNA repair through the PI3K/Akt/FOXO3a pathway in human epithelial cells.J Virol. 2008; 82: 8124-8137Crossref PubMed Scopus (45) Google Scholar, 22Nakae J. Barr V. Accili D. Differential regulation of gene expression by insulin and IGF-1 receptors correlates with phosphorylation of a single amino acid residue in the forkhead transcription factor FKHR.EMBO J. 2000; 19: 989-996Crossref PubMed Scopus (254) Google Scholar, 23Tang E.D. Nuñez G. Barr F.G. Guan K.L. Negative regulation of the forkhead transcription factor FKHR by Akt.J Biol Chem. 1999; 274: 16741-16746Crossref PubMed Scopus (662) Google Scholar We found that β1 integrin–collagen interaction suppresses FoxO3a function in a PTEN–PI3K–Akt–dependent fashion.18Nho R.S. Kahm J. β1-Integrin-collagen interaction suppresses FoxO3a by the coordination of Akt and PP2A.J Biol Chem. 2010; 285: 14195-14209Crossref PubMed Scopus (20) Google Scholar Herein, we demonstrate that, in IPF fibroblasts, the FoxO3a transcription factor, which inhibits cell cycle progression by up-regulating the cell cycle inhibitor p27, is largely inactive. We show that inappropriately high Akt activity owing to low PTEN function phosphorylates and inactivates FoxO3a in IPF fibroblasts. We found that, in IPF fibroblasts, inactive FoxO3a is associated with low p27 levels. More important, we demonstrate that this inappropriately high Akt activity inactivates FoxO3a and confers IPF fibroblasts with the ability to circumvent the proliferation-suppressive effects of polymerized collagen. Eight control and seven IPF primary fibroblast lines were established and analyzed for this study. Cell lines were derived from lungs removed at transplantation or death. The diagnosis of IPF was supported by medical history, physical examination findings, pulmonary function test results, and typical high-resolution chest computed tomography findings of IPF. In all cases, the diagnosis of IPF was confirmed by microscopic analysis of lung tissue and demonstrated the characteristic morphological findings of usual interstitial pneumonia. All patients fulfilled the criteria for the diagnosis of IPF as established by the American Thoracic Society and the European Respiratory Society. Eight control primary adult human lung fibroblast lines were established from histologically normal lung tissue adjacent to carcinoid tumor or adjacent to radiation-induced fibrotic lung tissue. Primary lung fibroblast lines were generated by explant culture and cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. Fibroblasts were used between passages five and seven. Cells were characterized as fibroblasts as previously described.8Xia H. Diebold D. Nho R. Perlman D. Kleidon J. Kahm J. Avdulov S. Peterson M. Nerva J. Bitterman P. Henke C. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (175) Google Scholar Use of human tissues was approved by the Institutional Review Board at the University of Minnesota, Minneapolis. FoxO3a−/− mouse embryonic fibroblasts were obtained from Dr. Noboru Motoyama (National Center for Geriatrics and Gerontology, Obu, Aichi, Japan). PTEN−/− and PTEN+/+ mouse fibroblasts were provided by Deane F. Mosher (University of Wisconsin–Madison). These cells were maintained in DMEM plus 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Type I collagen solution (Vitrogen 1000) was obtained from Cohesion (Palo Alto, CA). Three-dimensional polymerized collagen matrices (final concentration, 2 mg/mL) were prepared by neutralizing the collagen solution with a one-sixth volume of six times DMEM medium, diluting to a final volume with one times DMEM, and incubating the solution at 37°C for 3 to 4 hours before use.18Nho R.S. Kahm J. β1-Integrin-collagen interaction suppresses FoxO3a by the coordination of Akt and PP2A.J Biol Chem. 2010; 285: 14195-14209Crossref PubMed Scopus (20) Google Scholar For Western blot analysis, anti-PTEN antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Akt antibody was obtained from Cell Signaling Technologies (Danvers, MA). FoxO3a and phosphorylated FoxO3a (p-FoxO3a; Ser253) antibodies were obtained from Millipore (Billerica, MA) and Cell Signaling Technologies. The p27 antibody was obtained from Chemicon International (Temecula, CA). For immunohistochemistry (IHC) and immunofluorescence, p-FoxO3a antibody was obtained from Santa Cruz Biotechnology. α-Smooth muscle actin antibody was obtained from Vector Laboratories (Burlingame, CA). β1 Integrin blocking antibody P5D2 was also produced from hybridoma culture. β1 Integrin–activating monoclonal antibody TS2/16 was produced from hybridoma culture (American Type Culture Collection, HB-243). Akt inhibitor was obtained from Calbiochem (San Diego, CA). For PTEN adenovirus construction, wild-type (WT) PTEN cDNA was generated from normal human lung fibroblasts (HLF-210) by RT-PCR. The forward and reverse primers used for WT PTEN (PW), which span the entire coding region of PTEN, are 5′-CTACTCGAGGCTCCCAGACATGAC-3′ and 5′-ACGCTCGAGATAAAAAAAAATTCAG-3′, respectively. The lipid and protein phosphatase truncated mutant was generated by PCR with forward and reverse primers 5′-CAGCTCGAGGGACGAACTGGTGTAA-3′ and 5′-ACGCTCGAGATAAAAAAAAAT TCAG-3′, respectively. The amplified products were cloned into MIGR1-IRES–green fluorescent protein (GFP), followed by SalI and EcoRI digestion. The PW MIGR1-IRES-GFP fragments were then cloned into the adenoviral vector, pAxCAwt (obtained from Dakara Bio. Inc., Otsu, Shiga, Japan), and adenovirus titer was measured by the plaque method in 0.5% soft agar. The cells were infected with adenoviral vectors at a multiplicity of infection of 1:20. Adenovirus-expressing HA-tagged Akt with a c-Src myristolyation sequence fused in frame to the N-terminus [hyperactive Akt (HA)], HA-tagged Akt dominant-negative mutant (T308A and S473A), and WT FoxO3a were obtained from Vector Biolabs, Eagleville, PA. Adenovirus-expressing GFP-tagged WT FoxO3a, dominant-negative FoxO3a (DF) with the deletion of the transactivation domain from the C-terminus, and empty vector were obtained from Vector Biolabs. Control or IPF fibroblasts were infected with adenovirus expressing PTEN, Akt, or empty vector in 2 mL of tissue culture dishes. A total of 1 × 105 to 2 × 105 control or IPF fibroblasts per milliliter of DMEM were infected with 1 × 106 plaque-forming units of each adenovirus for assay. For the separation of cytoplasmic and nuclear fractions, NE-PER Nuclear and Cytoplasmic extraction reagents from Thermo Scientific (Pittsburgh, PA), were used. Briefly, control and IPF fibroblasts were collected and suspended in 100 μL of cytoplasmic extraction reagent I solution (Thermo Scientific). Tube-containing cells were then vortex mixed, and 5.5 μL of cytoplasmic extraction reagent II was added, followed by incubation in ice for 5 minutes. Cytoplasmic extract was then collected by centrifugation at maximum speed for 5 minutes. The insoluble nuclear fraction was resuspended in 50 μL of nuclear extraction reagent buffer and vortex mixed for 15 seconds every 10 minutes. The nuclear fraction was finally collected by centrifugation at maximum speed (16,000 × g) for 10 minutes for analysis. Human lung tissue was prepared as previously described.8Xia H. Diebold D. Nho R. Perlman D. Kleidon J. Kahm J. Avdulov S. Peterson M. Nerva J. Bitterman P. Henke C. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (175) Google Scholar, 9Xia H. Khalil W. Kahm J. Jessurun J. Kleidon J. Henke C.A. Pathologic caveolin-1 regulation of PTEN in idiopathic pulmonary fibrosis.Am J Pathol. 2010; 176: 2626-2637Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar After embedding in paraffin, sections (5-μm thick) were cut and mounted onto polylysine-coated slides. The paraffin sections were deparaffinized in xylene, rehydrated through a graded series of methanol, and placed in a 95°C to 100°C water bath for 30 minutes in citrate buffer (pH 6.0) for antigen retrieval. The sections were quenched with 3% hydrogen peroxide in PBS and incubated in normal goat serum for 30 minutes to block nonspecific binding of secondary antibodies. Endogenous avidin and biotin binding sites were blocked by sequential incubation for 15 minutes. The sections were then incubated overnight at 4°C with p-FoxO3a or FoxO3a in PBS (polyclonal antibody, 1:50; Santa Cruz Biotechnology). IHC was performed by the avidin-biotin peroxidase complex method. The Vectastain Elite ABC kit was used for all experiments, as instructed by the manufacturer (Vector Laboratories). Immunoreactivity was detected with 3,3′-diaminobenzidine as a peroxidase substrate, and the sections were counterstained with hematoxylin. The Leica Leitz DMRB microscope (Leica, Wetzlar, Germany) was used to acquire all images. Photo-Shop CS5 (Adobe, San Jose, CA) was used for image processing. For the immunofluorescence assay, IPF fibroblasts cultured on polymerized collagen were fixed for 10 minutes in cold methanol, rinsed with PBS, and blocked in 5% donkey serum in PBS buffer for 30 minutes at room temperature. Cells were then incubated with p-FoxO3a primary antibody overnight at 4°C, washed three times with PBS, and incubated in donkey anti-rabbit Cy 2 (Jackson Immunoresearch, West Grove, PA) secondary antibody diluted in PBS for 45 minutes at room temperature in the dark. After rinsing with PBS, the slides were incubated in DAPI in PBS at room temperature for 20 minutes in the dark. After washing three times, ProLong Gold antifade reagent (Life Technologies, Eugene, OR) was added to the coverslip and the slides were sealed with nail polish. Specimens were imaged with the Zeiss Axiovert 200 microscope (Zeiss, Gottigen, Germany). The number of viable proliferating cells was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; Promega (Madison, WI)]. Briefly, normal human lung or IPF fibroblasts were infected with WT, DF, HA, or dominant-negative Akt (DA) for 24 hours. A total of 2 × 103 human lung fibroblasts were then grown in 100 μL of DMEM on 96-well plates overnight. CellTiter 96 Aqueous One Solution Reagent, 20 μL, was added to each well of the 96-well plate, followed by incubation for 2 hours at 37°C in a humidified 5% CO2 atmosphere. The absorbance at 490 nm, using a 96-well plate reader, was measured. For the cell counting assay, cell numbers were counted using the Coulter counter (Beckman Coulter, Miami, FL). Attached cells were then trypsinized, collected, and counted. Data are expressed as the mean ± SD. Experiments were performed three times. Paired evaluations were made for experimental and control conditions within each experiment, and significance was determined by the Student's t-test. The significance level was set at P < 0.05. We have previously shown that when normal fibroblasts attach to polymerized collagen, high PTEN activity inhibits their proliferation.8Xia H. Diebold D. Nho R. Perlman D. Kleidon J. Kahm J. Avdulov S. Peterson M. Nerva J. Bitterman P. Henke C. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (175) Google Scholar In contrast, IPF fibroblasts display inappropriately high proliferation on polymerized collagen because of aberrantly high Akt activity, resulting from low membrane PTEN expression. Because Akt suppresses FoxO3a by phosphorylating the Ser253 residue, we hypothesized that FoxO3a activity is abnormally low in IPF fibroblasts on polymerized collagen, thereby promoting fibroblast proliferation. To examine this hypothesis, we first measured the level of phosphorylated (inactive) FoxO3 in control (n = 5) and IPF (n = 5) fibroblasts on polymerized collagen. The level of p-FoxO3a has been used as a surrogate marker of FoxO3a activity. The p-FoxO3a levels were high in IPF compared with control fibroblasts when cultured on polymerized collagen (Figure 1A). p-FoxO3a–actin expression (n = 8 control fibroblasts, and n = 7 IPF fibroblasts) was overall more than threefold higher in IPF compared with control fibroblasts (Figure 1B). These data indicate that FoxO3a activity is low in response to IPF fibroblast attachment to collagen. Furthermore, the overall level of FoxO3a expression was high in control compared with IPF fibroblasts on collagen matrix (Figure 1, C and D). Our data demonstrate that, in response to IPF fibroblast attachment to collagen, FoxO3a activity is aberrantly low. Because the level of p-FoxO3a is high in IPF fibroblasts, we also sought to address whether high Akt activity is responsible for phosphorylation and inactivation of FoxO3a in IPF fibroblasts. We first measured the level of FoxO3a phosphorylation in the presence or absence of an Akt inhibitor. For this assay, IPF fibroblasts were pre-incubated with various concentrations of an Akt inhibitor or dimethylsulfoxide control and plated on type I collagen, and the level of p-FoxO3a was measured. p-FoxO3a levels were progressively diminished in a dose-dependent fashion (Figure 2A). We also examined FoxO3a levels using an antibody that recognizes FoxO3a expression in general (not specific for p-FoxO3a). Unlike p-FoxO3a expression, we found that FoxO3a levels were maintained high when a high dose of Akt inhibitor was used (Figure 2A). These data suggest that during IPF fibroblast interaction with collagen, Akt phosphorylates and suppresses FoxO3a activity. Because IPF fibroblasts display inappropriately low PTEN activity, resulting in high Akt activity, we next examined the role of PTEN in regulating FoxO3a via Akt. To begin to address this issue, we first used PTEN−/− cells to examine FoxO3a activity. PTEN−/− cells were reconstituted with PW or mutant PTEN (PD) protein, and the levels of FoxO3a and p-FoxO3a were assessed. Overexpression of PW completely suppressed the expression of p-FoxO3a, despite an increase in the overall level of FoxO3a (Figure 2B). In contrast, mutant PTEN failed to inhibit FoxO3a activity. To verify this finding, control lung fibroblasts were first infected with an adenoviral vector expressing PW, phosphatase-dead mutant PTEN, or empty vector (GFP), and the level of p-FoxO3a was measured. The level of p-FoxO3a was barely detectable when PW was overexpressed in control fibroblasts (Figure 2C). In contrast, the overall level of FoxO3a remained high when PW was overexpressed. However, when mutant PTEN was overexpressed, the level of p-FoxO3a was increased compared with control, whereas the FoxO3a level was markedly diminished (lane 2). Interestingly, the FoxO3a/p-FoxO3a expression ratio was not significantly altered when PW was expressed in control fibroblasts (Figure 2C), suggesting that further induction of PTEN protein does not significantly increase FoxO3a activity, presumably because of the presence of high baseline PTEN activity when these cells interact with polymerized collagen. We next analyzed the level of FoxO3a phosphorylation in control fibroblasts using HA or DA constructs to further examine the effect of Akt on FoxO3a function. HA increased p-FoxO3a levels compared with control (Figure 2D). Interestingly, the level of p-FoxO3a remained suppressed in control fibroblasts. This suggests that DA cannot further suppress the already low level of Akt activity resulting from high PTEN activity in control fibroblasts on collagen (Figure 2D). We next examined the role of the PTEN-Akt axis in regulating p-FoxO3a levels in IPF fibroblasts interacting with polymerized collagen. The overexpression of PW in IPF fibroblasts decreased the level of p-FoxO3a, whereas mutant PTEN had a minimal effect on the already high level of p-FoxO3a in IPF fibroblasts compared with the GFP control (Figure 2E). Furthermore, the analysis of the FoxO3a/p-FoxO3a expression ratio showed that, compared with control, HA did not significantly increase the level of p-FoxO3a (Figure 2F). In contrast, the FoxO3a/p-FoxO3a expression ratio was high when DA was overexpressed in IPF fibroblasts on collagen matrix (lane 2). Taken together, these data demonstrate that the PTEN-Akt axis regulates FoxO3a function in response to fibroblast attachment to polymerized collagen. Our data indicate that when IPF fibroblasts interact with polymerized collagen, low PTEN function inappropriately activates Akt, thereby phosphorylating and inactivating FoxO3a. On activation, the FoxO3a transcription factor translocates from the cytoplasm into the nucleus. In contrast, FoxO3a that has been phosphorylated by Akt is located predominantly in the cytoplasm and is inactive.20Yusuf L. Zhu X. Kharas M.G. Chen J. Fruman D.A. Optimal B-cell proliferation requires phosphoinositide 3-kinase-dependent inactivation of FOXO transcription factors.Blood. 2004; 104: 784-787Crossref PubMed Scopus (114) Google Scholar, 21Chen Y.R. Liu M.T. Chang Y.T. Wu C.C. Hu C.Y. Chen J.Y. Epstein-Barr virus latent membrane protein 1 represses DNA repair through the PI3K/Akt/FOXO3a pathway in human epithelial cells.J Virol. 2008; 82: 8124-8137Crossref PubMed Scopus (45) Google Scholar Because IPF fibroblasts display low FoxO3a activity, we hypothesized that FoxO3a will be largely located in the cytoplasm when IPF fibroblasts interact with polymerized collagen. To examine this hypothesis, we isolated and measured the nuclear and cytoplasmic fractions of FoxO3a and p-FoxO3a in control and IPF fibroblasts cultured on polymerized collagen. Consistent with our finding that FoxO3a is largely inactive in IPF fibroblasts, we found that FoxO3a was barely detectable in the nucleus of IPF fibroblasts (Figure 3A), whereas FoxO3a expression was high in the nuclear fraction of control fibroblasts (lane 3). The calculation of the FoxO3a/p-FoxO3a expression ratio using densitometry illustrates that nuclear FoxO3a is low in IPF fibroblasts compared with control (Figure 3B). In contrast, we found that the level of phosphorylated or inactive FoxO3a was elevated in the cytoplasm of IPF fibroblasts cultured on polymerized collagen compared with that of control fibroblasts (Figure 3A). The ratio of FoxO3a/p-FoxO3a in the cytoplasmic fraction of IPF fibroblasts was approximately 32% lower compared with that of control fibroblasts (Figure 3B). To verify our Western blot analysis data, we further examined the location of p-FoxO3a in IPF fibroblasts on collagen using immunofluorescence. We found that p-FoxO3a is mainly located in the cytoplasm but that some p-FoxO3a can also be found in the nucleus (see Supplemental Figure S1A at http://ajp.amjpathol.org). Our results indicate that when IPF fibroblasts interact with polymerized collagen, FoxO3a is predominantly inactivated and largely located in the cytoplasm, whereas there is little active FoxO3a in the nucleus. We next examined the role of Akt in regulating the cellular distribution of FoxO3a in IPF fibroblasts cultured on collagen. For this experiment, we assessed the level of p-FoxO3a in the cytoplasm in IPF fibroblasts infected with an adenovirus expressing DA and HA. Inhibition of Akt by DA reduced the level of p-FoxO3a in t
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