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• W2023974524 abstract "Transforming growth factor-β (TGF-β) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-β and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-β causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-β to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-β, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-β enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-β signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis. Transforming growth factor-β (TGF-β) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-β and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-β causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-β to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-β, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-β enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-β signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis. Scleroderma or systemic sclerosis (SSc), a potentially fatal disease of unknown cause, is characterized by fibrosis with collagenous scar tissue formation in the skin and lungs.1Jimenez SA Derk CT Following the molecular pathways toward an understanding of the pathogenesis of systemic sclerosis.Ann Intern Med. 2004; 140: 37-45Crossref PubMed Google Scholar The pathogenesis of fibrosis remains incompletely understood, and effective treatments are lacking.2Varga J Abraham D Systemic sclerosis: a prototypic multisystem fibrotic disorder.J Clin Invest. 2007; 117: 557-567Crossref PubMed Scopus (944) Google Scholar As the primary collagen-producing cells activated by paracrine and autocrine transforming growth factor-β (TGF-β), fibroblasts are the key cellular effectors of fibrosis.3Mauviel A Transforming growth factor-beta: a key mediator of fibrosis.Methods Mol Med. 2005; 117: 69-80PubMed Google Scholar Fibroblast responses elicited by TGF-β involve both Smad-dependent and -independent signal transduction. In the Smad pathway, activated ALK5 phosphorylates Smad2/3, inducing interaction with Smad4, translocation into the nucleus, and binding to consensus Smad-binding DNA elements of target genes, such as COL1A2.4Massagué J Seoane J Wotton D Smad transcription factors (review).Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1927) Google Scholar In addition, TGF-β also activates non-Smad pathways in a cell- and context-dependent manner.5Moustakas A Heldin CH Non-Smad TGF-beta signals (review).J Cell Sci. 2005; 118: 3573-3584Crossref PubMed Scopus (910) Google Scholar The early growth response-1 (Egr-1) transcription factor was originally identified as an early-immediate gene product induced by environmental stress.6Thiel G Cibelli G Regulation of life and death by the zinc finger transcription factor Eg-1 (review).J Cell Physiol. 2002; 193: 287-292Crossref PubMed Scopus (506) Google Scholar In addition to Egr-1, the early response gene family also includes Egr-2, Egr-3, and Egr-4, along with their endogenous inhibitors NAB1 and NAB2. The Egr-1 gene codes for an 82-kDa zinc finger protein that binds to GC-rich regulatory DNA elements found in the promoter region of many genes. Although negligible in normal cells, Egr-1 expression is rapidly and transiently induced during acute injury by growth factors, cytokines, oxidant stress, hormones, neurotransmitters, UV light, and mechanical forces.7Gashler A Sukhatme VP Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors.Prog Nucleic Acid Res Mol Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar, 8Khachigian LM Early growth response-1 in cardiovascular pathobiology.Circ Res. 2006; 98: 186-191Crossref PubMed Scopus (230) Google Scholar As a transcriptional master regulator that integrates converging signaling pathways, Egr-1 has a central role in orchestrating adaptive cellular response to injury.9Ngiam N Post M Kavanagh BP Early growth response factor-1 in acute lung injury.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L1089-L1091Crossref PubMed Scopus (30) Google Scholar Because of its significance in cardiovascular pathobiology, the regulation of Egr-1 in the vascular system has been investigated extensively. In vascular smooth muscle cells, TGF-β was shown to induce Egr-1 via the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) pathway.10Fu M Zhang J Lin Y Zhu X Zhao L Ahmad M Ehrengruber MU Chen YE Early stimulation and late inhibition of peroxisome proliferator-activated receptor gamma (PPARγ) gene expression by transforming growth factor beta in human aortic smooth muscle cells: role of early growth-response factor-1 (Egr-1), activator protein 1 (AP1) and Smads.Biochem J. 2003; 370: 1019-1025Crossref PubMed Scopus (70) Google Scholar In contrast, the regulation of Egr-1 expression in the context of fibrogenesis has not been characterized to date, despite an emerging recognition of the importance of Egr-1 in the pathogenesis of fibrosis. We had previously demonstrated that TGF-β stimulation of normal fibroblasts resulted in enhanced Egr-1 DNA-binding activity specific for a GC-rich sequence within the promoter region of COL1A2.11Chen SJ 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 Furthermore, Egr-1 was shown to be both necessary and sufficient to stimulate type I collagen production in vitro, suggesting a fundamental role for Egr-1 in mediating TGF-β-dependent profibrotic responses. In accordance with these observations, Egr-1-null mice were shown to be protected from pulmonary fibrosis induced by TGF-β.12Lee CG Cho SJ Kang MJ Chapoval SP Lee PJ Noble PW Yehualaeshet T Lu B Flavell RA Milbrandt J Homer RJ Elias JA Early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fibrosis.J Exp Med. 2004; 200: 377-389Crossref PubMed Scopus (317) Google Scholar In the present study, we investigated the mechanism of Egr-1 stimulation by TGF-β. The results indicate that TGF-β caused rapid and transient up-regulation of Egr-1 via a Smad-independent pathway involving mitogen-activated protein kinase kinase 1 (MEK1)-ERK1/2- Elk-1. The expression of Egr-1 was markedly up-regulated in fibrotic lesions in a mouse model of scleroderma, and elevated Egr-1 was noted in skin and lung biopsies from patients with active scleroderma. Because Egr-1 is known to mediate stimulation of collagen by TGF-β and to induce the production of fibrogenic signals and their receptors, our findings suggest that sustained up-regulation of Egr-1 in scleroderma might contribute to amplification or persistence of the TGF-β-driven fibrotic process. Accordingly, targeting Egr-1 regulation and biological activity represents a novel therapeutic strategy to block TGF-β-dependent pathological fibrogenesis. Cultures of normal human dermal fibroblasts were established by explant from neonatal foreskin or forearm skin biopsies of healthy adults and studied at early (<8) passage. Dermal fibroblasts were obtained by biopsy of the affected dorsal forearm and/or from clinically unaffected skin from four patients with early diffuse cutaneous SSc13Mori Y Chen SJ Varga J Modulation of endogenous Smad expression in normal skin fibroblasts by transforming growth factor-beta.Exp Cell Res. 2000; 258: 374-383Crossref PubMed Scopus (100) Google Scholar and studied in parallel with fibroblasts from three healthy donors. All patients fulfilled the American College of Rheumatology criteria for SSc. Cultures of murine dermal fibroblasts were established from newborn female Smad3-null mice and their wild-type littermates.14Lakos G Takagawa S Chen SJ Ferreira AM Han G Masuda K Wang XJ DiPietro LA Varga J Targeted disruption of TGF-beta/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma.Am J Pathol. 2004; 165: 203-217Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Murine NIH3T3 fibroblasts were from the American Type Culture Collection (Manassas, VA). All fibroblasts were maintained in modified Eagle's minimal essential medium or Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% vitamins, and 2 mmol/L l-glutamine and studied at early confluence. Culture media and tissue culture reagents were from Biowhittaker (Walkersville, MD). For experiments, fresh serum-free media containing 0.1% bovine serum albumin was added to cultures for 24 hours before TGF-β. In selected experiments, fibroblasts were preincubated in media containing the kinase inhibitors SB431542 (Sigma-Aldrich, St. Louis, MO) or U0126 (Cell Signaling, Beverly, MA) for 30 minutes, followed by 12.5 ng/ml TGF-β1 (PeproTech, Rocky Hill, NJ) for up to 48 hours. Expression plasmids for constitutively active MEK1, Gal4-dbd-Elk-1, and its control vector and the reporter plasmid 5xGAL4-luciferase were all from Stratagene (La Jolla, CA). Total RNA was isolated from confluent fibroblasts using TRIzol Reagent (Life Technologies, Grand Island, NY). For Northern analysis, total RNA was examined using [α-32P]dCTP-labeled cDNA probes for human Egr-1, glyceraldehyde-3-phosphate dehydrogenase, and 18S. After extensive washing of the nitrocellulose membranes, cDNA-mRNA hybrids were visualized by autoradiography. Signal intensities were quantitated by densitometry, and results were normalized with levels for glyceraldehyde-3-phosphate dehydrogenase mRNA or 18S RNA in each sample. For PCR analysis, total RNA (1 μg) from cultured fibroblasts or lesional skin tissue from mice was reverse-transcribed to cDNA using AMV Reverse Transcriptase System (Promega, Madison, WI) and subjected to amplification. PCR products were separated by electrophoresis in 1.5% agarose gels. For real-time quantitative PCR analysis, total RNA was reverse-transcribed, and the products (50 ng) were amplified with the oligonucleotide primers shown in Table 1 using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on the ABI 7500 Prism Sequence Detection System machine.Table 1Sequences Used for RT-PCR and Real-Time Quantitative PCRGenecDNA sequenceRT-PCR Egr-1Forward: 5′-GACAGCAACCTTTTCTCC CAGG-3′Reverse: 5′-GTTAGGTCCTCACTTGGG GGAA-3′ Egr-2Forward: 5′-CCGCCAAGGCCGTAGACA AAA-3′Reverse: 5′-GGGTCAATGGAGAACTT GCCC-3′ Egr-3Forward: 5′-GAGAAGCTGCCGGTGACC ATGA-3′Reverse: 5′-AGTTGGAAGGGGAGTCGAA GGC-3′ Nab1Forward: 5′-TGCTGACAAGAAGAGA TGAG-3′Reverse: 5′-TCCTGGTTTCCACAGA CTAC-3′Quantitative PCR Egr-1Forward: 5′-TGCGGCAGAAGGACAAGA AAGC-3′Reverse: 5′-TGAGGAAGGGAAGCTGCT GACC-3′ Egr-2Forward: 5′-CCGCCAAGGCCGTAGACA AAAT-3′Reverse: 5′-GGGTCAATGGAGAACTTG CCCA-3′ Egr-3Forward: 5′-GAGAAGCTGCCGGTGACC ATGA-3′Reverse: 5′-AGTTGGAAGGGGAGTCGA AGGC-3′ Nab2Forward: 5′-TGACAGCCAGAAGGAAG AGGA-3′Reverse: 5′-AGGTGCTCTCTCTCGGGCT ACTT-3′ ActinForward: 5′-AATGTCGCGGAGGACTT TGAT-3′Reverse: 5′-AGGATGGCAAGGGACTT CCTG-3′ Mouse Egr-1Forward: 5′-TTTGCCTCCGTTCCA CCTGC-3′Reverse: 5′-TGCCAACTTGATGGTCAT GCGC-3′ Open table in a new tab At the end of the experiments, fibroblasts were harvested, whole-cell lysates were prepared, and equal amounts of proteins (20 to 50 μg/lane) were subjected to electrophoresis in 8% SDS polyacrylamide gels. Proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA), and membranes were probed sequentially with primary antibodies specific for Egr-1 (C19), MEK1 (C18), phospho-MEK1(Ser298), and Actin (C2) (all from Santa Cruz Biotechnology, Santa Cruz, CA); Smad3 (Zymed Laboratories, South San Francisco, CA); or phospho-ERK1/2 (Thr202 Tyr204), ERK1/2, or phospho-Smad2 (Ser465,467) (all from Cell Signaling), as indicated. Membranes were then incubated with appropriate secondary antibodies and subjected to enhanced chemiluminescence detection using ECL Reagent (Amersham Pharmacia, Piscataway, NJ). Plasmids harboring 1.2 kb of the mouse Egr-1 gene promoter or 5′-truncated fragments of the promoter15Sukhatme VP Cao XM Chang LC Tsai-Morris CH Stamenkovich D Ferreira PC Cohen DR Edwards SA Shows TB Curran T A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization.Cell. 1988; 53: 37-43Abstract Full Text PDF PubMed Scopus (1026) Google Scholar fused to luciferase gene were used for transient transfection assays. At early confluence, fibroblasts were transfected using SuperFect Transfection kit (Qiagen, Valencia, CA). Twenty-four hours later, fresh serum-free media containing 0.1% bovine serum albumin and indicated kinase inhibitors (10 μmol/L) were added, followed 30 minutes later by TGF-β1. At the end of the experiments cultures were harvested, and cell lysates were assayed for their luciferase activities using the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase pRL-TK (Promega) was cotransfected along with the reporter constructs, and luciferase activity was used to monitor transfection efficiency. Experiments were performed in triplicate, and the results were normalized for protein concentrations. To assess Elk-1 transactivation, NIH3T3 fibroblasts were cotransfected with Gal4-dbd-Elk-1 along with 5xGAL4-luciferase reporter plasmids, and after incubation with TGF-β, luciferase activities were quantified as above. The regulation of endogenous Egr-1 expression was further examined by immunocytochemistry and fluorescence confocal microscopy.16Mori Y Chen SJ Varga J Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts.Arthritis Rheum. 2003; 48: 1964-1978Crossref PubMed Scopus (171) Google Scholar Briefly, foreskin fibroblasts (104 cells/well) incubated in serum-free media with 0.1% bovine serum albumin were exposed to 1 ng/ml TGF-β1 for 30 minutes. Cultures were then fixed with 100% methanol and incubated with rat anti-human Egr-1 antibodies (C19) at a 1:100 dilution, followed by fluorescein isothiocyanate-labeled secondary antibodies. Nuclei were identified using 4,6-diamidino-2-phenylindone. Subcellular distribution of immunofluorescence was then evaluated under a Zeiss LSHS10 laser scanning confocal microscope. Six-week-old female Smad3-null mice and wild-type Back Swiss ×129 SJ littermates (Taconic, Germantown, NY) weighing 15 to 20 g were administered 250 ng TGF-β1 s.c. or PBS s.c. One or 24 hours later, mice were euthanized, and the injected skin was processed for immunohistochemical analysis.17Lakos G Takagawa S Varga J Animal models of scleroderma.Methods Mol Med. 2004; 102: 377-393PubMed Google Scholar Four-micrometer sections from lesional skin were deparaffinized, rehydrated, and immersed in Tris-buffered saline-0.1%Tween 20 buffer followed by target retrieval solution (DAKO, Carpinteria, CA). After incubation of the slides with rabbit anti-mouse Egr-1 polyclonal antibodies (C-19; Santa Cruz Biotechnology) at a dilution of 1:300, donkey anti-rabbit Ig as secondary antibodies (Promega) at 1:1000 dilution were applied. Bound antibodies were detected using DAKO Envision + System according to the manufacturer's instructions. After counterstaining with hematoxylin, sections were mounted with Permount (Fisher Scientific, Pittsburgh, PA) and viewed under an BH-2 microscope (Olympus, Tokyo, Japan). Skin samples from Egr-1-null C57/BL6 mice18Lee SL Sadovsky Y Swirnoff AH Polish JA 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 (441) Google Scholar were stained in parallel as negative controls. An additional negative control consisted of substituting nonspecific rabbit IgG (Invitrogen, Carlsbad, CA) for the primary antibody. To quantify Egr-1 expression in lesional skin, at least 30 fibroblastic cells (identified based on their characteristic spindle-shaped morphology) from multiple microscopic fields were scored as clearly immunopositive or negative for Egr-1 by three independent examiners blinded to the treatment, and the ratio of positive cells to total cells was calculated. In other experiments, 6-week-old BALB/c mice weighing 20 g were given s.c. bleomycin or PBS daily for up to 28 days.17Lakos G Takagawa S Varga J Animal models of scleroderma.Methods Mol Med. 2004; 102: 377-393PubMed Google Scholar At the end of the experiments, mice were sacrificed, and lesional skin was processed for quantitative PCR or for immunohistochemistry as above. Each experimental group contained at least five mice, and three separate experiments were performed with similar results. All animal studies were performed using protocols and guidelines approved by the Northwestern University Animal Care and Use Committee. Skin tissue samples were obtained by biopsy from the affected forearm from 23 patients with SSc (16 patients with disease duration <3 years, early stage; and seven patients with disease duration ≥3 years, late stage) and five healthy adult controls. The protocols for tissue collection were approved by the Institutional Review Boards for Human Studies at Northwestern University, the University of Pittsburgh, and Kanazawa University. Some of the SSc tissues were processed following exactly the same procedure as described under Egr-1 Expression in Mouse Skin in Vivo, where the slides were counterstained with hematoxylin. For other tissues, methyl green was used for counterstain. Paraffin-embedded or frozen tissue sections were subjected to immunostaining using primary anti-Egr-1 antibody (C19; Santa Cruz Biotechnology) at 1:300 dilution, followed by incubation with biotinylated anti-rabbit IgG secondary antibodies (Santa Cruz sc-2089) at 1:100 dilution. Slides were then reacted with avidin-biotin peroxidase (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and peroxidase activity was detected with diaminobenzidine substrate (brown color). To quantify Egr-1 in lesional dermis, Egr-1-positive fibroblasts were counted by blinded examiners, and the ratio of positive cells to total cells was calculated. Tissue sections from prostate cancer were used as positive control, and substitution of primary antibody with nonspecific rabbit IgG (Invitrogen) served as negative control. Lung tissue was obtained from four patients with SSc-associated pulmonary fibrosis who were undergoing lung transplant surgery, and from unused normal donor lungs from three patients. Tissues were processed as above, the slides were counterstained with hematoxylin. To quantify Egr-1 expression, the numbers of cells in several microscopic fields were scored as clearly immunopositive or negative for Egr-1 by two independent examiners, and the ratio of positive cells to total cells was calculated. Tissue sections from a colon cancer were used as a positive control, and nonspecific rabbit IgG was used as a negative control. Statistical significance was determined using the unpaired Student's t-test. Values of P ≤ 0.05 were considered significant. To characterize Egr-1 regulation by TGF-β, quiescent foreskin fibroblasts were stimulated with increasing concentrations of TGF-β1. The results of Western and Northern analysis showed rapid (30 minutes) and dose-dependent increase in Egr-1 protein (Figure 1A, top) and mRNA (Figure 1B, top) expression. Immunofluorescence confocal microscopy indicated little detectable Egr-1 in the absence of TGF-β and marked accumulation in both cytoplasm and nucleus in TGF-β-treated fibroblasts (Figure 1A, bottom). Real-time quantitative PCR analysis confirmed the dose-dependent stimulation of Egr-1 mRNA in neonatal and adult dermal fibroblasts (Figure 1B, bottom). To characterize the level of regulation of Egr-1, quiescent fibroblasts were transiently transfected with a plasmid containing the proximal 1.2 kb of the human Egr-1 gene promoter linked to the luciferase reporter and then incubated in media with increasing concentrations of TGF-β1. Twenty-four hours later, the cultures were harvested, and cell lysates were assayed for their luciferase activities. The results showed that TGF-β induced a dose-dependent increase in Egr-1 promoter activity in transfected primary foreskin fibroblasts (Figure 1C, left) and in NIH3T3 fibroblasts (Figure 1C, right). In both human and mouse fibroblasts, TGF-β at a concentration of 10 ng/ml induced a ∼threefold stimulation of Egr-1 promoter activity. In addition to Egr-1, the family of early growth response transcription factors also includes Egr-2, Egr-3, and Egr-4. Because little is known about the function or regulation of the Egr moieties in the context of fibrosis, foreskin fibroblasts were incubated with TGF-β for indicated periods, and mRNA expression was evaluated by RT PCR analysis. The results showed that TGF-β1 caused rapid stimulation of Egr-3 and Egr-1 mRNA in these cells (Figure 2). Marked increase was seen at 60 minutes, followed by decline after 120 minutes. The expression of Egr-2 mRNA was also stimulated, but in contrast to Egr-1, the response was relatively sustained, with elevated mRNA expression persisting at 4 hours. We also examined TGF-β regulation of Nab1 and Nab2, which are inhibitory members of the early growth response family that bind to Egr-1 and regulate its activity.19Svaren J Sevetson BR Apel ED Zimonjic DB Popescu NC Milbrandt J NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli.Mol Cell Biol. 1996; 16: 3545-3553Crossref PubMed Scopus (326) Google Scholar The results of PCR analysis showed that Nab1 was constitutively expressed in fibroblasts, as it is in most cell types, and was further increased by TGF-β. In contrast, Nab2 mRNA was undetectable in the absence of stimulation but was strongly induced by TGF-β; however, compared with the rapid stimulation of Egr-1 and Egr-3, the Nab2 response was delayed, peaking at 4 hours (Figure 2). Thus, endogenous inhibitors of Egr-1 are induced by the same environmental signals that stimulate Egr-1 and, at least in the case of Nab2, show delayed response compatible with a negative feedback function. The stimulation of mRNA of each of the Egr moieties by TGF-β was confirmed by real-time quantitative PCR (data not shown). As illustrated schematically (Figure 3, left), the proximal promoter of the Egr-1 gene harbors multiple potential regulatory elements. These include binding sites for AP-1, GC-rich elements, and five serum-responsive elements (SREs). Transient transfection assays were performed to localize the functional TGF-β response regions of the Egr-1 gene. For this purpose, mouse NIH3T3 fibroblasts were transfected with the indicated deletion or truncation mutants of the mouse Egr-1 promoter linked to the luciferase reporter gene. Incubation of transfected fibroblasts with TGF-β resulted in ∼twofold stimulation of luciferase activity driven by the full-length Egr-1 promoter (Figure 3). The activity of luciferase fused to a promoter mutant lacking all five SRE sites (B2) showed a substantial decrease in basal activity and complete lack of stimulation by TGF-β. Deletion of neither the region containing the distal AP-1 and Sp1 binding sites (C21) nor the two proximal SRE sites (D7) significantly affected either basal promoter activity or the TGF-β response. In contrast, truncation of the three distal SRE sites (AA) resulted in complete abrogation of TGF-β response, indicating that the distal SRE sites mediate TGF-β-induced Egr-1 promoter transactivation. Downstream targets for activated ALK5, Smad2, and Smad3 are the primary signal transducers for most TGF-β responses in fibroblasts.20Chen S-J Yuan W Mori Y Levenson A Trojanowska M Varga J Stimulation of type I collagen transcription in human skin fibroblasts by TGF-β: involvement of Smad 3.J Invest Dermatol. 1999; 112: 49-57Crossref PubMed Scopus (373) Google Scholar To evaluate their role as potential mediators of Egr-1 stimulation, SB431542, a potent and specific inhibitor of ALK5-dependent TGF-β signaling, was used.21Mori Y Ishida W Bhattacharyya S Li Y Platanias LC Varga J Selective inhibition of activin receptor-like kinase 5 signaling blocks profibrotic transforming growth factor beta responses in skin fibroblasts.Arthritis Rheum. 2004; 50: 4008-4021Crossref PubMed Scopus (98) Google Scholar Confluent serum-starved human fibroblasts were preincubated with 10 μmol/L SB431542 for 30 minutes, followed by TGF-β1 for indicated periods. The results of Northern analysis showed that SB431542 pretreatment failed to prevent rapid and transient stimulation of Egr-1 mRNA induced by TGF-β (Figure 4A). Under these experimental conditions, SB431542 significantly prevented the phosphorylation of Smad2 induced by TGF-β (Figure 4B, right). Complementary experiments used primary dermal fibroblasts from Smad3-null mice. We have shown previously that Smad3-null fibroblasts displayed impaired TGF-β responses in vitro, with substantially reduced stimulation of collagen.14Lakos G Takagawa S Chen SJ Ferreira AM Han G Masuda K Wang XJ DiPietro LA Varga J Targeted disruption of TGF-beta/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma.Am J Pathol. 2004; 165: 203-217Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Confluent fibroblasts from Smad3-null mice and their wild-type littermates were incubated in parallel with TGF-β1 (12.5 ng) for up to 120 minutes. The results of Western analysis showed that Egr-1 was rapidly induced by TGF-β1 in wild-type murine fibroblasts in a manner comparable with that observed in normal human fibroblasts (Figure 4B). Smad3-null fibroblasts showed virtually identical early induction of Egr-1. Interestingly, basal levels of Egr-1 were found to be modestly elevated. In agreement with results from Western analysis, examination of Egr-1 mRNA levels showed that TGF-β1 caused comparable stimulation in both wild-type fibroblasts and Smad3-null fibroblasts (data not shown; Figure 5B). Taken together, the results from experiments using ALK5 inhibition and Smad3-deficient mouse fibroblasts indicated that stimulation of Egr-1 by TGF-β occurred largely independent of the ALK5/Smad3 signaling axis. Because stimulation of Egr-1 appeared to be a Smad3-independent TGF-β response, we proceeded to examine the functional role of non-Smad TGF-β signaling pathways. For this purpose, confluent foreskin fibroblasts were pretreated with the selective MEK1/2 kinase inhibitor U0126 for 30 minutes, followed by incubation with TGF-β1 for up to 120 minutes. Results from Northern analysis showed that the rapid stimulation of Egr-1 mRNA was completely abolished by pretreatment of the cultures with U0126 (Figure 5A, left). Under the experimental conditions used here, U0126 had no demonstrable cellular toxicity. Consistent results were obtained in separate experiments using real-time quantitative PCR analysis (Figure 5A, right). Furthermore, in contrast to the ALK5 inhibitor SB431542 (Figure 5B) or inhibitors of p38 (data not shown), pretreatment of the cultures with U0126 abrogated the stimulation of Egr-1 in both wild-type and Smad3-null dermal fibroblasts (Figure 5B). These results suggested that MEK1/2, which is upstream of, and directly activates, ERK1/2, and which is induced by a variety of growth factors and cytokines, might serve as a mediator for TGF-β stimulation of Egr-1. To examine the effect of TGF-β on MEK1/2, serum-starved fibroblasts were incubated with TGF-β for indicated periods and subjected to Western analysis. The results showed that TGF-β stimulati" @default.
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• W2023974524 title "Smad-Independent Transforming Growth Factor-β Regulation of Early Growth Response-1 and Sustained Expression in Fibrosis" @default.
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