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• W2061203824 abstract "Although the early growth response-2 (Egr-2, alias Krox20) protein shows structural and functional similarities to Egr-1, these two related early-immediate transcription factors are nonredundant. Egr-2 plays essential roles in peripheral nerve myelination, adipogenesis, and immune tolerance; however, its regulation and role in tissue repair and fibrosis remain poorly understood. We show herein that transforming growth factor (TGF)-β induced a Smad3-dependent sustained stimulation of Egr2 gene expression in normal fibroblasts. Overexpression of Egr-2 was sufficient to stimulate collagen gene expression and myofibroblast differentiation, whereas these profibrotic TGF-β responses were attenuated in Egr-2–depleted fibroblasts. Genomewide transcriptional profiling revealed that multiple genes associated with tissue remodeling and wound healing were up-regulated by Egr-2, but the Egr-2–regulated gene expression profile overlapped only partially with the Egr-1–regulated gene profile. Levels of Egr-2 were elevated in lesional tissue from mice with bleomycin-induced scleroderma. Moreover, elevated Egr-2 was noted in biopsy specimens of skin and lung from patients with systemic sclerosis. These results provide the first evidence that Egr-2 is a functionally distinct transcription factor that is both necessary and sufficient for TGF-β–induced profibrotic responses and is aberrantly expressed in lesional tissue in systemic sclerosis and in a murine model of scleroderma. Together, these findings suggest that Egr-2 plays an important nonredundant role in the pathogenesis of fibrosis. Targeting Egr-2 might represent a novel therapeutic strategy to control fibrosis. Although the early growth response-2 (Egr-2, alias Krox20) protein shows structural and functional similarities to Egr-1, these two related early-immediate transcription factors are nonredundant. Egr-2 plays essential roles in peripheral nerve myelination, adipogenesis, and immune tolerance; however, its regulation and role in tissue repair and fibrosis remain poorly understood. We show herein that transforming growth factor (TGF)-β induced a Smad3-dependent sustained stimulation of Egr2 gene expression in normal fibroblasts. Overexpression of Egr-2 was sufficient to stimulate collagen gene expression and myofibroblast differentiation, whereas these profibrotic TGF-β responses were attenuated in Egr-2–depleted fibroblasts. Genomewide transcriptional profiling revealed that multiple genes associated with tissue remodeling and wound healing were up-regulated by Egr-2, but the Egr-2–regulated gene expression profile overlapped only partially with the Egr-1–regulated gene profile. Levels of Egr-2 were elevated in lesional tissue from mice with bleomycin-induced scleroderma. Moreover, elevated Egr-2 was noted in biopsy specimens of skin and lung from patients with systemic sclerosis. These results provide the first evidence that Egr-2 is a functionally distinct transcription factor that is both necessary and sufficient for TGF-β–induced profibrotic responses and is aberrantly expressed in lesional tissue in systemic sclerosis and in a murine model of scleroderma. Together, these findings suggest that Egr-2 plays an important nonredundant role in the pathogenesis of fibrosis. Targeting Egr-2 might represent a novel therapeutic strategy to control fibrosis. Scleroderma or systemic sclerosis (SSc) is an acquired connective tissue disease of unknown etiology associated with progressive fibrosis in the skin and internal organs.1Jimenez S.A. Derk C.T. Following the molecular pathways toward an understanding of the pathogenesis of systemic sclerosis.Ann Intern Med. 2004; 140: 37-50Crossref PubMed Google Scholar, 2Gabrielli A. Avvedimento E.V. Krieg T. Scleroderma.N Engl J Med. 2009; 360: 1989-2003Crossref PubMed Scopus (1138) Google Scholar, 3Abraham D.J. Varga J. Scleroderma: from cell and molecular mechanisms to disease models.Trends Immunol. 2005; 26: 587-595Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar There is no effective therapy to prevent or control the progression of fibrosis in SSc. Transforming growth factor (TGF)-β is a potent inducer of collagen synthesis, myofibroblast differentiation, and epithelial-mesenchymal transition; and is implicated in both physiological and pathological tissue repair.4Varga J. Pasche B. Antitransforming growth factor-beta therapy in fibrosis: recent progress and implications for systemic sclerosis.Curr Opin Rheumatol. 2008; 20: 720-728Crossref PubMed Scopus (87) Google Scholar, 5Heldin C.H. Landstrom M. Moustakas A. Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition.Curr Opin Cell Biol. 2009; 21: 166-176Crossref PubMed Scopus (524) Google Scholar The intracellular signaling pathways that mediate fibrotic TGF-β responses remain incompletely understood. The early growth response (Egr) transcription factors belong to a family comprising Egr-1 (alias Krox-20), Egr-2 (alias Krox-24), Egr-3, and Egr-4, along with their endogenous inhibitors, NGFI-A binding protein (NAB1 and NAB2).6Thiel G. Cibelli G. Regulation of life and death by the zinc finger transcription factor Egr-1.J Cell Physiol. 2002; 193: 287-292Crossref PubMed Scopus (505) Google Scholar, 7O'Donovan K.J. Tourtellotte W.G. Millbrandt J. Baraban J.M. The EGR family of transcription-regulatory factors: progress at the interface of molecular and systems neuroscience.Trends Neurosci. 1999; 22: 167-173Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar These pleiotropic early-immediate gene products are induced in response to multiple extracellular signals, such as growth factors, cytokines, hypoxia, and mechanical forces associated with injury and cellular stress. Egr-1, Egr-2, and Egr-3 share conserved zinc finger DNA-binding domains that recognize a 9-bp GC-rich DNA sequence called Egr-binding element (EBS) found in multiple target gene promoters.8Christy B. Nathans D. DNA binding site of the growth factor-inducible protein Zif268.Proc Natl Acad Sci U S A. 1989; 86: 8737-8741Crossref PubMed Scopus (455) Google Scholar The induction of Egr-1 is characteristically rapid and transient.9Sukhatme V.P. Kartha S. Toback F.G. Taub R. Hoover R.G. Tsai-Morris C.H. A novel early growth response gene rapidly induced by fibroblast, epithelial cell and lymphocyte mitogens.Oncogene Res. 1987; 1: 343-355PubMed Google Scholar In contrast to Egr-1, which has been extensively investigated, the regulation and function of Egr-2 and Egr-3 remain less well characterized. These transcription factors show a high degree of homology to each other and to Egr-1 but are regulated by distinct intracellular signaling pathways in a cell type–specific manner. Moreover, it is becoming clear that individual members of the Egr gene family serve nonredundant and occasionally even opposing biological functions. The functional divergence is highlighted by genetic studies that showed that, although Egr-1–null mice have no spontaneous phenotype,10Lee S.L. Sadovsky Y. Swirnoff A.H. Polish J.A. Goda P. Gavrilina G. Milbrandt J. Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1).Science. 1996; 273: 1219-1221Crossref PubMed Scopus (440) Google Scholar targeting Egr-2 resulted in early mouse lethality due to defective myelination.11Le N. Nagarajan R. Wang J.Y. Araki T. Schmidt R.E. Milbrandt J. Analysis of congenital hypomyelinating Egr2Lo/Lo nerves identifies Sox2 as an inhibitor of Schwann cell differentiation and myelination.Proc Natl Acad Sci U S A. 2005; 102: 2596-2601Crossref PubMed Scopus (242) Google Scholar Functional divergence is also evident in immune regulation: Egr-2 appears to be required for immune tolerance, whereas Egr-1 enhances T-cell activity in response to T-cell receptor engagement.12Collins S. Lutz M.A. Zarek P.E. Anders R.A. Kersh G.J. Powell J.D. Opposing regulation of T cell function by Egr-1/NAB2 and Egr-2/Egr-3.Eur J Immunol. 2008; 38: 528-536Crossref PubMed Scopus (86) Google Scholar, 13Basson M.A. Wilson T.J. Legname G.A. Sarner N. Tomlinson P.D. Tybulewicz V.L. Zamoyska R. Early growth response (Egr)-1 gene induction in the thymus in response to TCR ligation during early steps in positive selection is not required for CD8 lineage commitment.J Immunol. 2000; 165: 2444-2450PubMed Google Scholar These and similar observations underline the significant differences that exist between these two structurally related members of the Egr transcription factor family. In previous studies14Bhattacharyya S. Chen S.J. Wu M. Warner-Blankenship M. Ning H. Lakos G. Mori Y. Chang E. Nihijima C. Takehara K. Feghali-Bostwick C. Varga J. Smad-independent transforming growth factor-beta regulation of early growth response-1 and sustained expression in fibrosis: implications for scleroderma.Am J Pathol. 2008; 173: 1085-1099Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar investigating the expression and function of Egr-1 in the context of fibrogenesis, we showed that Egr-1 was required for TGF-β–induced fibroblast activation and played an important role in both physiological and pathological fibrogenesis. The expression of Egr-1 was rapidly induced by TGF-β in skin and lung fibroblasts, and Egr-1 was sufficient to stimulate the transcription of COL1A2 and other TGF-β–inducible genes.14Bhattacharyya S. Chen S.J. Wu M. Warner-Blankenship M. Ning H. Lakos G. Mori Y. Chang E. Nihijima C. Takehara K. Feghali-Bostwick C. Varga J. Smad-independent transforming growth factor-beta regulation of early growth response-1 and sustained expression in fibrosis: implications for scleroderma.Am J Pathol. 2008; 173: 1085-1099Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Chen S.J. Ning H. Ishida W. Sodin-Semrl S. Takagawa S. Mori Y. Varga J. The early-immediate gene EGR-1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression.J Biol Chem. 2006; 281: 21183-21197Crossref PubMed Scopus (142) Google Scholar Moreover, mice with targeted deletion of Egr-1 were protected from bleomycin-induced fibrosis.16Wu M. Melichian D.S. de la Garza M. Gruner K. Bhattacharyya S. Barr L. Nair A. Shahrara S. Sporn P.H. Mustoe T.A. Tourtellotte W.G. Varga J. Essential roles for early growth response transcription factor Egr-1 in tissue fibrosis and wound healing.Am J Pathol. 2009; 175: 1041-1055Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar In contrast to Egr-1, the expression, regulation, and physiological roles of Egr-2 in connective tissue metabolism remain incompletely understood. Therefore, the present studies focused on Egr-2 in the context of TGF-β–mediated fibrogenesis in vivo and in vitro. The results demonstrate that Egr-2 is markedly elevated in skin and lung biopsy specimens from patients with SSc and in a mouse model of scleroderma. In explanted normal fibroblasts, TGF-β stimulated the expression of Egr-2; however, the kinetics and signal transduction pathways were distinct from those associated with Egr-1. Overexpression of Egr-2 in these cells was associated with induction of profibrotic gene responses. Taken together, these findings identify Egr-2 as a novel TGF-β–inducible mediator of profibrotic responses that is aberrantly expressed in SSc and murine fibrosis and suggest an important nonredundant functional role for Egr-2 in pathogenesis. Primary cultures of skin fibroblasts were established by explantation from biopsy specimens of healthy adults (n = 3) or patients with diffuse cutaneous SSc (n = 3) or from neonatal foreskin specimens.17Varga J. Brenner D. Phan S.H. Fibrosis Research: Methods and Protocols. Humana Press, Totowa, NJ2005Crossref Google Scholar The protocols for skin biopsies were approved by the Institutional Review Board at Northwestern University, Chicago, IL. Fibroblasts were maintained in Eagle's minimum essential medium or Dulbecco's modified Eagle medium, supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland), 50 μg/mL penicillin, and 50 μg/mL streptomycin, in a humidified atmosphere of 5% CO2 at 37°C; and studied between passages 2 and 8.17Varga J. Brenner D. Phan S.H. Fibrosis Research: Methods and Protocols. Humana Press, Totowa, NJ2005Crossref Google Scholar At confluence, serum-free media supplemented with 0.1% bovine serum albumin (BSA) were added to the cultures for 16 hours before the addition of TGF-β2 or TGF-β1 (both from Peprotech, Rocky Hill, NJ). Mouse embryonic fibroblasts explanted from Smad3−/− or wild-type embryos were maintained in Dulbecco's modified Eagle medium. In selected experiments, LY294002 (Cell Signaling, Danvers, MA), SB431542 (Glaxo Smith Kline, King of Prussia, PA), or U0126 (Cell Signaling) was added to the cultures. At the end of each experiment, cultures were harvested and RNA was isolated using minikits (RNeasy Plus; Qiagen, Valencia, CA) and examined by real-time quantitative PCR (qPCR).18Fang F. Flegler A.J. Du P. Lin S. Clevenger C.V. Expression of cyclophilin B is associated with malignant progression and regulation of genes implicated in the pathogenesis of breast cancer.Am J Pathol. 2009; 174: 297-308Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar RNA, 1 μg, was used for cDNA synthesis in a 20-μL reaction volume using supermix (cDNA Synthesis Supermix; Quanta Biosciences, Gaithersburg, MD): 8 μL cDNA, 2 μL primers (2 μmol/L each), and 10 μL ×2 mix (Power SYBR Mastermix; Applied Biosystems, Foster City, CA). qPCR was performed in triplicate on a thermocycler (ABI 7300; Applied Biosystems). Data were normalized to 18S RNA, and fold change was represented as follows: 2−ΔΔCt (2−[(Ct target-Ct 18S)treatment-(Ct target-Ct 18S)nontreatment]). The primers used for qPCR are listed in Table 1.18Fang F. Flegler A.J. Du P. Lin S. Clevenger C.V. Expression of cyclophilin B is associated with malignant progression and regulation of genes implicated in the pathogenesis of breast cancer.Am J Pathol. 2009; 174: 297-308Abstract Full Text Full Text PDF PubMed Scopus (61) Google ScholarTable 1Primers Used for qPCRGene namePrimer sequencesRNA accession no.18S ribosomal RNAOlg1 5′-CCCCATGAACGAGGGAATT-3′NR_003286Olg2 5′-GGGACTTAATCAACGCAAGCTT-3′GAPDHOlg3 5′-CATGAGAAGTATGACAACAGCCT-3′NM_002046Olg4 5′-AGTCCTTCCACGATACCAAAGT-3′COL1A2Olg10 5′-CGGACGACCTGGTGAGAGA-3′NM_000089Olg11 5′-CATTGTGTCCCCTAATGCCTT-3′ACTA2 (α-SMA)Olg20 5′-CAGGGCTGTTTTCCCATCCAT-3′NM_001613Olg21 5′-GCCATGTTCTATCGGGTACTTC-3′CTGF (CCN2)Olg18 5′-AGCTGACCTGGAAGAGAACATTAAG-3′NM_001901Olg19 5′-GATAGGCTTGGAGATTTTGGGAGTA-3′Egr-1Olga 5′-TGCGGCAGAAGGACAAGAAAGC-3′NM_001964Olgb 5′-TGAGGAAGGGAAGCTGCTGACC-3′Egr-2Olg26 5′-AACGGAGTGGCCGGAGAT-3′NM_000399Olg27 5′-ATGGGAGATCCAACGACCTCTT-3′Egr-3Olg31 5′-GCGACCTCTACTCAGAGCC-3′NM_004430Olg32 5′-ATGGGGAAGAGATTGCTGTCC-3′COL1A1Olg149 5′-GCTGGTGTGATGGGATTC-3′NM_000088Olg150 5′-GGGAACACCTCGCTCT-3′Pai1Olg151 5′-CGCCTCTTCCACAAATCA-3′NM_000602Olg162 5′-GCAGTTCCAGGATGTCGTA-3′Plod2Olg179 5′-CTTTAGTGTGGATGCAGATGTTG-3′NM_182943Olg180 5′-GACTCAATGCTCCCCAGAAA-3′Col5A1Olg230 5′-TCGCTTACAGAGTCACCAAAG-3′NM_000093Olg231 5′-GTTGTAGATGGAGACCAGGAAG-3′Itgb1Olg222 5′-CAGCGCATATCTGGAAATTTGG-3′NM_002211Olg223 5′-TCTCCAGCAAAGTGAAACCC-3′Primer sequences were obtained using computer software (Primer Express 3.0; Primerbank Boston, MA) or were from published sequences.14Bhattacharyya S. Chen S.J. Wu M. Warner-Blankenship M. Ning H. Lakos G. Mori Y. Chang E. Nihijima C. Takehara K. Feghali-Bostwick C. Varga J. Smad-independent transforming growth factor-beta regulation of early growth response-1 and sustained expression in fibrosis: implications for scleroderma.Am J Pathol. 2008; 173: 1085-1099Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Fang F. Flegler A.J. Du P. Lin S. Clevenger C.V. Expression of cyclophilin B is associated with malignant progression and regulation of genes implicated in the pathogenesis of breast cancer.Am J Pathol. 2009; 174: 297-308Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Most primer pairs span two exons, except the intronless 18S ribosomal RNA. Primers were tested by dissociation curve analysis to ensure only a single amplicon. The base pairs that the forward primer starts and reverse primer ends are based on RNA sequence. All primers are located inside the coding sequence. Open table in a new tab Primer sequences were obtained using computer software (Primer Express 3.0; Primerbank Boston, MA) or were from published sequences.14Bhattacharyya S. Chen S.J. Wu M. Warner-Blankenship M. Ning H. Lakos G. Mori Y. Chang E. Nihijima C. Takehara K. Feghali-Bostwick C. Varga J. Smad-independent transforming growth factor-beta regulation of early growth response-1 and sustained expression in fibrosis: implications for scleroderma.Am J Pathol. 2008; 173: 1085-1099Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Fang F. Flegler A.J. Du P. Lin S. Clevenger C.V. Expression of cyclophilin B is associated with malignant progression and regulation of genes implicated in the pathogenesis of breast cancer.Am J Pathol. 2009; 174: 297-308Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Most primer pairs span two exons, except the intronless 18S ribosomal RNA. Primers were tested by dissociation curve analysis to ensure only a single amplicon. The base pairs that the forward primer starts and reverse primer ends are based on RNA sequence. All primers are located inside the coding sequence. The Egr–2–luciferase (luc) construct harbors a truncated human Egr2 gene promoter with 5′ ends at –1.3 kb fused to firefly luc.19Kao S.C. Wu H. Xie J. Chang C.P. Ranish J.A. Graef I.A. Crabtree G.R. Calcineurin/NFAT signaling is required for neuregulin-regulated Schwann cell differentiation.Science. 2009; 323: 651-654Crossref PubMed Scopus (169) Google Scholar pCMV Egr-1 or pCMV Egr-2 contains the full-length Egr-1 or Egr-2 coding sequence, respectively.20Tan L. Peng H. Osaki M. Choy B.K. Auron P.E. Sandell L.J. Goldring M.B. Egr-1 mediates transcriptional repression of COL2A1 promoter activity by interleukin-1beta.J Biol Chem. 2003; 278: 17688-17700Crossref PubMed Scopus (110) Google Scholar 772COL1A2–CAT harbors the truncated human COL1A2 promoter with 5′ ends at −772 bp fused to chloramphenicol acetyltransferase (CAT).21Ihn H. Ohnishi K. Tamaki T. LeRoy E.C. Trojanowska M. Transcriptional regulation of the human alpha2(I) collagen gene: combined action of upstream stimulatory and inhibitory cis-acting elements.J Biol Chem. 1996; 271: 26717-26723Crossref PubMed Scopus (120) Google Scholar p[EBS]4-luc harbors four copies of the minimal Egr-1–responsive element linked to luc.22Thiel G. Kaufmann K. Magin A. Lietz M. Bach K. Cramer M. The human transcriptional repressor protein NAB1: expression and biological activity.Biochim Biophys Acta. 2000; 1493: 289-301Crossref PubMed Scopus (101) Google Scholar ASMA–luc harbors a 1.1-kb promoter fragment from the mouse alpha smooth muscle actin (ASMA; gene symbol, ACTA) gene upstream of luc.23Min B.H. Foster D.N. Strauch A.R. The 5′-flanking region of the mouse vascular smooth muscle alpha-actin gene contains evolutionarily conserved sequence motifs within a functional promoter.J Biol Chem. 1990; 265: 16667-16675Abstract Full Text PDF PubMed Google Scholar Fibroblasts at early confluence were transfected with the indicated constructs using a kit (SuperFect Transfection kit; Qiagen). Cultures were incubated in serum-free media containing 0.1% BSA for 16 hours, followed by TGF-β2 for a further 24 hours. Cultures were harvested and whole cell lysates were assayed for their chloramphenicol acetyltransferase activities by liquid scintillation counting or luc activities using the dual-luc reporter assay system (Promega, Madison, WI). In each experiment, Renilla luc pRL-TK (Promega) was cotransfected as a control for transfection efficiency.24Fang F. Antico G. Zheng J. Clevenger C.V. Quantification of PRL/Stat5 signaling with a novel pGL4-CISH reporter.BMC Biotechnol. 2008; 8: 11Crossref PubMed Scopus (32) Google Scholar Transient transfection experiments were performed in triplicate and repeated at least twice, with consistent results. Duplex oligo short-interfering RNAs (siRNAs) specific to human Smad3 (Pierce, Rockford, IL) or Egr-2 (Dharmacon, Lafayette, CO) and scrambled control siRNA were used to deplete endogenous Smad3 or Egr-2. Skin fibroblasts in serum-free media containing 0.1% BSA were transfected with indicated siRNA at a final concentration of 10 nmol/L. Sixteen hours later, fresh media containing TGF-β were added and the incubations were continued for a further 24 hours. Knockdown efficiency was determined by measuring endogenous protein or mRNA levels by Western analysis or real-time qPCR. For adenovirus (Ad) infection, confluent fibroblasts in serum-free media were infected with Ad–Egr-2 (Vector Biolabs, Philadelphia, PA) or Ad GFP (green fluorescent protein) and incubated for up to 48 hours before harvesting. Fibroblasts (10,000 cells per well) were seeded onto eight-well chamber-glass slides (Lab-Tek II; Nalge Nunc International, Naperville, IL) and incubated in serum-free Eagle's minimum essential medium with 0.1% BSA for 16 hours. Fresh media with TGF-β2 (10 ng/mL) were added, and the incubations were continued for a further 1 to 24 hours. At the end of the experiments, cells were fixed, permeabilized, and incubated with primary antibodies to Egr-2 at 1:200 dilution (Covance, Princeton, NJ) or to type I collagen at 1:100 dilution (Southern Biotech, Birmingham, AL).14Bhattacharyya S. Chen S.J. Wu M. Warner-Blankenship M. Ning H. Lakos G. Mori Y. Chang E. Nihijima C. Takehara K. Feghali-Bostwick C. Varga J. Smad-independent transforming growth factor-beta regulation of early growth response-1 and sustained expression in fibrosis: implications for scleroderma.Am J Pathol. 2008; 173: 1085-1099Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Cells were then washed with PBS and incubated with secondary antibodies at 1:200 dilution (Alexa Fluor 488 and 594; Invitrogen, Carlsbad, CA) and viewed under a confocal microscope (Nikon C1Si).25Wu M. Melichian D.S. Chang E. Warner-Blankenship M. Ghosh A.K. Varga J. Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.Am J Pathol. 2009; 174: 519-533Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar At the end of each experiment, fibroblasts were harvested, lysed in radioimmunoprecipitation assay buffer supplemented with 1× protease inhibitor cocktail and whole cell lysates (100 μg), and subjected to Western analysis using antibodies to Egr-2 (Covance), type I collagen (Southern Biotech), and glyceraldehyde-3-phosphate dehydrogenase (Zymed, San Francisco, CA). Images were detected by electrochemiluminescence reagents (Pierce) and developed on film. To examine genomewide changes in gene expression induced by Egr-2, serum-starved confluent fibroblasts were infected with Ad–Egr-1, Ad–Egr-2, or Ad-GFP using multiplicity of infection 50, previously shown to be optimal for high-level expression. Close to 100% of infected fibroblasts showed strong GFP expression by immunofluorescence microscopy. After 48 hours of incubation, total RNA was isolated from three independent cultures using kits (RNeasy Mini Plus Kits; Qiagen). The integrity of RNA was determined using a bioanalyzer (Agilent, Santa Clara, CA). Fluorescently labeled cDNA was prepared using labeling kits (Ambion, Austin, TX), followed by hybridization to microarray chips (Illumina Human HT-12 Version 3) containing 44,000 probes (Illumina, San Diego, CA). Raw signal intensities for each probe were obtained using data analysis software (Beadstudio; Illumina) and imported to the Bioconductor lumi package for transformation and normalization.26Du P. Kibbe W.A. Lin S.M. nuID: a universal naming scheme of oligonucleotides for illumina, affymetrix, and other microarrays.Biol Direct. 2007; 2: 16Crossref PubMed Scopus (70) Google Scholar, 27Du P. Kibbe W.A. Lin S.M. lumi: a pipeline for processing Illumina microarray.Bioinformatics. 2008; 24: 1547-1548Crossref PubMed Scopus (1621) Google Scholar, 28Lin S.M. Du P. Huber W. Kibbe W.A. Model-based variance-stabilizing transformation for Illumina microarray data.Nucleic Acids Res. 2008; 36: e11Crossref PubMed Scopus (402) Google Scholar The data were preprocessed using a variance stabilization transformation method,28Lin S.M. Du P. Huber W. Kibbe W.A. Model-based variance-stabilizing transformation for Illumina microarray data.Nucleic Acids Res. 2008; 36: e11Crossref PubMed Scopus (402) Google Scholar followed by quantile normalization. Data from probes that produced signals near or lower than background levels (estimated based on Illumina negative control probes) with all samples were discarded. Genes that showed greater than twofold up- or down-regulation in Egr-2–expressing fibroblasts compared with controls at 48 hours were subjected to further analysis. To examine the tissue expression of Egr-2 in vivo, scleroderma was induced in mice by bleomycin injections (Jackson Laboratory, Bar Harbor, ME).29Takagawa S. Lakos G. Mori Y. Yamamoto T. Nishioka K. Varga J. Sustained activation of fibroblast transforming growth factor-beta/Smad signaling in a murine model of scleroderma.J Invest Dermatol. 2003; 121: 41-50Crossref PubMed Scopus (100) Google Scholar The animal protocols were institutionally approved by the Northwestern University Animal Care and Use Committee. Filter-sterilized bleomycin (20 μg per mouse dissolved in PBS) (Mayne Pharma, Paramus, NJ) or PBS was administered by daily s.c. injections into the shaved backs of 6- to 8-week-old female BALB/c mice.30Lakos G. Takagawa S. Varga J. Animal models of scleroderma.Methods Mol Med. 2004; 102: 377-393PubMed Google Scholar Each experimental group consisted of at least five mice. At the end of the experiments, mice were sacrificed and lesional skin was harvested.25Wu M. Melichian D.S. Chang E. Warner-Blankenship M. Ghosh A.K. Varga J. Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.Am J Pathol. 2009; 174: 519-533Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Sections (4 μm) were deparaffinized, rehydrated, and immersed in TBS–T buffer (Tris-buffered saline and 0.1% Tween-20); and treated with target retrieval solution (DAKO, Carpinteria, CA) at 95°C for 10 minutes, followed by incubation with primary antibodies against Egr-2 (Covance) at 1:200 dilution (anti–Egr-2 antibody was used for both mouse and human sections). Secondary antibodies (Histomouse-Max kit; Zymed) at 1:200 dilution were used to detect bound antibodies. Substitution of the primary antibodies with isotype-matched irrelevant IgG served as negative controls. After counterstaining with hematoxylin, sections were mounted (Permount; Fisher Scientific, Pittsburgh, PA) and viewed under a microscope (Olympus BH-2).25Wu M. Melichian D.S. Chang E. Warner-Blankenship M. Ghosh A.K. Varga J. Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.Am J Pathol. 2009; 174: 519-533Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Unambiguously immunopositive fibroblastic cells were counted by a blinded observed. Results are shown as the mean ± SEM from a representative section from each of five mice per experimental group. Skin biopsy specimens from the affected forearm from patients with early diffuse cutaneous SSc or healthy adult control subjects were obtained under protocols approved by the Institutional Review Boards for Human Studies at Thomas Jefferson University, Philadelphia, PA, or Northwestern University. Paraffin-embedded or frozen sections (4-μm thick) were immunostained with the same primary antibody to Egr-2 (Covance) as used in the mouse studies (previously described), followed by incubation with rabbit IgG secondary antibodies (Alexa Fluor 488; Invitrogen). The entire skin section was scanned at ×20 magnification at four different microscopic fields spanning the dermis under a confocal laser scanning microscope (Zeiss LSM 510 META). The computer then generated 2.5-dimensional images plotting the fluorescence intensity of each microscopic field. Sections were analyzed with software (Image J) that calculated the sum of the intensity of each pixel in a given microscopic field as the integrated density of fluorescence. Each biopsy specimen is represented as the mean of four integrated density of fluorescence values per specimen.31Galdo F.D. Sotgia F. de Almeida C.J. Jasmin J.F. Musick M. Lisanti M.P. Jimenez S.A. Decreased expression of caveolin 1 in patients with systemic sclerosis: crucial role in the pathogenesis of tissue fibrosis.Arthritis Rheum. 2008; 58: 2854-2865Crossref PubMed Scopus (138) Google Scholar Egr-2 expression was also examined in lungs from patients with SSc-associated pulmonary fibrosis undergoing lung transplantation and in normal donor lungs. The biopsy protocols were approved by the Institutional Review Board for Human Studies at the University of Pittsburgh, Pittsburgh. Paraffin-embedded sections of lung tissue (6 μm) were immunostained with antibodies to Egr-2 (Covance) at 1:200 dilution, followed by incubation with biotinylated anti-rabbit IgG secondary antibodies (sc-2089; Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 dilution. Tissue sections from a lung tumor were used as positive control, and substitution of nonspecific rabbit IgG for Egr-2 antibody was used as a negative control.32Hsu E. Feghali-Bostwick C.A. Insulin-like growth factor-II is increa" @default.
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• W2061203824 title "The Early Growth Response Gene Egr2 (Alias Krox20) Is a Novel Transcriptional Target of Transforming Growth Factor-β that Is Up-Regulated in Systemic Sclerosis and Mediates Profibrotic Responses" @default.
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