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Role of Early Growth Response-1 (Egr-1) in Interleukin-13-induced Inflammation and Remodeling

2006; Elsevier BV; Volume: 281; Issue: 12 Linguagem: Inglês

10.1074/jbc.m506770200

1083-351X

Soo Jung Cho, Min‐Jong Kang, Robert Homer, Hye‐Ryun Kang, Xuchen Zhang, Patty J. Lee, Jack A. Elias, Chun Geun Lee,

Eosinophilic Esophagitis

IL-13 is an important stimulator of inflammation and tissue remodeling at sites of Th2 inflammation, which plays a key role in the pathogenesis of a variety of human disorders. We hypothesized that the ubiquitous transcription factor, early growth response-1 (Egr-1), plays a key role in IL-13-induced tissue responses. To test this hypothesis we compared the expression of Egr-1 and related moieties in lungs from wild type mice and transgenic mice in which IL-13 was overexpressed in a lung-specific fashion. We simultaneously characterized the effects of a null mutation of Egr-1 on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 stimulates Egr-1 via an Erk1/2-independent Stat6-dependent pathway(s). They also demonstrate that IL-13 is a potent stimulator of eosinophil- and mononuclear cell-rich inflammation, alveolar remodeling, and tissue fibrosis in mice with wild type Egr-1 loci and that these alterations are ameliorated in the absence of Egr-1. Lastly, they provide insights into the mechanisms of these processes by demonstrating that IL-13 stimulates select CC and CXC chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL2/3, MCP-1/CCL-2, MCP-2/CCL-8, MCP-3/CCL-7, MCP-5/CCL-12, KC/CXCL-1, and Lix/CXCL-5), matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and apoptosis regulators (caspase-3, -6, -8, and -9 and Bax) and activates transforming growth factor-β1 and pulmonary caspases via Egr-1-dependent pathways. These studies demonstrate that Egr-1 plays a key role in the pathogenesis of IL-13-induced inflammatory and remodeling responses. IL-13 is an important stimulator of inflammation and tissue remodeling at sites of Th2 inflammation, which plays a key role in the pathogenesis of a variety of human disorders. We hypothesized that the ubiquitous transcription factor, early growth response-1 (Egr-1), plays a key role in IL-13-induced tissue responses. To test this hypothesis we compared the expression of Egr-1 and related moieties in lungs from wild type mice and transgenic mice in which IL-13 was overexpressed in a lung-specific fashion. We simultaneously characterized the effects of a null mutation of Egr-1 on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 stimulates Egr-1 via an Erk1/2-independent Stat6-dependent pathway(s). They also demonstrate that IL-13 is a potent stimulator of eosinophil- and mononuclear cell-rich inflammation, alveolar remodeling, and tissue fibrosis in mice with wild type Egr-1 loci and that these alterations are ameliorated in the absence of Egr-1. Lastly, they provide insights into the mechanisms of these processes by demonstrating that IL-13 stimulates select CC and CXC chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL2/3, MCP-1/CCL-2, MCP-2/CCL-8, MCP-3/CCL-7, MCP-5/CCL-12, KC/CXCL-1, and Lix/CXCL-5), matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and apoptosis regulators (caspase-3, -6, -8, and -9 and Bax) and activates transforming growth factor-β1 and pulmonary caspases via Egr-1-dependent pathways. These studies demonstrate that Egr-1 plays a key role in the pathogenesis of IL-13-induced inflammatory and remodeling responses. Interleukin (IL 2The abbreviations used are: IL, interleukin; MMP, matrix metalloproteinase; TGF, transforming growth factor; uPA, urinary plasminogen activator; BAL, bronchoalveolar lavage; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PARP, poly(ADP-ribose) polymerase; ICAD, inhibitor of caspase-activated DNase; Erk, extracellular signal-related kinase; Tg, transgenic; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; TSP-1, thrombospondin-1; 2The abbreviations used are: IL, interleukin; MMP, matrix metalloproteinase; TGF, transforming growth factor; uPA, urinary plasminogen activator; BAL, bronchoalveolar lavage; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PARP, poly(ADP-ribose) polymerase; ICAD, inhibitor of caspase-activated DNase; Erk, extracellular signal-related kinase; Tg, transgenic; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; TSP-1, thrombospondin-1;)-13 is a 12-kDa product of a gene on chromosome 5q31 that is produced in large quantities by stimulated Th2 cells. It was originally described as an IL-4-like molecule based on shared effector properties including the ability to stimulate IgE production. Subsequent studies demonstrated that IL-13 and IL-4 play distinct roles in biology with IL-4 contributing to Th2 cell differentiation and response generation, whereas IL-13 contributes as a major effector of Th2 inflammation and tissue remodeling (1Wynn T.A. Nat. Rev. Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1202) Google Scholar, 2Wills-Karp M. Curr. Allergy Asthma Rep. 2004; 4: 123-131Crossref PubMed Scopus (78) Google Scholar, 3Elias J.A. Lee C.G. Zheng T. Ma B. Homer R.J. Zhu Z. J. Clin. Investig. 2003; 111: 291-297Crossref PubMed Scopus (380) Google Scholar, 4Corry D.B. Kheradmand F. Am. J. Respir. Med. 2002; 1: 185-193Crossref PubMed Scopus (46) Google Scholar). The latter is nicely illustrated in studies from our laboratory and others that demonstrate that IL-13 is a potent stimulator of eosinophil-, macrophage- and lymphocyte-rich inflammation, mucus metaplasia, tissue fibrosis, and parenchymal proteolysis (5Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Investig. 1999; 103: 779-788Crossref PubMed Scopus (1481) Google Scholar, 6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar, 7Hancock A. Armstrong L. Gama R. Millar A. Am. J. Respir. Cell Mol. Biol. 1998; 18: 60-65Crossref PubMed Scopus (231) Google Scholar). In accord with these observations, IL-13 dysregulation has been documented, and IL-13 has been implicated in the pathogenesis of a variety of diseases characterized by inflammation and tissue remodeling including asthma, scleroderma, idiopathic pulmonary fibrosis, viral pneumonia, hepatic fibrosis, nodular sclerosing Hodgkin's disease, and chronic obstructive pulmonary diseases (COPD) (1Wynn T.A. Nat. Rev. Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1202) Google Scholar, 2Wills-Karp M. Curr. Allergy Asthma Rep. 2004; 4: 123-131Crossref PubMed Scopus (78) Google Scholar, 3Elias J.A. Lee C.G. Zheng T. Ma B. Homer R.J. Zhu Z. J. Clin. Investig. 2003; 111: 291-297Crossref PubMed Scopus (380) Google Scholar, 4Corry D.B. Kheradmand F. Am. J. Respir. Med. 2002; 1: 185-193Crossref PubMed Scopus (46) Google Scholar, 7Hancock A. Armstrong L. Gama R. Millar A. Am. J. Respir. Cell Mol. Biol. 1998; 18: 60-65Crossref PubMed Scopus (231) Google Scholar, 8Belperio J.A. Dy M. 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Studies from our laboratory and others have demonstrated that IL-13 mediates its tissue effects by activating a broad array of downstream target genes including chemokines, matrix metalloproteinases (MMPs), transforming growth factor (TGF)-β1, and chitinases (6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar, 14Zhu Z. Ma B. Zheng T. Homer R.J. Lee C.G. Charo I.F. Noble P. Elias J.A. J. Immunol. 2002; 168: 2953-2962Crossref PubMed Scopus (174) Google Scholar, 15Zhu Z. Zheng T. Homer R.J. Kim Y.K. Chen N.Y. Cohn L. Hamid Q. Elias J.A. Science. 2004; 304: 1678-1682Crossref PubMed Scopus (680) Google Scholar, 16Lee C.G. Homer R.J. Zhu Z. Lanone S. Wang X. Koteliansky V. Shipley J.M. Gotwals P. Noble P.W. Senior R.M. Elias J.A. J. Exp. Med. 2001; 194: 809-821Crossref PubMed Scopus (769) Google Scholar, 17Lanone S. Zheng T. Zhu Z. Liu W. Lee C.G. Ma B. Chen Q. Homer R.J. Wang J. Rabach L.A. Rabach M.E. Shipley J.M. Shapiro S.D. Senior R.M. Elias J.A. J. Clin. Investig. 2002; 110: 463-474Crossref PubMed Scopus (283) Google Scholar). The mechanisms that underlie many of these responses, however, have not been adequately defined.Egr-1 is an 80-82 kDa-inducible zinc finger transcription factor that has also been identified as nerve growth factor-induced A, Krox-24, ZIF-268, ETR-103, and TIS-8 (18Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (552) Google Scholar, 19Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (476) Google Scholar, 20Silverman E.S. Collins T. Am. J. Pathol. 1999; 154: 665-670Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). It is the prototype of the Egr family that includes Egr-1, Egr-2, Egr-3, Egr-4, and the Wilms' tumor product. Members of this family have been implicated in commitments to proliferation, differentiation, and the activation of cell death pathways. Egr-1 can be induced, both acutely and chronically, at sites of injury and repair by a variety of stimuli including cytokines, oxidized lipids, angiotensin II, hypoxia, H2O2, and mechanical injury (18Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (552) Google Scholar, 19Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (476) Google Scholar, 20Silverman E.S. Collins T. Am. J. Pathol. 1999; 154: 665-670Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 21Du B. Fu C. Kent K.C. Bush Jr., H. Schulick A.H. Kreiger K. Collins T. McCaffrey T.A. J. Biol. Chem. 2000; 275: 39039-39047Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22Okada M. Wang C.Y. Hwang D.W. Sakaguchi T. Olson K.E. Yoshikawa Y. Minamoto K. Mazer S.P. Yan S.F. Pinsky D.J. Circ. Res. 2002; 91: 135-142Crossref PubMed Scopus (39) Google Scholar). It mediates its effects by regulating the transcription of a wide array of downstream genes involved in inflammation, matrix formation, thrombosis, apoptosis, and remodeling. Prominent targets include the A and B chains of platelet-derived growth factor, fibroblast growth factor-2, vascular endothelial growth factor, CD44, tissue factor, fibronectin, MMPs, plasminogen activator inhibitor-1, and urinary plasminogen activator (uPA), p53, tumor necrosis factor, Fas, and FasL (18Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (552) Google Scholar, 19Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (476) Google Scholar, 20Silverman E.S. Collins T. Am. J. Pathol. 1999; 154: 665-670Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 23Virolle T. Adamson E.D. Baron V. Birle D. Mercola D. Mustelin T. de Belle I. Nat. Cell Biol. 2001; 3: 1124-1128Crossref PubMed Scopus (349) Google Scholar, 24Das A. Chendil D. Dey S. Mohiuddin M. Milbrandt J. Rangnekar V.M. Ahmed M.M. J. Biol. Chem. 2001; 276: 3279-3286Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In accord with these findings, Egr-1 is an important mediator of tissue inflammation and remodeling. Surprisingly, the regulation of Egr-1 by Th2 tissue responses and the role of Egr-1 in the pathogenesis of Th2-induced inflammation and remodeling have not been assessed.We hypothesized that Egr-1 is a critical mediator of IL-13 induced tissue responses. To test this hypothesis we characterized the expression of Egr-1 and related moieties in lungs from wild type mice and mice in which IL-13 was overexpressed in a lung-specific fashion. We also characterized the effects of a null mutation of Egr-1 on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of Egr-1. They also demonstrate that Egr-1 plays a key role in IL-13-induced inflammation, fibrosis, and alveolar remodeling. Lastly, they provide insights into the mechanisms of these processes by demonstrating that IL-13 stimulates chemokines, MMPs, and antiproteases, activates TGF-β1, and induces cell death via Egr-1-dependent pathways.MATERIALS AND METHODSOverexpression Transgenic Mice—CC10-IL-13 transgenic mice were generated in our laboratory, bred onto a C57BL/6 background, and used in these studies. These mice utilize the Clara cell 10-kDa protein (CC10) promoter to target IL-13 to the lung. The methods that were used to generate and characterize these mice were described previously (5Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Investig. 1999; 103: 779-788Crossref PubMed Scopus (1481) Google Scholar). In this modeling system, IL-13 caused a mononuclear cell- and eosinophil-rich tissue inflammatory response, alveolar enlargement, subepithelial and parenchymal fibrosis, mucus metaplasia, and respiratory failure and death as previously described (5Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Investig. 1999; 103: 779-788Crossref PubMed Scopus (1481) Google Scholar, 6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar, 14Zhu Z. Ma B. Zheng T. Homer R.J. Lee C.G. Charo I.F. Noble P. Elias J.A. J. Immunol. 2002; 168: 2953-2962Crossref PubMed Scopus (174) Google Scholar).Breeding to Egr-1 Null Mutant (-/-), Stat6 Null, and Dominant-negative MEK-1 Overexpressing Mice—CC10-IL-13 transgenic animals were bred with mice with wild type and null Egr-1 or Stat6 loci. Egr-1(-/-) mice were a generous gift from Dr. Jeffrey Milbrandt, Washington University, St. Louis, MO; Stat6(-/-) mice were purchased from Jackson laboratory (Bar Harbor MA) (25Lee S.L. Sadovsky Y. Swirnoff A.H. Polish J.A. Goda P. Gavrilina G. Milbrandt J. Science. 1996; 273: 1219-1221Crossref PubMed Scopus (434) Google Scholar). In all cases these mice had been bred for >10 generations onto a C57BL/6 genetic background. As a result of these crosses, CC10-IL-13 animals with (+/+) and (-/-) Egr-1 or Stat6 loci and CC10-IL-13 mice with (+) and without (-) the dominant-negative MEK-1 transgene were generated. Genotyping was accomplished as previously described (5Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Investig. 1999; 103: 779-788Crossref PubMed Scopus (1481) Google Scholar, 26Nandurkar H.H. Robb L. Tarlinton D. Barnett L. Kontgen F. Begley C.G. Blood. 1997; 90: 2148-2159Crossref PubMed Google Scholar). The phenotypes of these mice were compared as described below.In Vivo Administration of PD98059—Wild type and CC10-IL-13 animals were exposed to the MEK/Erk1/2 inhibitor PD98059 (Calbiochem) (5 mg/kg/day, via an intraperitoneal route) or its vehicle control for 14 days.Bronchoalveolar Lavage (BAL)—Lung inflammation was assessed by BAL as described previously (6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar, 27Wang J. Homer R.J. Hong L. Cohn L. Lee C.G. Jung S. Elias J.A. J. Immunol. 2000; 165: 2222-2231Crossref PubMed Scopus (53) Google Scholar). The BAL samples from each animal were pooled and centrifuged. The number and types of cells in the cell pellet were determined with light microscopy. The supernatants were stored at -20 °C until used.Lung Volume and Morphometric Assessments—Animals were anesthetized, the trachea was cannulated, and the lungs were removed and inflated with phosphate-buffered saline at 25 cm. The size of each lung was evaluated via volume displacement, and alveolar size was estimated from the mean chord length of the airspace as previously described by our laboratory (6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar). Chord length increases with alveolar enlargement.Histologic Evaluation—Animals were sacrificed, a median sternotomy was performed, and right heart perfusion was accomplished with calcium and magnesium-free phosphate-buffered saline. The heart and lungs were then removed en bloc inflated at 25 cm pressure with neutral buffered 10% formalin, fixed in 10% formalin, embedded in paraffin, sectioned, and stained. Hematoxylin and eosin, Mallory's trichrome, and periodic acid-Schiff with diastase stains were performed in the Research Histology Laboratory of the Department of Pathology at Yale University School of Medicine.mRNA Analysis—mRNA levels were evaluated by conventional reverse transcription PCR analysis as described previously (6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar, 28He C.H. Waxman A.B. Lee C.G. Link H. Rabach M.E. Ma B. Chen Q. Zhu Z. Zhong M. Nakayama K. Nakayama K.I. Homer R. Elias J.A. J. Clin. Investig. 2005; 115: 1039-1048Crossref PubMed Scopus (76) Google Scholar). The primers that were employed have been described (6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar, 14Zhu Z. Ma B. Zheng T. Homer R.J. Lee C.G. Charo I.F. Noble P. Elias J.A. J. Immunol. 2002; 168: 2953-2962Crossref PubMed Scopus (174) Google Scholar, 16Lee C.G. Homer R.J. Zhu Z. Lanone S. Wang X. Koteliansky V. Shipley J.M. Gotwals P. Noble P.W. Senior R.M. Elias J.A. J. Exp. Med. 2001; 194: 809-821Crossref PubMed Scopus (769) Google Scholar, 17Lanone S. Zheng T. Zhu Z. Liu W. Lee C.G. Ma B. Chen Q. Homer R.J. Wang J. Rabach L.A. Rabach M.E. Shipley J.M. Shapiro S.D. Senior R.M. Elias J.A. J. Clin. Investig. 2002; 110: 463-474Crossref PubMed Scopus (283) Google Scholar). For each cytokine, the optimal numbers of cycles that will produce a quantity of cytokine product that is directly proportional to the quantity of input mRNA was determined experimentally. β-Actin was used as an internal standard. Amplified PCR products were detected using ethidium bromide gel electrophoresis, quantitated electronically, and confirmed by nucleotide sequencing. In selected experiments, real time reverse transcription PCR was used as previously described (28He C.H. Waxman A.B. Lee C.G. Link H. Rabach M.E. Ma B. Chen Q. Zhu Z. Zhong M. Nakayama K. Nakayama K.I. Homer R. Elias J.A. J. Clin. Investig. 2005; 115: 1039-1048Crossref PubMed Scopus (76) Google Scholar), and the data are presented in the supplemental materials.Quantification of IL-13, TGF-β, and Chemokines—BAL IL-13, TGF-β, and chemokine levels were quantitated using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Inc., Minneapolis, MN) per the manufacturer's instructions.Quantification of Lung Collagen—Collagen content was determined biochemically by quantifying total soluble collagen using the Sircol Collagen Assay kit (Biocolor, Northern Ireland) according to the manufacture's instructions (16Lee C.G. Homer R.J. Zhu Z. Lanone S. Wang X. Koteliansky V. Shipley J.M. Gotwals P. Noble P.W. Senior R.M. Elias J.A. J. Exp. Med. 2001; 194: 809-821Crossref PubMed Scopus (769) Google Scholar). The data are expressed as the collagen content of the entire right lung.TUNEL Evaluations—End labeling of exposed 3′-OH ends of DNA fragments was undertaken with the TUNEL in situ cell death detection kit AP (Roche Diagnostics) as described by the manufacturer. After staining, 20 fields of alveoli were randomly chosen for examination. The labeled cells were expressed as a percentage of total nuclei.Immunoblott Analysis—Lung lysates were prepared, and Western analysis was undertaken with antibodies that reacted selectively with Egr-1, caspase-3, caspase-7, caspase-8, poly(ADP-ribose) polymerase (PARP), β-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), and inhibitor of caspase-activated DNase (ICAD) (Chemicon, Temecula, CA) as previously described (30Lee C.G. Cho S.J. Kang M.J. Chapoval S.P. Lee P.J. Noble P.W. Yehualaeshet T. Lu B. Flavell R.A. Milbrandt J. Homer R.J. Elias J.A. J. Exp. Med. 2004; 200: 377-389Crossref PubMed Scopus (312) Google Scholar).Statistics—Normally distributed data are expressed as means ± S.E. and assessed for significance by Student's t test or analysis of variance as appropriate. Data that were not normally distributed were assessed for significance using the Wilcoxon rank sum test.RESULTSIL-13 Regulation of Egr-1—To begin to understand the importance of Egr-1 in the pathogenesis of IL-13-induced tissue alterations, studies were undertaken to determine whether IL-13 regulated the expression and/or production of Egr-1 and related moieties. These experiments demonstrate that transgenic IL-13 is a potent stimulator of Egr-1 mRNA (Fig. 1A) and Egr-1 protein (Fig. 1B). These effects were not specific for Egr-1 because Egr-2 and Egr-3 are similarly regulated. However, the Egr-1-binding proteins NAB-1 and NAB-2 were not similarly altered (Fig. 1A).Role of Stat6 and Erk1/2 in IL-13 Stimulation of Egr-1—The signaling pathways that mediate the stimulatory effects of IL-13 were also evaluated. In these experiments we initially evaluated the role of Stat6, the canonical signaling pathway for IL-13. This was done by comparing the effects of transgenic IL-13 in mice with wild type and null Stat6 loci. Because Egr-1 expression can also be regulated via Erk1/2-dependent pathways (31De Sousa L.P. Brasil B.S. Silva B.M. Freitas M.H. Nogueira S.V. Ferreira P.C. Kroon E.G. Bonjardim C.A. Biochem. Biophys. Res. Commun. 2005; 329: 237-245Crossref PubMed Scopus (30) Google Scholar, 32Jones N. Agani F.H. J. Cell. Physiol. 2003; 196: 326-333Crossref PubMed Scopus (46) Google Scholar, 33Revest J.M. Di Blasi F. Kitchener P. Rouge-Pont F. Desmedt A. Turiault M. Tronche F. Piazza P.V. Nat. Neurosci. 2005; 8: 664-672Crossref PubMed Scopus (190) Google Scholar), we also evaluated the role of Erk1/2 signaling in the stimulation of Egr-1 in IL-13 transgenic mice (Tg). This was done by crossing IL-13 Tg mice with mice in which a dominant-negative MEK-1 construct was overexpressed in a lung-specific fashion. As previously reported by our laboratory (34Lee P.J. Zhang X. Shan P. Ma B. Lee C.G. Homer R.J. Zhu Z. Rincon M. Mossman B.T. Elias J.A. J. Clin. Investig. 2005; 116: 163-173Crossref PubMed Scopus (106) Google Scholar) these mice have a defect in Erk1/2 activation and significant alterations in IL-13 effector pathway activation. These results were compared with the results that were obtained with the specific pharmacologic Erk inhibitor (PD98059). IL-13 induction of Egr-1 was not significantly altered in IL-13 Tg mice in which dominant-negative MEK-1 was expressed (Fig. 1C). PD98059 treatment also did not alter IL-13 stimulation of Egr-1 when compared with vehicle-treated Tg (+) animals (Fig. 1C). In contrast, IL-13 stimulation of EGR-1 was completely abrogated by the null mutation of Stat6 (Fig. 1C). Collectively, these results demonstrate that IL-13 induces Egr-1 via an Erk1/2-independent and Stat6-dependent pathway(s).Role of Egr-1 on IL-13-induced Inflammation—Studies were next undertaken to define the role of Egr-1 plays in IL-13-induced inflammation. In these experiments we compared the BAL and tissue inflammatory responses in transgene(Tg) (-) and Tg (+) mice with wild type (+/+) and null mutant (-/-) Egr-1 loci. As previously reported, transgenic IL-13 caused BAL and tissue inflammation with enhanced total cell, eosinophil, and lymphocyte responses (Fig. 2, A and B, data not shown). Egr-1 appeared to play an important role in these responses because tissue (Fig. 2A) and BAL inflammation were all significantly diminished in Tg (+) mice with null mutant Egr-1 loci (Fig. 2B). These studies demonstrate that Egr-1 plays a critical role in IL-13-induced pulmonary inflammation.FIGURE 2Role of Egr-1 in IL-13-induced inflammation. 2-month-old Tg (+) and TG (-) littermate controls with (+/+) and (-/-) Egr-1 loci were generated. The histologic appearance of their lungs on hematoxylin and eosin evaluations (A, 10× original magnification) and BAL cell recovery (B) are compared. A is representative of a minimum of five similar evaluations, and in B the values represent the mean ± S.E. of evaluations in a minimum of five animals (*, p < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Role of Egr-1 in IL-13-induced Pulmonary Fibrosis—Because IL-13 is a major fibrogenic mediator at sites of Th2 inflammation (1Wynn T.A. Nat. Rev. Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1202) Google Scholar, 3Elias J.A. Lee C.G. Zheng T. Ma B. Homer R.J. Zhu Z. J. Clin. Investig. 2003; 111: 291-297Crossref PubMed Scopus (380) Google Scholar), studies were undertaken to define the role of Egr-1 in the IL-13-induced fibrotic response. In these studies we used trichrome evaluations and lung collagen assessments to characterize the fibrotic response in Tg (-) and Tg (+) mice with (+/+) and (-/-) Egr-1 loci. As previously reported by our laboratory (5Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Investig. 1999; 103: 779-788Crossref PubMed Scopus (1481) Google Scholar), transgenic IL-13 caused peribronchial and interstitial fibrosis in Tg (+) mice with (+/+) Egr-1 loci (Fig. 3A). This induction was readily appreciated in trichrome evaluations (Fig. 3A) and biochemical assays (Fig. 3B). This fibrotic response was Egr-1-dependent because trichrome and biochemical assays demonstrated marked decreases in collagen accumulation in Tg (+) mice with null mutant versus (+/+) Egr-1 loci (Fig. 3, A and B). Thus, IL-13-induced tissue fibrosis is mediated via a mechanism that is, at least in part, Egr-1-dependent.FIGURE 3Role of Egr-1 in IL-13-induced fibrosis. The collagen content of lungs from 3-month-old IL-13 Tg (-) and (+) mice with (+/+) and (-/-) Egr-1 loci were compared using Mallory's trichrome (A) and sircol (B) collagen evaluations. A is representative of a minimum of five similar evaluations. In B, each value represents the mean ± S.E. of evaluations in a minimum of four mice (*, p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Role of Egr-1 in IL-13-induced Alveolar Remodeling—Previous studies from our laboratory highlighted the ability of IL-13 to induce an alveolar remodeling response (6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar). Thus, studies were undertaken to define the role of Egr-1 in these responses. In accord with our previous observations in Egr-1-sufficient mice (6Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Investig. 2000; 106: 1081-1093Crossref PubMed Scopus (539) Google Scholar), transgenic IL-13 caused impressive increases in pulmonary compliance and alveolar enlargement after pressure fixation (Fig. 4). Egr-1 played an important role in these responses because IL-13-induced alveolar remodeling (Fig. 4A) and alveolar enlargement (Fig. 4, A and B) were diminished in Tg (+) mice with null mutant Egr-1 loci. These studies demonstrate that IL-13 induces alveolar remodeling via a mechanism that is, at least in part, Egr-1-dependent.FIGURE 4Role of Egr-1 in IL-13-induced alveolar remodeling. Lungs were obtained from Tg (-) and Tg (+) mice with (+/+) and (-/-) Egr-1 loci, fixed to pressure and hematoxylin and eosin histologic stains (A, 20× original magnification) and chord length (B) assessments were undertaken. A is representative of a minimum of five similar experiments. In B, the values represent the mean ± S.E. of evaluations in a minimum of five mice (*, p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Egr-1 Deficiency on IL-13 Elaboration—A deficiency of Egr-1 could modify IL-13-induced tissue responses by altering IL-13 production or modifying IL-13 effector functions. To differentiate among these options, we compared the levels of BAL IL-13 in Tg (-) and Tg (+) mice with wild type and null mutant Egr-1 loci. IL-13 was not readily apparent in BAL fluids from Tg (-) mice with wild type or null mutant Egr-1 loci. In contrast, significant levels of BAL of IL-13 were noted in doxycycline-treated Tg (+) animals. These levels, however, were similar in Tg (+) mice with wild type and null mutant Egr-1 loci (data not shown). This demonstrates that the ablation of Egr-1 alters the IL-13 phenotype by modifying IL-1