Gene Expression Profiling of Pulmonary Fibrosis Identifies Twist1 as an Antiapoptotic Molecular “Rectifier” of Growth Factor Signaling
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080954
ISSN1525-2191
AutoresRobert S. Bridges, Daniel J. Kass, Katrina Loh, Carlota Glackin, Alain Borczuk, Steven M. Greenberg,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoIdiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease. To gain insight into IPF pathogenesis, we performed gene expression profiling of IPF lungs. Twist1, a basic helix-loop-helix protein, was found among the most consistently and highly up-regulated genes and was expressed in nuclei of type II epithelial cells, macrophages, and fibroblasts in IPF lungs. We studied the function of Twist1 in fibroblasts further, because they are the major effector cells in this disease and persist despite an ambient proapoptotic environment. Twist1 was induced by the profibrotic growth factors (GFs) basic fibroblast growth factor, platelet-derived growth factor, and epidermal growth factor in primary rat lung fibroblasts (RLFs). Suppression of Twist1 expression resulted in decreased RLF accumulation due to increased apoptosis, whereas Twist1 overexpression protected RLFs against several apoptotic stimuli. Addition of platelet-derived growth factor in combination with other GFs led to an increase in proliferation. When Twist1 was depleted, GFs continued to act as mitogens but caused a marked increase in cell death. The increase in apoptosis under basal or growth factor-stimulated conditions was partly mediated by up-regulation of the proapoptotic Bcl-2 family members, Bim and PUMA. These findings indicate that Twist1 promotes survival and accumulation of fibroblasts by shaping their responsiveness to growth factor stimulation. We propose that Twist1 represents one of the factors that promotes pathogenic accumulation of fibroblasts in fibrotic lung disease. Idiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease. To gain insight into IPF pathogenesis, we performed gene expression profiling of IPF lungs. Twist1, a basic helix-loop-helix protein, was found among the most consistently and highly up-regulated genes and was expressed in nuclei of type II epithelial cells, macrophages, and fibroblasts in IPF lungs. We studied the function of Twist1 in fibroblasts further, because they are the major effector cells in this disease and persist despite an ambient proapoptotic environment. Twist1 was induced by the profibrotic growth factors (GFs) basic fibroblast growth factor, platelet-derived growth factor, and epidermal growth factor in primary rat lung fibroblasts (RLFs). Suppression of Twist1 expression resulted in decreased RLF accumulation due to increased apoptosis, whereas Twist1 overexpression protected RLFs against several apoptotic stimuli. Addition of platelet-derived growth factor in combination with other GFs led to an increase in proliferation. When Twist1 was depleted, GFs continued to act as mitogens but caused a marked increase in cell death. The increase in apoptosis under basal or growth factor-stimulated conditions was partly mediated by up-regulation of the proapoptotic Bcl-2 family members, Bim and PUMA. These findings indicate that Twist1 promotes survival and accumulation of fibroblasts by shaping their responsiveness to growth factor stimulation. We propose that Twist1 represents one of the factors that promotes pathogenic accumulation of fibroblasts in fibrotic lung disease. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and typically fatal lung disease characterized by excess deposition of extracellular matrix proteins. Fibroblasts accumulate in IPF lungs and are viewed as a major effector cell type in the disease, because they are a principal source of extracellular matrix proteins that contribute to irreversible scarring (reviewed in Refs. 1Meltzer EB Noble PW Idiopathic pulmonary fibrosis.Orphanet J Rare Dis. 2008; 3: 8Crossref PubMed Scopus (293) Google Scholar, 2Thannickal VJ Horowitz JC Evolving concepts of apoptosis in idiopathic pulmonary fibrosis.Proc Am Thorac Soc. 2006; 3: 350-356Crossref PubMed Scopus (269) Google Scholar). The source of fibroblasts in IPF is unclear but likely includes a combination of recruitment of circulating fibroblast precursors,3Phillips RJ Burdick MD Hong K Lutz MA Murray LA Xue YY Belperio JA Keane MP Strieter RM Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (911) Google Scholar, 4Andersson-Sjoland A de Alba CG Nihlberg K Becerril C Ramirez R Pardo A Westergren-Thorsson G Selman M Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis.Int J Biochem Cell Biol. 2008; 40: 2129-2140Crossref PubMed Scopus (286) Google Scholar, 5Hashimoto N Jin H Liu T Chensue SW Phan SH Bone marrow-derived progenitor cells in pulmonary fibrosis.J Clin Invest. 2004; 113: 243-252Crossref PubMed Scopus (643) Google Scholar transdifferentiation of epithelial or endothelial cells into fibroblasts (e.g., epithelial-mesenchymal transformation; EMT),6Kim KK Kugler MC Wolters PJ Robillard L Galvez MG Brumwell AN Sheppard D Chapman HA Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix.Proc Natl Acad Sci USA. 2006; 103: 13180-13185Crossref PubMed Scopus (998) Google Scholar, 7Willis BC Liebler JM Luby-Phelps K Nicholson AG Crandall ED du Bois RM Borok Z Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-β1: potential role in idiopathic pulmonary fibrosis.Am J Pathol. 2005; 166: 1321-1332Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar and proliferation of resident lung fibroblasts.8Kass D Bridges RS Borczuk A Greenberg S Methionine aminopeptidase-2 as a selective target of myofibroblasts in pulmonary fibrosis.Am J Respir Cell Mol Biol. 2007; 37: 193-201Crossref PubMed Scopus (12) Google Scholar, 9Selman M Ruiz V Cabrera S Segura L Ramirez R Barrios R Pardo A TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis: a prevailing nondegradative lung microenvironment?.Am J Physiol. 2000; 279: L562-L574Google Scholar Regardless of their proximate source, fibroblasts in IPF lungs accumulate despite the presence of a hostile, proapoptotic environment. Because these cells exhibit little evidence of apoptosis,10Uhal BD Joshi I Hughes WF Ramos C Pardo A Selman M Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung.Am J Physiol Lung Cell Mol Physiol. 1998; 275: L1192-L1199Google Scholar this suggests that they possess highly effective mechanisms to evade apoptotic stimuli.To identify potential mediators of fibroblast accumulation and survival in IPF, we performed gene expression profiling of IPF lungs. Our analysis revealed Twist1 as the most highly up-regulated transcription factor in diseased lungs. Twist1, which is a member of a large family of basic helix-loop-helix proteins, has been implicated in bone and cartilage development,11Bialek P Kern B Yang X Schrock M Sosic D Hong N Wu H Yu K Ornitz DM Olson EN Justice MJ Karsenty G A twist code determines the onset of osteoblast differentiation.Dev Cell. 2004; 6: 423-435Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 12Rice DP Aberg T Chan Y Tang Z Kettunen PJ Pakarinen L Maxson RE Thesleff I Integration of FGF and TWIST in calvarial bone and suture development.Development. 2000; 127: 1845-1855PubMed Google Scholar regulation of inflammation,13Sharif MN Sosic D Rothlin CV Kelly E Lemke G Olson EN Ivashkiv LB Twist mediates suppression of inflammation by type I IFNs and Axl.J Exp Med. 2006; 203: 1891-1901Crossref PubMed Scopus (185) Google Scholar, 14Sosic D Richardson JA Yu K Ornitz DM Olson EN Twist regulates cytokine gene expression through a negative feedback loop that represses NF-κB activity.Cell. 2003; 112: 169-180Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar tumor progression and metastasis,15Maestro R Dei Tos AP Hamamori Y Krasnokutsky S Sartorelli V Kedes L Doglioni C Beach DH Hannon GJ Twist is a potential oncogene that inhibits apoptosis.Genes Dev. 1999; 13: 2207-2217Crossref PubMed Scopus (452) Google Scholar, 16Cheng GZ Chan J Wang Q Zhang W Sun CD Wang LH Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel.Cancer Res. 2007; 67: 1979-1987Crossref PubMed Scopus (452) Google Scholar, 17Yang J Mani SA Donaher JL Ramaswamy S Itzykson RA Come C Savagner P Gitelman I Richardson A Weinberg RA Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis.Cell. 2004; 117: 927-939Abstract Full Text Full Text PDF PubMed Scopus (3035) Google Scholar and resistance of tumor cells to apoptosis induced by chemotherapeutic agents.16Cheng GZ Chan J Wang Q Zhang W Sun CD Wang LH Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel.Cancer Res. 2007; 67: 1979-1987Crossref PubMed Scopus (452) Google Scholar, 18Wang X Ling MT Guan XY Tsao SW Cheung HW Lee DT Wong YC Identification of a novel function of TWIST, a bHLH protein, in the development of acquired Taxol resistance in human cancer cells.Oncogene. 2004; 23: 474-482Crossref PubMed Scopus (198) Google Scholar Thus, we reasoned that Twist1 may play an analogous role in primary lung fibroblasts subjected to the harsh proapoptotic conditions that characterize IPF lungs. In this study, we tested whether Twist1 expression affects proliferation, survival, or both of primary lung fibroblasts exposed to various GFs and apoptotic stimuli present in IPF lungs.Materials and MethodsReagentsDulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). Rat platelet-derived growth factor (PDGF)-BB and human epidermal growth factor (EGF) were from R&D Systems (Minneapolis, MN). Human basic fibroblast growth factor (bFGF) and human soluble Fas ligand were from PeproTech (Rocky Hill, NJ). 4-Hydroxynonenal was from Cayman Chemicals (Ann Arbor, MI). Insulin-selenium-transferrin supplement, human endothelin-1, and thapsigargin were from Sigma-Aldrich (St. Louis, MO). Protein S from human plasma was from Calbiochem/EMD Biosciences (Darmstadt, Germany). Antibodies against Bim (numbers 2819 and 2933), PUMA (number 4976), Bak (number 3814S), Bad (number 9292), and Bik (number 4592) were from Cell Signaling Technology (Beverley, MA). Antibodies against Bik (E-20), actin (I-19), Bcl-Xl (H5); Mcl-1 (S-19), Bax (N-20), and Bcl-2 (C-21), were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal Ab against 5-bromo-2′-deoxyuridine (BrdU) (clone BMC9318) was from Roche Diagnostics (Indianapolis, IN). Monoclonal Ab against 4-hydroxy-2-nonenal (4-HNE) (clone N45.1) was from the Japan Institute for the Control of Aging (Shizuoka, Japan). Monoclonal Ab against FLAG (clone M2) was from Sigma- Aldrich. Monoclonal Ab against tubulin (clone E7) was from the Developmental Hybridoma Bank (University of Iowa, Iowa City, IA). We generated antibodies against Twist1 using the following peptide from rat Twist1: DSLSNSEEEPDRQQPASGKRGARKR. Peptide synthesis, conjugation to keyhole limpet hemocyanin, injection into rabbits, and serum collection were performed by GenScript (Piscataway, NJ). We affinity-purified IgG using a peptide affinity column. Horseradish peroxidase-, fluorescein isothiocyanate-, rhodamine-, and Cy5-conjugated F(ab′)2 fragments of donkey anti-goat, -rabbit, and -mouse IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). Alexa Fluor 488- or 594-conjugated anti-mouse, anti-rabbit, or anti-goat IgG, and 4′,6-diamidino-2-phenylindole diacetate (DAPI) were from Molecular Probes/Invitrogen (Carlsbad, CA). A plasmid-encoding Twist1-FLAG and vector control were a gift from E. Olson (University of Texas Southwestern Medical Center, Dallas, TX). The PUMA-FLAG construct was from the laboratory of Dr. L. Greene (Columbia Medical Center, New York, NY). All short hairpin RNA (shRNA) constructs were cloned into the pSIREN-RetroQ-zsGreen vector (BD Clontech, Mountain View, CA). The shLUC negative control (from BD Clontech, sequence not provided) and shBim pSIREN (target sequence: 5′-GACAGAGAAGGTGGACAATTG-3′) were also from Dr. L. Greene. The target sequence for PUMA was 5′-CGGAGACAAGAAGAGCAACAT-3′.Gene Expression ProfilingTotal RNA extraction: mRNA samples were taken from surgical lung biopsies from 7 normal and 10 IPF samples, with 3 of the IPF samples from microdissected fibroblastic foci (Table 1). For extraction from whole-lung biopsies, tissue sections were homogenized with a rotor-stator homogenizer before purification. For microdissected samples, serial 8 μmol/L sections of frozen tissue were obtained. The first section was stained with H&E to ascertain areas containing fibroblastic foci, and foci from the remainder were stained with eosin, microdissected with a 20-gauge needle and collected in a pipette tip under vacuum. Total RNA was extracted using RNEasy columns (Qiagen, Valencia, CA), quantitated by spectrophotometry, and agarose gel electrophoresis was performed to confirm lack of RNA degradation.Table 1Demographic Data of Patients Used in This StudySample no.ClassificationAge (yr)/SexSpecimen typeTissue diagnosis1Normal70/FLobectomyAdenocarcinoma2Normal81/MLobectomyNSCLC3Normal65/MLobectomySCC4Normal69/FLobectomyNSCLC5Normal66/FLobectomyAdenocarcinoma6Normal66/FLobectomyAdenocarcinoma7Normal70/FLobectomyNSCLC8IPF53/FExplantIPF9IPF59/FExplantIPF10IPF68/MBiopsyIPF11IPF53/FExplantIPF12IPF59/FExplantIPF13IPF68/MBiopsyIPF14IPF43/FExplantIPF15IPF61/MBiopsyIPF16IPF58/MBiopsyIPF17IPF61/FExplantIPFSamples 1 to 7 are from pathologically normal tissue taken from surgical lobectomies of lung cancer patients. Samples 8 to 10 are derived from microdissected fibroblastic foci taken from samples 11 to 13. Samples 11 to 17 are homogenates of grossly abnormal lung, selected for abundance of fibroblastic foci. NSLC, non-small cell lung carcinoma; SCC, Small cell lung carcinoma. Open table in a new tab Probe Preparation and HybridizationTwo micrograms of total RNA was reverse transcribed using the Superscript Choice reverse transcriptase and a T7 oligo dT primer (Invitrogen). After second strand synthesis, cDNA was assessed for quality by agarose gel electrophoresis. Biotinylated probe RNA was generated using the Enzo high efficiency bioarray kit (Enzo Biochem, New York, NY). After chemical fragmentation at 95°C, 15 μg of labeled RNA was used for hybridization on a U95Av2 chip (Affymetrix, Santa Clara, CA). Hybridization and array scanning were performed by the Columbia Genome Center. The quality of the microarray was assessed by visual inspection of the .cel images by examining the scaling factor, and by noting a consistent number of present, absent, and marginal calls across comparable samples. The 3′ to 5′ ratio of glyceraldehyde-3-phosphate dehydrogenase was used as an additional quality control parameter, with a goal of 1 to 3 for samples to be acceptable.Data AnalysisThe array was first analyzed using the Affymetrix MAS 5.0 software. An average intensity value was calculated for each probe cell, excluding bordering pixels and using a 75% percentile value of the remaining pixels. Once background and noise values were subtracted, the image was stored in .cel files. Positive and negative calls were generated for each probe pair (perfect match and mismatch), and then a present, absent, or marginal call was assigned to the entire probe set. These calls, as well as raw intensity values, were recorded in a spreadsheet and imported into GeneSpring 5.0 software (Agilent Technologies, Santa Clara, CA). After normalization, data were globally filtered to exclude genes that showed low expression across all samples, and then a second global filter was used for genes that showed excessive variability across the samples. This 7814 gene list was used for unsupervised clustering. In addition, a supervised approach was used to determine whether sets of genes were statistically associated with IPF. A permutation test and neighborhood analysis was performed using Cluster and TreeView software (http://rana.lbl.gov/EisenSoftware.htm). The significance score for each gene was assessed by nearest neighbor t-test analysis after randomly permuting the class assignments 1000 times. This analysis determined that ∼10% of the 7814 genes had statistically significant (P < 0.05) changes in expression in IPF.Tissue ImmunohistochemistryFor immunohistochemistry, lungs were fixed in 10% formaldehyde and embedded in paraffin. Five-micrometer sections were cut and mounted on positively charged glass slides. Sections were deparaffinized with xylene, followed by rehydration with a graded ethanol series and stained with the indicated antibodies. All antigens were developed with the Vectastain ABC Universal kit and diaminobenzidine (Vector Laboratories, Burlingame, CA). Sections were counterstained with Harris’ hematoxylin (Sigma-Aldrich), dehydrated through graded alcohol series, and mounted with Permount (Fisher, Fairlawn, NJ). Sections were visualized on an Olympus CHS microscope (Olympus, Tokyo, Japan).Isolation, Culture, and Transfection of Rat Lung FibroblastsFemale Sprague-Dawley rats (150–250 g) were purchased from Harlan (Indianapolis, IN). Rats were sacrificed with Euthanasia-5 solution. The lungs were perfused with cold PBS, and the parenchyma were excised. Large airways were carefully removed, and lungs were minced and digested in a 1.25% trypsinizing solution for 90 minutes. After passage through sterile gauze, the cell suspension was pelleted and the cells were maintained in DMEM supplemented with 10% FBS, 5 ng/ml PDGF-BB, 10 ng/ml EGF and insulin-transferrin-selenite liquid supplement. The RLFs were incubated in 5% CO2 at 37°C, and used for experiments during passages 2–6.Small Interfering RNARLFs were plated in DMEM with 10% FBS and immediately transfected with 37.5 ng of RNA oligomers and 3 μl of Hiperfect (Qiagen) according to the fast-forward protocol in the manufacturer’s instructions. One day after initial transfection, the cells were washed once with DMEM and placed in DMEM with the indicated serum concentrations, and fresh transfection complexes were added. Target sequences for small interfering (si)RNA complexes were as follows: nontargeting control small interfering RNA (siRNA), 5′-ACACGAGGUACGCCGACAA-3′; Twist-siRNA1, 5′-ACGAGGAGCUGCAGACACA-3′ (designed and synthesized by Dharmacon); and Twist-siRNA2, 5′-ACUCCAAGAUGGCAAGCUG-3′ (synthesized by Qiagen).Plasmid TransfectionRLFs were plated 1 day before transfection in DMEM and 10% FBS. The next day, transfection complexes containing 0.5 μg of plasmid and 1.5 μl of Lipofectamine LTX (Invitrogen) were added to the cells according to the manufacturer’s instructions. After 5 hours, the cells were washed and incubated overnight in DMEM and 10% FBS. For Twist1 overexpression experiments, cells were incubated overnight in DMEM and 0.5% FBS before adding the indicated reagents. Where applicable, plasmids were cotransfected with an enhanced green fluorescent protein (GFP) expression vector to visualize transfected cells. We routinely observed >90% cotransfection efficiency. We determined that cotransfection of multiple siRNA oligomers was inefficient. Instead, we used a combination of plasmid-based shRNA and siRNA oligomers to suppress multiple genes. For experiments with dual transfection of plasmid shRNA and siRNA oligomers, we plated 1.5 × 105 RLFs in 6-well plates and performed shRNA transfection 1 day later with Lipofectamine LTX as described above. The cells were incubated further at 37°C for 18–24 hours, trypsinized, replated, and transfected with siRNA oligomers as described above.Quantitative Real-Time PCRmRNA was isolated from human surgical lung biopsies or whole mouse lungs by tissue disruption with a rotor-stator homogenizer followed by purification with the RNEasy mini kit (Qiagen). For experiments with RLFs, cells were plated at 1.2 × 104 in duplicate or triplicate in 24-well plates and treated as described. mRNA was reverse-transcribed to cDNA using the Quantitect RT kit (Qiagen) and qPCR was performed using the POWER SYBR Green PCR Master Mix (Applied Biosystems) and a LightCycler 2.0 RealTime PCR System (Roche Diagnostics). Copies of cDNA were normalized to expression of housekeeping genes glyceraldehyde-3-phosphate dehydrogenase or peptidyl-prolyl isomerase (PPIA). We confirmed specific amplification of the target genes by analyzing dissociation curves and agarose gel electrophoresis following PCR. Primers had the following sequences: human Twist1, forward, 5′-CGGGTCATGGCTAACGTG-3′, and reverse, 5′-CAGCTTGCCATCTTGGAGTC-3′17Yang J Mani SA Donaher JL Ramaswamy S Itzykson RA Come C Savagner P Gitelman I Richardson A Weinberg RA Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis.Cell. 2004; 117: 927-939Abstract Full Text Full Text PDF PubMed Scopus (3035) Google Scholar; rat Twist1, forward, 5′-CGGACAAGCTGAGCAAGATT-3′, and reverse, 5′-CCTTCTCTGGAAACAATGAC-3′; and glyceraldehyde-3-phosphate dehydrogenase, forward, 5′-GACCCCTTCATTGACCTCAAC-3′, and reverse, 5′-CTTCTCCATGGTGGTGAAGA-3′. Primers for Bad, Bid1, Bid3 (Hrk), Bik, Bim, BMF, NOXA, PPIA, and PUMA were Quantitect primers from Qiagen (sequences not provided).Cell-Based Fluorescence Microscopy, Cell Viability, and Proliferation AnalysisFor immunofluorescence, cells were fixed in 3.7% formaldehyde for 5 minutes, followed by permeabilization in 0.2% Triton X-100. Cells were incubated with indicated primary antibody for 30 minutes at room temperature or overnight at 4°C in a humid chamber, washed, and incubated with a fluorophore-conjugated secondary antibody for 30 minutes at room temperature. Following mounting, cells were examined using a Nikon Eclipse TE200-inverted fluorescence microscope equipped with a ×60 Plan Apochromat objective (NA 1.4) and an Orca-S100 Hamamatsu CCD camera. Imaging was performed using MetaMorph software. Quantitation of Twist1 and Bim expression of unprocessed images was performed by microspectrofluorimetry. Fluorescence intensity associated with Twist1 immunoreactivity was determined by calculating average raw pixel intensity of a circular region completely within the nucleus and corrected for background fluorescence of each field, as well as nonspecific IgG staining measured with nonimmune rabbit IgG. RLFs were cotransfected with enhanced GFP and Twist-FLAG or control. Bim is localized to mitochondria and stains in a perinuclear distribution. Fluorescent intensity associated with Bim immunoreactivity was determined in all GFP+ cells per coverslip by calculating average raw pixel intensity in a circular region completely within this perinuclear area of staining. All comparison images were exposed for the same amount of time. For viability experiments, cells were fixed and stained with DAPI without permeabilizing. Viability was assessed by examining up to 200 GFP-positive cells per replicate for classical markers of apoptosis, including nuclear condensation and morphological changes. To analyze proliferation by BrdU incorporation, RLFs were treated as described and incubated with 10 μmol/L BrdU for the last 12 to 18 hours before fixation. After fixation and permeabilization, the cells were incubated in DNase buffer (100 U/ml DNase, 1 mmol/L Ca2+, and 1 mmol/L Mg2+ in PBS) for 45 minutes at 37°C in a humid chamber and then stained with an anti-BrdU mAb. At least 200 cells per replicate in two to three independent experiments were assessed for BrdU incorporation.Fluorescence-Based Cell Counting and Caspase-3 AssaysTo facilitate counting cells, cells were fixed in 3.7% formaldehyde, permeabilized in 0.2% Triton X-100, and stained with the DNA-sensitive dye Sybr green I (Invitrogen). Sybr green fluorescence was quantitated with a plate-reading spectrofluorimeter. Pilot experiments indicated that fluorescence intensity correlated with RLF cell counts in a hemacytometer. All data represent fold changes in cell number following background subtraction. Assays for active caspase-3 were done with the EnzChek Caspase-3 Assay Kit number 2 (Invitrogen) based on the caspase-3 substrate Z-DEVD conjugated to rhodamine 110. RLFs were plated at 0.2 to 0.5 × 104 cells per well in triplicate in 48-well plates. At the indicated time points, all but 50 μl of culture media were removed, and cells were lysed by adding 2.5 μl of 20× lysis buffer to the remaining media and one cycle of freezing and thawing. We then added samples to 2× caspase-3 assay buffer in 96-well plates, incubated the reaction 24 hours at room temperature in the dark, and measured fluorescence intensity on a plate-reading spectrofluorimeter. We confirmed that all fluorescence was specific to caspase-3 by including the specific inhibitor Ac-DEVD-CHO in some samples. After subtracting background, caspase-3 activity was normalized to cell number.ImmunoblottingFor immunoblotting, RLFs were plated at 1.5 × 105 each in three wells of 6-well plates/condition and treated as described previously. At the indicated times, cells were lifted for 5 minutes in trypsin, neutralized in 0.5% FBS, and pelleted at 400 × g for 5 minutes. Cells were lysed in radioimmunoprecipitation assay buffer (150 mmol/L sodium chloride, 0.5% IPEGAL CA-630, 0.5% sodium deoxycholate, 0.05% SDS, 1 mmol/L EDTA, 50 mmol/L sodium fluoride, 1 mmol/L phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 50 mmol/L Tris-HCl (pH 7.4)) at 4°C for 10 minutes, followed by sonication using a tip-probe sonicator and centrifugation at 13,000 × g for 5 minutes to remove insoluble debris. Loading onto SDS-PAGE gels was normalized using the detergent compatible protein assay (Bio-Rad, Hercules, CA). All lysates were boiled in Laemmli buffer for 5 minutes before resolving by SDS-PAGE, and transferring onto nitrocellulose membranes. Equal loading onto polyacrylamide gels was confirmed by Ponceau S staining. Proteins were immunoblotted with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were developed using enhanced chemiluminescence (Pierce, Rockford, IL).Statistical AnalysisData analysis of experiments using RLFs was performed using two-tailed Student’s t-tests or one- or two-way analysis of variance, followed by Bonferroni post hoc testing. For experiments comparing categorical data, analysis was performed by creating contingency tables and applying χ2Thannickal VJ Horowitz JC Evolving concepts of apoptosis in idiopathic pulmonary fibrosis.Proc Am Thorac Soc. 2006; 3: 350-356Crossref PubMed Scopus (269) Google Scholar or Fisher’s exact tests. All computations were performed with Microsoft Excel and GraphPad Prism software version 4.0c.ResultsGene Expression Profiling Identifies Twist1 as the Most Consistently Up-Regulated Transcription Factor in IPF LungsIPF tissue samples were selected for presence of fibroblastic foci. Unsupervised clustering of gene expression data revealed two primary branches that corresponded to either IPF or control lungs (data not shown), indicating that IPF lungs exhibited a unique gene expression signature. Among the 30 most consistently up-regulated genes were several proteins previously associated with IPF, such as several extracellular matrix proteins and IGFBP-5(Table 1).19Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding proteins 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar One of the most consistently up-regulated genes was twist1 (Table 2 and Figure 1A). Interestingly, although earlier gene expression profiling studies of this disease did not identify twist1 as being up-regulated,20Pardo A Gibson K Cisneros J Richards TJ Yang Y Becerril C Yousem S Herrera I Ruiz V Selman M Kaminski N Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis.PLoS Med. 2005; 2: e251Crossref PubMed Scopus (354) Google Scholar, 21Zuo F Kaminski N Eugui E Allard J Yakhini Z Ben-Dor A Lollini L Morris D Kim Y DeLustro B Sheppard D Pardo A Selman M Heller RA Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans.Proc Natl Acad Sci USA. 2002; 99: 6292-6297Crossref PubMed Scopus (515) Google Scholar, 22Yang IV Burch LH Steele MP Savov JD Hollingsworth JW McElvania-Tekippe E Berman KG Speer MC Sporn TA Brown KK Schwarz MI Schwartz DA Gene expression profiling of familial and sporadic interstitial pneumonia.Am J Respir Crit Care Med. 2007; 175: 45-54Crossref PubMed Scopus (142) Google Scholar a recent reanalysis of microarray data did identify twist1 as being-up-regulated, although this result was not explored further.23Selman M Pardo A Kaminski N Idiopathic pulmonary fibrosis: aberrant recapitulation of developmental programs?.PLoS Med. 2008; 5: e62Crossref PubMed Scopus (266) Google Scholar Quantitative RT-PCR (qRT-PCR) confirmed these results (Figure 1B). Strong immunoreactivity against Twist1 protein was present in the nuclei of multiple cells types in IPF but not control lungs, particularly interstitial cells within fibroblastic foci as well as type II alveolar epithelial cells (Figure 1C).Table 2Thirty Most Significantly Up-Regulated Genes in IPF SamplesAffymetrix IDGene symbolGene nameEntrez gene IDFold increase32249_atCFHR1Complement factor H-related 130784.140328_atTwist1Twist homolog (acrocephalosyndactyly 3; Saethre-Chotzen syndrome)72915.51396_atIGFBP5Insulin-like growth factor-binding protein 534883.038722_atCOL6A1Collagen, type VI, α112912.038111_atVCANChondroitin sulfate proteoglycan 2 (versican)14623.938650_atIGFBP5Insulin-like growth factor-binding protein 534882
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