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• W2068661207 abstract "Glial cell line-derived neurotrophic factor (Gdnf) promotes neurite outgrowth and survival of neuronal cells, but its transcriptional regulation is poorly understood. Here, we sought to investigate the mechanism underlying fibroblast growth factor-2 (FGF2) induction of Gdnf expression in astrocytes. We found that FGF2 stimulation of rat astrocytes induced expression of Egr-1 at a high level. Sequence analysis of the rat Gdnf gene identified three overlapping Egr-1-binding sites between positions −185 and −163 of the rat Gdnf promoter. Transfection studies using a series of deleted Gdnf promoters revealed that these Egr-1-binding sites are required for maximal activation of the Gdnf promoter by FGF2. Chromatin immunoprecipitation analysis indicated that Egr-1 binds to the Gdnf promoter. Furthermore, the induction of Gdnf expression by FGF2 is strongly attenuated both in C6 glioma cells stably expressing Egr-1-specific small interfering RNA and in primary cultured astrocytes from the Egr-1 knock-out mouse. Additionally, we found that stimulation of the ERK and JNK pathways by FGF2 is functionally linked to Gdnf expression through the induction of Egr-1. These data demonstrate that FGF2-induced Gdnf expression is mediated by the induction of Egr-1 through activation of the ERK and JNK/Elk-1 signaling pathways. Glial cell line-derived neurotrophic factor (Gdnf) promotes neurite outgrowth and survival of neuronal cells, but its transcriptional regulation is poorly understood. Here, we sought to investigate the mechanism underlying fibroblast growth factor-2 (FGF2) induction of Gdnf expression in astrocytes. We found that FGF2 stimulation of rat astrocytes induced expression of Egr-1 at a high level. Sequence analysis of the rat Gdnf gene identified three overlapping Egr-1-binding sites between positions −185 and −163 of the rat Gdnf promoter. Transfection studies using a series of deleted Gdnf promoters revealed that these Egr-1-binding sites are required for maximal activation of the Gdnf promoter by FGF2. Chromatin immunoprecipitation analysis indicated that Egr-1 binds to the Gdnf promoter. Furthermore, the induction of Gdnf expression by FGF2 is strongly attenuated both in C6 glioma cells stably expressing Egr-1-specific small interfering RNA and in primary cultured astrocytes from the Egr-1 knock-out mouse. Additionally, we found that stimulation of the ERK and JNK pathways by FGF2 is functionally linked to Gdnf expression through the induction of Egr-1. These data demonstrate that FGF2-induced Gdnf expression is mediated by the induction of Egr-1 through activation of the ERK and JNK/Elk-1 signaling pathways. Astrocytes play important roles in the regulation of neurogenesis and synaptogenesis of the central nervous system, as well as in the regulation of its ionic and neurotransmitter environments (1Ullian E.M. Christopherson K.S. Barres B.A. Glia. 2004; 47: 209-216Crossref PubMed Scopus (310) Google Scholar). By releasing various growth factors, such as fibroblast growth factor-2 (FGF2), 2The abbreviations used are: FGF2fibroblast growth factor-2Gdnfglial cell line-derived neurotrophic factorERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinasesiRNAsmall interfering RNADNdominant negativeDAdominant activeRTreverse transcriptaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseshRNAsmall hairpin RNAChIPchromatin immunoprecipitationMAPKmitogen-activated protein kinaseCAcornu ammonisCREcAMP-response elementCREBcAMP response element-binding protein. glial cell line-derived neurotrophic factor (Gdnf), nerve growth factor, and ciliary neurotrophic factor, astrocytes also provide neuroprotection against various types of neuronal damage (2Darlington C.L. Curr. Opin. Investig. Drug. 2005; 6: 700-703PubMed Google Scholar). fibroblast growth factor-2 glial cell line-derived neurotrophic factor extracellular signal-regulated kinase c-Jun NH2-terminal kinase small interfering RNA dominant negative dominant active reverse transcriptase glyceraldehyde-3-phosphate dehydrogenase small hairpin RNA chromatin immunoprecipitation mitogen-activated protein kinase cornu ammonis cAMP-response element cAMP response element-binding protein. Gdnf is a neurotrophic factor that promotes neurite outgrowth and the survival and differentiation of distinct populations of neuronal cells, as well as astrocyte proliferation (3Lin L.F. Doherty D.H. Lile J.D. Bektesh S. Collins F. Science. 1993; 260: 1130-1132Crossref PubMed Scopus (2875) Google Scholar, 4Pozas E. Ibáñez C.F. Neuron. 2005; 45: 701-713Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Targeted disruption of the mouse Gdnf gene causes various defects in sensory and enteric neuron subpopulations, renal abnormalities, and a failure of spermatogenesis (5Moore M.W. Klein R.D. Fariñas I. Sauer H. Armanini M. Phillips H. Reichardt L.F. Ryan A.M. Carver-Moore K. Rosenthal A. Nature. 1996; 382: 76-79Crossref PubMed Scopus (1089) Google Scholar, 6Pichel J.G. Shen L. Sheng H.Z. Granholm A.C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. Sariola H. Westphal H. Nature. 1996; 382: 73-76Crossref PubMed Scopus (1011) Google Scholar, 7Sánchez M.P. Silos-Santiago I. Frisén J. He B. Lira S.A. Barbacid M. Nature. 1996; 382: 70-73Crossref PubMed Scopus (1047) Google Scholar, 8Naughton C.K. Jain S. Strickland A.M. Gupta A. Milbrandt J. Biol. Reprod. 2006; 74: 314-321Crossref PubMed Scopus (301) Google Scholar). Gdnf gene expression is elevated during embryogenesis and continues at a low level in adults (9Strömberg I. Björklund L. Johansson M. Tomac A. Collins F. Olson L. Hoffer B. Humpel C. Exp. Neurol. 1993; 124: 401-412Crossref PubMed Scopus (324) Google Scholar). Gdnf is rapidly up-regulated in response to brain injury (10Kitagawa H. Hayashi T. Mitsumoto Y. Koga N. Itoyama Y. Abe K. Stroke. 1998; 29: 1417-1422Crossref PubMed Scopus (204) Google Scholar) and specific pharmacological treatments. However, the details of the mechanism underlying the transcriptional regulation of Gdnf expression are not clearly understood. Egr-1 (also known as nerve growth factor-induced-A (NGFI-A), zinc-finger clone 28 (zif268), Krüppel box 24 (krox24), and TPA-induced sequence 8 (Tis8)) is a transcription factor encoded by an immediate-early response gene. Egr-1 is present at high concentrations in neurons of the cerebral cortex, hippocampus, thalamus, amygdaloid nuclei, and striatum (11Mack K. Day M. Milbrandt J. Gottlieb D.I. Brain Res. Mol. Brain Res. 1990; 8: 177-180Crossref PubMed Scopus (49) Google Scholar, 12Schlingensiepen K.H. Lüno K. Brysch W. Neurosci. Lett. 1991; 122: 67-70Crossref PubMed Scopus (77) Google Scholar) and is maintained by synaptic plasticity occurring in response to physiological stimuli (13Beckmann A.M. Wilce P.A. Neurochem. Int. 1997; 31: 477-510Crossref PubMed Scopus (280) Google Scholar), suggesting that Egr-1 may play a role in the nervous system. The regulatory regions of the murine and human Gdnf genes contain putative consensus sequences for Egr-1 binding. Because Egr-1 (14Gentilella A. Passiatore G. Deshmane S. Turco M.C. Khalili K. Oncogene. 2008; 27: 5011-5018Crossref PubMed Scopus (39) Google Scholar) and Gdnf (15Suter-Crazzolara C. Unsicker K. Brain Res. Mol. Brain Res. 1996; 41: 175-182Crossref PubMed Scopus (79) Google Scholar) can be induced by FGF2, we investigated whether Egr-1 is involved in Gdnf expression. Here, we show that Egr-1 directly binds to the Gdnf promoter and promotes transcriptional activation of the Gdnf gene. FGF2 induces Egr-1 expression through the ERK or JNK pathways. Inhibition of either of these pathways abrogates FGF2-induced expression of Egr-1 and Gdnf. An important role for Egr-1 in Gdnf expression is supported by the demonstration that FGF2-induced GDNF expression is attenuated both in C6 glioma cells stably expressing Egr-1-specific small interfering RNA (siRNA) and in primary cultured astrocytes derived from the Egr-1 knock-out mouse. These findings suggest a novel role for Egr-1 in the regulation of Gdnf transcription by FGF2 in astrocytes. Egr-1 knock-out mice developed on a C57BL6 background were from Dr. Jeffrey Milbrandt (Washington University, St. Louis, MO) and have been described elsewhere (16Lee S.L. Wang Y. Milbrandt J. Mol. Cell Biol. 1996; 16: 4566-4572Crossref PubMed Google Scholar). Mouse recombinant FGF2 was purchased from Calbiochem (San Diego, CA). The firefly and Renilla Dual-GloTM Luciferase Assay System was purchased from Promega (Madison, WI). The plasmid pRL-null, which encodes Renilla luciferase, was purchased from Promega. Plasmid pFA2-Elk1, which encodes a fusion protein consisting of the yeast Gal4 DNA-binding domain (amino acids 1–147) and the activation domain of Elk-1 (amino acids 307–427), and plasmid pFR-Luc, which contains five Gal4-binding element repeats upstream of the luciferase gene, were purchased from Stratagene (La Jolla, CA). Plasmids expressing dominant negative (DN)-MEK1 (pCGN1/MEK DN), DN-ERK2 (pHA-ERK2 K52R), DN-JNK1 (pSRα/HA-JNK T183A/Y185F), and kinase-dead p38 kinase (pCDNA3/FLAG-p38 T180A/Y182F) were kindly provided by Dr. D. S. Min (Department of Molecular Biology, College of Natural Science, Pusan National University, Korea). The pCDNA3.1/Egr-1(I293F) plasmid, which expresses dominant-active (DA)-Egr-1, was generated as previously described (17Russo M.W. Matheny C. Milbrandt J. Mol. Cell Biol. 1993; 13: 6858-6865Crossref PubMed Scopus (89) Google Scholar). The rat glioma cell line C6 was obtained from the American Type Culture Collection (Manassas, VA). C6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT). Primary rat astrocytes and astrocytes from Egr-1 knock-out (Egr-1−/−) and heterozygous (Egr-1+/−) mice were prepared from cerebral cortices of newborn pup littermates as previously described (18Lee J.M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar) and cultured in modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone). The Egr-1 genotype was determined by PCR amplification from genomic DNA using the forward primer 5′-aaccggcccagcaagacacc-3′ and the reverse primer 5′-ctcgtgctttacggtatcgc-3′. Astrocyte purity was determined by immunostaining with anti-glial fibrillary acidic protein antibody (1:500, Dako Corp., Carpinteria, CA). Total RNA was extracted using a TRIzol RNA extraction kit (Invitrogen) from cells stimulated with FGF2 (10 ng/ml) for various lengths of time. The first-strand cDNA was synthesized from 500 ng of total RNA by using an iScript cDNA synthesis kit (Bio-Rad). Quantitative real time PCR was performed an icycler iQTM system (Bio-Rad), using the TaqMan-iQTM supermix kit (Bio-Rad) according to the manufacturer's recommendation. The TaqManTM fluogenic probes and PCR primers for rat Gdnf and glyceraldehyde-3-phosphate dehydrogenase (gapdh) were designed by Metabion Int. (Martinsreid, Deutschland). The sequences of primers were 5′-CTGCCCGCCGGTAAGAGG-3′ (forward for Gdnf) and 5′-CGTCATCAAACTGGTCAGGATAATC-3′ (reverse for Gdnf), 5′-GTGATGCTGGTGCTGAGTATGTC-3′ (forward for gapdh), and 5′-GCGGAAGGGGCGGAGATG-3′ (reverse for gapdh). The TaqMan fluorogenic probes used were 5′-FAM-CGCCCGCCGAAGACCACTCCCTC-BHQ-3′ (for Gdnf) and 5′-Yakima Yellow TM-ACCCTTCAGGTGAGCCCCAGCCTT-BHQ-1-3′ (for gapdh). Threshold cycle, Ct, which correlates inversely with the target mRNA levels, was measured as the cycle number at which the reporter fluorescent emission increases above a threshold level. The relative changes in Gdnf mRNA levels were normalized for gapdh mRNA in the same samples. For RT-PCR analysis, gene-specific primers for murine Gdnf (forward, 5′-GGTCTACGGAGAGACCGATCCGAGGTGC-3′; reverse, 5′-TCTCTGGAGCCAGGGTCAGATACATC-3′) and actin (forward, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′; reverse, 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′) were used. The PCR conditions for all primers were as follows: hold for 5 min at 94 °C, followed by 30 cycles consisting of denaturation at 94 °C (30 s), annealing at 55 °C (30 s), and elongation at 72 °C (1 min). The amplified products were subjected to electrophoresis on a 1% agarose gel. A Gdnf promoter fragment spanning nucleotides −983 to +3 was synthesized from rat genomic DNA (Promega) by PCR using primers 5′-ccggtaccTCCATCAAAGTCCAACTTGGCAAATA-3′ (forward) and 5′-gaagatctTCTCAGCTCTTAGGTCTCCTCTGCC-3′ (reverse). (KpnI and BglII restriction sites are indicated by lowercase letters.) The amplified PCR product was ligated into the KpnI-BglII site of the pGL3-basic vector (Promega) to yield pGDNF-Luc(−983/+3). A series of deletion constructs of rat Gdnf promoter fragments were synthesized by PCR using the pGDNF-Luc(−983/+3) plasmid as a template. The forward and reverse primers used in the PCR to generate deletion mutant promoters contained KpnI and BglII restriction enzyme sites, respectively, to facilitate subsequent ligation into the KpnI-BglII site of pGL3-basic. The forward primer sequences were 5′-ccggtaccCGCCCTCATGTCTTCAAGGG-3′ (−493 to +3) ccggtaccGAGGAGGTGCAGAGTGAGGC (−374 to +3) and 5′-ccggtaccCCGGTACCTGGATTGCGTGCTCTTGCTC-3′ (−114 to +3). One reverse primer (5′-gaagatctTCTCAGCTCTTAGGTCTCCTCTGCC-3′) was used to generate all deletion mutant promoters (lowercase letters in the forward and reverse primers indicate KpnI and BglII restriction sites, respectively). The PCR products were digested with KpnI and BglII and then ligated into the pGL3-basic vector. An internal deletion construct lacking Egr-1-binding motifs (−185 to −163) in pGDNF-Luc(−493/+3) was generated using two-step PCR yielding pGDNF-Luc(−493/+3ΔEgr1). The primers used for the first step were 5′-ccggtaccCGCCCTCATGTCTTCAAGGG-3′ (forward) and 5′-tctcgagAGGAGGGCGAAGGC-3′ (−493 to −185; reverse); 5′-actcgagACACGGTGGCCGC-3′ (forward) and 5′-gaagatctTCTCAGCTCTTAGGTCTCCTCTGCC-3′ (−163 to +3; reverse). (Lowercase letters indicate XhoI restriction sites.) The PCR products were digested with XhoI and ligated. In the second step, the primers were 5′-ccggtaccCGCCCTCATGTCTTCAAGGG-3′ (forward) and 5′-gaagatctTCTCAGCTCTTAGGTCTCCTCTGCC-3′ (reverse). All constructs were verified by DNA sequencing and restriction analysis. C6 cells or primary cultured astrocytes were seeded into 12-well plates and transfected with 0.5 μg of promoter-reporter construct using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's instructions. For Elk-1 trans-acting activity, cells were co-transfected with 50 ng of trans-activator plasmid (pFA2-Elk1) and 0.5 μg of reporter plasmid (pFR-Luc). To monitor transfection efficiency, 50 ng of the pRL-null plasmid encoding Renilla luciferase was included in all samples. Where indicated, 0.2 μg of mammalian expression vector was also included. At 24-h post-transfection, the levels of firefly and Renilla luciferase activity were measured sequentially from a single sample using the Dual-Glo Luciferase assay system (Promega) with a luminometer (Centro LB960; Berthold Tech, Bad Wildbad, Germany). Firefly luciferase activity was normalized to Renilla luciferase activity, and the relative amount of luciferase activity in the untreated cells was designated as 1. Cells were lysed in 20 mm HEPES (pH 7.2) containing 1% Triton X-100, 10% glycerol, 150 mm NaCl, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. The protein samples (20 μg/lane) were then separated by 10% SDS-PAGE and transferred onto nitrocellulose filters. Western blotting was performed according to standard procedures using antibodies specific for Egr-1 (1:1000 dilution), ERK2 (1:5000), or GAPDH (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA) or for phospho-Raf1 (1:1000), phospho-(Thr202/Tyr204)-ERK1/2 (1:1000), phospho-(Thr180/Tyr182)-p38 (1:1000), or phospho-(Thr183/Tyr185)-JNK (1:1000) (all from Cell Signaling Technology, Danvers, MA). The signals were developed using an enhanced chemiluminescence detection system (Amersham Biosciences Inc.). The small hairpin RNA (shRNA) plasmid targeting mouse Egr-1 mRNA (pSilencer/siEgr1) and the generation of C6 cells expressing Egr-1 siRNA (C6/SiEgr-1) or control siRNA (C6/Scramble) have been described previously (19Choi B.H. Kim C.G. Bae Y.S. Lim Y. Lee Y.H. Shin S.Y. Cancer Res. 2008; 68: 1369-1377Crossref PubMed Scopus (105) Google Scholar). C6 cells or primary cultured astrocytes plated on coverslips were treated with 0 or 10 ng/ml of FGF2 for 24 h, fixed with 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100 and 2% bovine serum albumin as described previously (20Shin S.Y. Bahk Y.Y. Ko J. Chung I.Y. Lee Y.S. Downward J. Eibel H. Sharma P.M. Olefsky J.M. Kim Y.H. Lee B. Lee Y.H. EMBO J. 2006; 25: 1093-1103Crossref PubMed Scopus (47) Google Scholar). The coverslips were co-stained with rabbit anti-Gdnf (1:100) and mouse anti-S100 (1:200) antibodies for the mouse astrocyte marker, for 90 min at room temperature and then incubated for 30 min with Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit secondary antibodies, yielding green and red signals, respectively. The labeled cells were examined under an Olympus FV-1000 Spectral confocal laser-scanning microscope with excitation at 488 nm and emission at 550 nm. Primary cultured astrocytes treated with FGF2 (10 ng/ml) were treated with 1% formaldehyde to cross-link the DNA, lysed, and chromatin immunoprecipitated using rabbit anti-Egr-1 antibody (Santa Cruz Biotechnology) or normal rabbit IgG as described previously (21Yang S.H. Sharrocks A.D. EMBO J. 2005; 24: 2161-2171Crossref PubMed Scopus (67) Google Scholar). The precipitated DNA was analyzed by standard RT-PCR methods. All procedures were carried out with the approval of the Konkuk University Institutional Animal Care and Use Committee. For preparation of samples, mice were anesthetized with a mixture of Zoletil (22.5 mg/kg; Virbac, Carros, France) and Xylazine (7.5 mg/kg; Bayer, KS) and perfused transcardially with a fixative solution containing 4% paraformaldehyde in 0.1 m phosphate-buffered saline (pH 7.4). Brains were post-fixed for 24 h in the same fixative solution and bisected sagitally at the midline. The samples were embedded in paraffin, cut into serial sections 4-μm thick, and stained with hematoxylin and eosin. Immunohistochemical analysis was carried out using a Vectastain Elite ABC kit (Vector Labs, Burlingame, CA). The paraffin sections were deparaffinized, hydrated, heated in 0.01 m sodium citrate for 10 min in a microwave to retrieve the antigen, and treated with 3% hydrogen peroxide in methanol. They were then incubated with blocking solution containing 1.5% anti-horse serum and 5% bovine serum albumin. The sections were incubated with anti-Gdnf antibody (1:50; Santa Cruz Biotechnology) overnight at 4 °C. After washing in phosphate-buffered saline, the sections were incubated with biotinylated secondary antibody for 60 min and then with a avidin-biotin peroxidase complex for 30 min at room temperature. Sections were treated with peroxidase substrate kits (Vector Labs) to visualize immunoreactivity, counterstained with hematoxylin, and dehydrated in ethanol. After clearing in xylene, sections were coverslipped and examined under a Olympus BX51 microscope (Olympus Optical, Tokyo, Japan). Immunoreactive cells were quantitatively analyzed by a computerized image analyzer MetaMorph 7.5 (Molecular Devices, Downington, PA). The immunopositive cells in three nonconsecutive sections per mouse were counted by two blinded independent investigators using the image analyzer at a magnification of ×200. Results are expressed as the mean number of cells per mm2 ± S.D. Statistical significance was evaluated by one-way analysis of variance using SPSS for Windows (version 14.0, SPSS Inc., Chicago, IL). For the assay of Gdnf release, Egr-1+/− or Egr-1−/− astrocytes were cultured with or without 10 ng/ml of FGF2 at a density of 13 × 104/cm2 on a 12-well plate. After 48 h treatment, conditioned medium was collected and stored at −80 °C until assayed. GDNF protein levels in cell-conditioned media were determined using a Gdnf enzyme-linked immunosorbent assay according to the manufacturer's instructions (Promega). To investigate the molecular mechanism of FGF2 regulation of Gdnf expression, we first determined the ability of FGF2 to induce the expression of Gdnf by quantitative real time-PCR using the TaqMan fluogenic probes in rat astrocytes. After 12 and 24 h of exposure to FGF2, Gdnf mRNA was increased 9.7- and 9.2-fold, respectively, as compared with control cells in a dissociated culture of rat astrocytes (Fig. 1A), and increased 4.4- and 5.6-fold, respectively, as compared with control cells in C6 glioma cells (Fig. 1B). All of the dissociated cultures of rat astrocytes were shown to express S-100, an astrocyte marker, by confocal microscopy. Notably, FGF2 treatment enhanced Gdnf immunoreactivity in the dissociated rat astrocyte culture (Fig. 1C). These data clearly demonstrate that FGF2 induces up-regulation of Gdnf expression in rat astrocytes. To identify the cis-acting region that mediates the responsiveness of the rat Gdnf gene to FGF2, progressively deleted Gdnf promoter-reporter constructs were made and transfected into rat primary astrocytes. The basal promoter activity of the −493/+3 and −114/+3 constructs was 10 and 5 times higher, respectively, than that of the −983/+3 construct (Fig. 1D), suggesting negative regulatory elements between nucleotides −983 and −492 control basal transcription. In contrast, FGF2-induced promoter activity approximately doubled in the −983/+3, −493/+3, and −374/+3 constructs but remained the same in the −114/+3 construct. Similar results were obtained in C6 glioma cells, although the observed increases in basal activity were much smaller than those seen in primary cultured astrocytes (Fig. 1E). These data suggest that a proximal region between −374 and −114 of the Gdnf promoter contains a cis-acting regulatory element responsive to FGF2. An analysis of the Gdnf promoter sequences spanning −374 to −114 performed using the program MatInspector revealed the presence of three putative binding motifs for Egr-1 proximal to the transcription start site at positions −185 to −163 (Fig. 2A). Egr-1 is a Cys2/His2-type zinc finger transcription factor that regulates cell growth, differentiation, and development (22Thiel G. Cibelli G. J. Cell. Physiol. 2002; 193: 287-292Crossref PubMed Scopus (512) Google Scholar). To assess whether Egr-1 activates the Gdnf promoter, we co-transfected the −493/+3 Gdnf promoter construct and the expression plasmid for DA-Egr-1, pcDNA3.1/Egr1(I293F), into rat primary cultured astrocytes. In the absence of FGF2 treatment, the forced expression of DA-Egr-1 caused basal reporter activity to increase as the amount of transfected DNA increased (Fig. 2B). To characterize whether the Egr-1-binding motifs were responsible for stimulation of the GDNF gene promoter by Egr-1, we generated an internal deletion construct lacking all three Egr-1-binding motifs (−493/+3ΔEgr1). As shown in Fig. 2C, transfection of DA-Egr-1 into rat primary astrocytes had no appreciable effect on the reporter activity of the −493/+3ΔEgr1 or −114/+3 constructs, which lack apparent Egr-1-binding sequences. To assess the contribution of the Egr-1-binding sequences to the activation of the GDNF promoter by FGF2, the transcriptional activity of these deletion constructs in response to FGF2 was evaluated in rat primary astrocytes. As shown in Fig. 2D, FGF2 approximately doubled the reporter activity in the −493/+3 construct, but not in the −493/+3ΔEgr1 or −114/+3 constructs. Similar results were obtained in C6 glioma cells (data not shown). These observations indicate that the Egr-1-binding motifs are cis-acting elements necessary for maximal activation of the GDNF promoter by FGF2. Egr-1 expression is induced by FGF2 in several cell lines (23Jin Y. Sheikh F. Detillieux K.A. Cattini P.A. Mol. Pharmacol. 2000; 57: 984-990PubMed Google Scholar, 24Santiago F.S. Lowe H.C. Day F.L. Chesterman C.N. Khachigian L.M. Am. J. Pathol. 1999; 154: 937-944Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 25Fahmy R.G. Dass C.R. Sun L.Q. Chesterman C.N. Khachigian L.M. Nat. Med. 2003; 9: 1026-1032Crossref PubMed Scopus (307) Google Scholar). Consistent with previous findings, Egr-1 expression was clearly up-regulated by FGF2 as a function of time in primary rat astrocytes (Fig. 3A) or in C6 glioma cells (Fig. 3B). In addition, FGF2 increased Egr-1 promoter activity in a concentration-dependent manner in primary rat astrocytes (Fig. 3C) and C6 cells (Fig. 3D). To determine whether Egr-1 protein binds to the Egr-1-binding sequences in vivo, we performed a ChIP assay. Cross-linked rat primary astrocytes were immunoprecipitated with rabbit anti-Egr-1 antibody or normal rabbit IgG. The resulting immunoprecipitates were analyzed by PCR assays using primers flanking the Egr-1-binding sequences (−324 to −88) of the Gdnf promoter. As shown in Fig. 3E, a noticeable increase in the intensity of the DNA band was observed for rabbit anti-Egr-1 antibody but not for the normal rabbit IgG. The off-target region (from −443 to −243) was not amplified, although positive results were obtained from input DNA. This result indicates that a physical interaction between Egr-1 and the Gdnf promoter occurs in vivo upon FGF2 treatment. In many cell systems, activation of MAPKs, including the ERK, JNK, and p38 MAPKs, induces Egr-1 promoter activity (26Kim C.G. Choi B.H. Son S.W. Yi S.J. Shin S.Y. Lee Y.H. Cell Signal. 2007; 19: 1290-1300Crossref PubMed Scopus (31) Google Scholar, 27Guha M. O'Connell M.A. Pawlinski R. Hollis A. McGovern P. Yan S.F. Stern D. Mackman N. Blood. 2001; 98: 1429-1439Crossref PubMed Scopus (331) Google Scholar, 28Tsai J.C. Liu L. Guan J. Aird W.C. Am. J. Physiol. Cell Physiol. 2000; 279: C1414-C1424Crossref PubMed Google Scholar, 29Lim C.P. Jain N. Cao X. Oncogene. 1998; 16: 2915-2926Crossref PubMed Scopus (145) Google Scholar, 30Cohen D.M. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 11242-11247Crossref PubMed Scopus (58) Google Scholar, 31Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar). To identify the signal transduction pathway that mediates FGF2-induced expression of Egr-1 in primary rat astrocytes, we examined the ability of FGF2 to stimulate MAPK phosphorylation (Fig. 4A). After serum-starved rat astrocytes were treated with FGF2 for various lengths of time, the activation status of MAPKs was assessed using phospho-specific antibodies. The levels of phosphorylated Raf, p38 kinase, JNK1/2, and ERK1/2 increased in a time-dependent manner upon FGF2 treatment. These increases in the phosphorylated levels were evident within 15 min after FGF2 treatment. Similar results were observed in C6 cells (Fig. 4B). To determine whether activation of these MAPKs was associated with FGF2-induced Egr-1 expression, the effects of chemical inhibitors were assessed. Pretreatment of primary rat astrocytes with the MEK inhibitor U0126 or the JNK inhibitor SP600125 completely inhibited FGF2-stimulated Egr-1 expression, whereas neither the phosphatidylinositol 3-kinase inhibitor LY294002 nor the p38 MAPK inhibitor SB203580 had any effect (Fig. 4C). Similar results were obtained in C6 cells (Fig. 4D). As a ternary complex factor, Elk-1 forms a complex with serum response factor on the serum response element (32Ling Y. Lakey J.H. Roberts C.E. Sharrocks A.D. EMBO J. 1997; 16: 2431-2440Crossref PubMed Scopus (68) Google Scholar). Elk-1 is activated by ERK, and JNK (33Yang S.H. Sharrocks A.D. Biochem. Soc. Symp. 2006; 73: 121-129Crossref PubMed Scopus (42) Google Scholar), and its transactivation plays a crucial role in extracellular signaling-induced transcription of Egr-1 (34Schratt G. Weinhold B. Lundberg A.S. Schuck S. Berger J. Schwarz H. Weinberg R.A. Rüther U. Nordheim A. Mol. Cell. Biol. 2001; 21: 2933-2943Crossref PubMed Scopus (125) Google Scholar, 35Watson D.K. Robinson L. Hodge D.R. Kola I. Papas T.S. Seth A. Oncogene. 1997; 14: 213-221Crossref PubMed Scopus (101) Google Scholar). To determine whether the ERK and JNK pathways induce Egr-1 expression via Elk-1 transactivation upon FGF2 stimulation in primary astrocytes, Elk-1 trans-acting activity was examined using a reporter assay system. Primary rat astrocytes were co-transfected with the trans-acting plasmid Gal4-Elk-1 and the pFR-Luc reporter plasmid containing Gal4-binding sequences. Treatment with FGF2 tripled the trans-acting activity of Gal4-Elk-1 (Fig. 5A). However, this activity was strongly inhibited in the presence of DN-MEK1, DN-ERK2, or DN-JNK1. Similarly, FGF2-induced Egr-1 promoter activity (Fig. 5B) and Gdnf promoter activity (Fig. 5C) were strongly attenuated by the expression of DN-MEK, DN-ERK2, or DN-JNK1. These data demonstrate that both ERK and JNK pathways are involved in FGF2-induced expression of Egr-1 and Gdnf. To determine whether Egr-1 plays a functional role in FGF2 regulation of Gdnf transcription, we used RNA interference. First, the knockdown of Egr-1 expression in primary rat astrocytes by transient transfection of shRNA plasmids generating Egr-1-specific siRNA (pSilencer/siEgr1) was verified (Fig. 6A). Then, FGF2-induced Gdnf expression was measured under these conditions using Western blotting. The transient expression of Egr-1 siRNA clearly attenuated activation of the Gdnf promoter (Fig. 6B) and accumulation of Gdnf mRNAs (Fig. 6C) by FGF2. To further evaluate the importance of Egr-1 expression in FGF2-mediated transcriptional activation of the Gdnf gene, we used C6 cell lines stably expressing Egr-1 siRNA (19Choi B.H. Kim C.G. Bae Y.S. Lim Y. Lee Y.H. Shin S.Y. Cancer Res. 2008; 68: 1369-1377Crossref PubMed Scopus (105) Google Scholar). The" @default.
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