FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2029537608> ?p ?o ?g. }

• W2029537608 endingPage "42793" @default.
• W2029537608 startingPage "42785" @default.
• W2029537608 abstract "The transcription factor Egr-1 regulates the expression of numerous genes involved in differentiation, growth, and in response to environmental signals. Egr-1 activity is modulated in part through the binding of corepressors Nab1 and Nab2. Nab2 appears crucial for controlling Egr-1-mediated transactivation because it is a delayed early response gene, induced by the same stimuli that induce the immediate early gene Egr-1. To identify important elements regulating Nab2 expression, we cloned the human Nab2 gene and investigated the 5′-region. The TATA- and initiator-less Nab2 promoter, located from –679 to –74 bp, contains a total of 11 Egr binding sites, including a cluster of multiple overlapping Egr/Sp1 sites between –329 and –260 bp. This region is critical for basal promoter activity as well as for maximum induction by phorbol esters. Electromobility shifts show that Sp1 binds to this region in normal and stimulated cells, whereas stimulation induces binding of Egr-1. In addition Egr-1 activates the Nab2 promoter in a pattern similar to phorbol esters, suggesting that Egr-1 is a major inducer of protein kinase C-mediated Nab2 induction. Depletion of Egr-1 by each of two distinct Egr-1 short-interfering RNAs reduces Nab2 expression and inducibility, confirming that Egr-1 is an important regulator of Nab2 expression. Transfection experiments show that Egr-1-induced Nab2 promoter activity is itself repressed by Nab2. These results indicate that Egr-1 mediates the induction of its own repressor, thereby preventing a permanent transactivation of Egr-1 target genes and a damaging overreaction in response to environmental signals. The transcription factor Egr-1 regulates the expression of numerous genes involved in differentiation, growth, and in response to environmental signals. Egr-1 activity is modulated in part through the binding of corepressors Nab1 and Nab2. Nab2 appears crucial for controlling Egr-1-mediated transactivation because it is a delayed early response gene, induced by the same stimuli that induce the immediate early gene Egr-1. To identify important elements regulating Nab2 expression, we cloned the human Nab2 gene and investigated the 5′-region. The TATA- and initiator-less Nab2 promoter, located from –679 to –74 bp, contains a total of 11 Egr binding sites, including a cluster of multiple overlapping Egr/Sp1 sites between –329 and –260 bp. This region is critical for basal promoter activity as well as for maximum induction by phorbol esters. Electromobility shifts show that Sp1 binds to this region in normal and stimulated cells, whereas stimulation induces binding of Egr-1. In addition Egr-1 activates the Nab2 promoter in a pattern similar to phorbol esters, suggesting that Egr-1 is a major inducer of protein kinase C-mediated Nab2 induction. Depletion of Egr-1 by each of two distinct Egr-1 short-interfering RNAs reduces Nab2 expression and inducibility, confirming that Egr-1 is an important regulator of Nab2 expression. Transfection experiments show that Egr-1-induced Nab2 promoter activity is itself repressed by Nab2. These results indicate that Egr-1 mediates the induction of its own repressor, thereby preventing a permanent transactivation of Egr-1 target genes and a damaging overreaction in response to environmental signals. The zinc finger transcription factor early growth response (Egr 5The abbreviations used are: Egrearly growth responseNabNGFI-A binding proteinAP2activator protein 2NCDNab conserved domainsiRNAshort-interfering RNAPMAphorbol myristate acetateEMSAelectrophoretic mobility shift assayPhlda-1pleckstrin homology-like domain A-1RT-PCRreverse transcription PCRGAPDHglyceraldehyde-3-phosphate dehydrogenase.5The abbreviations used are: Egrearly growth responseNabNGFI-A binding proteinAP2activator protein 2NCDNab conserved domainsiRNAshort-interfering RNAPMAphorbol myristate acetateEMSAelectrophoretic mobility shift assayPhlda-1pleckstrin homology-like domain A-1RT-PCRreverse transcription PCRGAPDHglyceraldehyde-3-phosphate dehydrogenase.)-1 is an immediate early response gene that couples extracellular signals to the induction of cellular programs of differentiation, growth, and cell death through changes in the expression of Egr-1 target genes. Although Egr-1 expression is low or undetectable in resting cells, it is rapidly and transiently induced by a wide variety of environmental signals including growth factors, cytokines, and toxic substances (1Thiel G. Cibelli G. J. Cell Physiol. 2002; 193: 287-292Crossref PubMed Scopus (500) Google Scholar). Egr-1 in turn activates the transcription of numerous target genes including growth factors that themselves induce Egr-1, as e.g. platelet-derived growth factor (PDGF), thereby establishing a positive autocrine feedback loop. Egr-1 activity is tightly regulated, in part through the function of two proteins, NGFI-A-binding protein (Nab)1 and Nab2, which prevents the permanent activation of Egr-1 target genes and signaling pathways such as those for PDGF (2Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), which could lead to cellular transformation. Nab proteins were first identified as Egr-1-binding proteins in a two-hybrid assay (3Svaren J. Sevetson B.R. Apel E.D. Zimonjic D.B. Popescu N.C. Milbrandt J. Mol. Cell Biol. 1996; 16: 3545-3553Crossref PubMed Scopus (322) Google Scholar, 4Russo M.W. Sevetson B.R. Milbrandt J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6873-6877Crossref PubMed Scopus (242) Google Scholar). Nab2, which is also known as mader (melanoma-associated delayed early response gene), was independently identified as a nuclear protein overexpressed in human malignant melanoma (5Kirsch K.H. Korradi Y. Johnson J.P. Oncogene. 1996; 12: 963-971PubMed Google Scholar). Binding of Nab1 and Nab2 to Egr-1 through interaction between the NCD1 (Nab conserved domain 1) and the R1 domain of Egr-1 has been shown to inhibit Egr-1 activity. early growth response NGFI-A binding protein activator protein 2 Nab conserved domain short-interfering RNA phorbol myristate acetate electrophoretic mobility shift assay pleckstrin homology-like domain A-1 reverse transcription PCR glyceraldehyde-3-phosphate dehydrogenase. early growth response NGFI-A binding protein activator protein 2 Nab conserved domain short-interfering RNA phorbol myristate acetate electrophoretic mobility shift assay pleckstrin homology-like domain A-1 reverse transcription PCR glyceraldehyde-3-phosphate dehydrogenase. Although Nab1 is constitutively expressed in most cells, Nab2 is a delayed early response gene and is induced by the same environmental signals that lead to Egr-1 expression. This suggests that Nab2 is the more important Egr-1 regulator, crucial for controlling Egr-1-mediated activation of transcription. Although this indicates the importance of understanding how the expression of Nab2 is regulated, little is known regarding the transcriptional regulation of the Nab2 gene. In the study presented here we show that Egr-1 has a significant influence on the regulation of the expression of its repressor protein Nab2. Depletion of Egr-1 by transfection of each of two distinct short-interfering (si)RNAs directed against Egr-1 reduced Nab2 expression. In addition the Nab2 promoter was shown to contain multiple putative Egr binding sites and a regulatory region, containing a cluster of multiple overlapping Egr/Sp1 sites, which is required for basal promoter activity and maximum phorbol ester-mediated induction. Although Sp1 was shown to bind to this region both in normal and phorbol ester-stimulated cells, stimulation with phorbol ester induced Egr-1 expression and Egr-1 binding to the Nab2 promoter. In addition Egr-1 activates the Nab2 promoter in a manner similar to that of phorbol ester. The Egr-1-mediated activation of Nab2 transcription was repressed by Nab2 in a dose-dependent manner, supporting the existence of an Egr-1-Nab2 negative feedback mechanism. Cell Lines and Materials—Cell lines were obtained from the ATCC (Manassas, VA), established in our laboratory or obtained through exchange. The human melanoma cell lines Mel 888 and Mel Ei, the human colon carcinoma cell lines Colo 320DM and LoVo and the human cervix carcinoma HeLa L cell line were cultured in RPMI 1640 Medium (Biochrom AG, Berlin, Germany) supplemented with 5% fetal calf serum, 1 mm sodium pyruvate, 2 mm l-glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin at 37 °C and 5% CO2. Cells were stimulated by the addition of phorbol myristate acetate (PMA, final concentration 10 ng/ml) obtained from Sigma-Aldrich for indicated times. All cells were routinely tested for mycoplasma contamination. The human Egr-1 cDNA, kindly provided by Erhard Hofer (Department of Vascular Biology and Thrombosis Research, Vienna International Research Cooperation Centre, University of Vienna (6Lucerna M. Mechtcheriakova D. Kadl A. Schabbauer G. Schafer R. Gruber F. Koshelnick Y. Muller H.D. Issbrucker K. Clauss M. Binder B.R. Hofer E. J. Biol. Chem. 2003; 278: 11433-11440Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar)), was subcloned into HindIII/EcoRI-digested pcDNA3 (Invitrogen). The IMAGE Clone 5721972, containing full-length human Nab1 cDNA, was purchased from MRC geneservice (Cambridge, UK) and was subcloned into EcoRI/NotI-digested pcDNA3. Human Nab2 cDNA, kindly provided by John Svaren (Department of Pathobiological Sciences, University of Wisconsin, Madison (3Svaren J. Sevetson B.R. Apel E.D. Zimonjic D.B. Popescu N.C. Milbrandt J. Mol. Cell Biol. 1996; 16: 3545-3553Crossref PubMed Scopus (322) Google Scholar)), was subcloned into HindIII-digested pcDNA3. The expression construct for human Phlda-1 was described previously (7Neef R. Kuske M.A. Prols E. Johnson J.P. Cancer Res. 2002; 62: 5920-5929PubMed Google Scholar). Inserts of all constructs were validated by sequencing. Jetstar 2.0 plasmid kit (Genomed, Löhne, Germany) was used for DNA preparation. Isolation and Characterization of the Nab2 Genomic Sequence— 2.5 × 105 independent clones of a lambda-Gem-11 human leukocyte genomic library (Promega) were screened with a 463-bp Nab2 cDNA fragment corresponding to base pairs 223–685 of the GenBank sequence NM_005967. The probe was labeled with [α-32P]dATP (Amersham Biosciences) by random priming and hybridization was performed as described (8Lehmann J.M. Riethmuller G. Johnson J.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9891-9895Crossref PubMed Scopus (388) Google Scholar) using Hybond N filters (Amersham Pharmacia Biotech). Purified DNA from two positive clones (m1, m4) was digested with restriction endonucleases and analyzed by Southern blotting for hybridization with the screening probe. Positive fragments were subcloned into pBluescriptII vectors for further analysis. The Nab2 genomic sequence was deposited with GenBank (AF268380). Identification of the potential promoter region and transcription factor binding sites was conducted using the Genomatix suite of programs (9Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2419) Google Scholar). Production of Nab2 5′-Region Deletion Reporter Constructs—All constructs were generated by PCR amplification using Pfu Turbo (Stratagene) with antisense primer Nab2-21 containing HindIII restriction site 5′-CGAAAGCTTGCTGCCCTTCTCGGTGCC-3′ plus the following sense primers containing KpnI restriction site: Nab2-872, 5′-CAAGGTACCCTGCAGAGCCCCACGC-3′; Nab2-679, 5′-CAAGGTACCGTCCGTGCGCTTCTGTG-3′; Nab2-558 5′-CAAGGTACCAAGAGGGCCGGCTGTG-3′; Nab2-468 5′-CACGGTACCTCGGTCACCTCGGTCCC-3′; Nab2-379 5′-CGAGGTACCTCTCCCAGTCTCTAAGC-3′; Nab2-263 5′-CAAGGTACCGGCAGCCAGCGCAGCTC-3′; Nab2-195, 5′-CAAGGTACCGCAAGCGCAGCCGGGG-3′; Nab2-74 5′-CAAGGTACCGGAAGAGGGCAGCAC-3′. Amplified fragments were subcloned into KpnI/HindIII-digested pGL3-basic luciferase reporter plasmid (Promega) and validated by sequencing. Transfections and Luciferase Assays—Each cell line was seeded at 1 × 104 cells/well (in 96-well plates) and cultured for 48 h prior to transfection. For each transfection 2–60 ng of empty and/or expression vector along with 60 or 100 ng of Nab2 promoter luciferase construct were transfected using MATra-A reagent (IBA, Goettingen, Germany) according to the manufacturer's recommendations. In one case cells were seeded at 3 × 105 cells/well (in a 6-well plate) and incubated for 24 h before transfection. Forty-eight hours or as indicated after transfection, cells were lysed with cell culture lysis reagent (Promega) for 30 min on a shaker at room temperature. Luciferase activity was measured in extracts from triplicate samples using a luciferase assay kit from Promega and normalized to protein concentration using Bio-Rad protein assay (Bio-Rad) based on the Bradford method. Each experiment was performed at least twice using different DNA preparations of each construct. siRNA Transfections—HeLa L cells were transfected with 50 μm siRNA using siPORT NeoFX (Ambion, Austin, Texas) according to the manufacturer's recommendations in 12-well plates and incubated for 48 h. After 24 h the medium was replaced with fresh growth medium. siRNAs directed against Egr-1 (siRNA ID number 115234 and ID number 146223) and negative control (catalog number 4706) were purchased from Ambion. The two distinct siRNAs specific for Egr-1 that do not interfere with RNAs of other members of the Egr family were Egr-1-1 (ID number 115234), 5′-GCAGACAAAAGUGUUGUGGtt-3′ (sense) and 5′-CCACAACACUUUUGUCUGCtt-3′ (antisense); Egr-1-2 (ID number 146223), 5′-CGACAGCAGUCCCAUUUACtt-3′ (sense) and 5′-GUAAAUGGGACUGCUGUCGtt-3′ (antisense). RT-PCR—Adherent cells were rinsed in phosphate-buffered saline and lysed in the culture flask on ice. RNA was extracted using RNAeasy kit from Qiagen (Hilden, Germany). 1 μg of total RNA was reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Fermentas, Lithuania) according to the manufacturer's recommendations. The PCR primers, which were designed to amplify cDNA and not genomic DNA, were human Egr-1, 5′-TCAGGCGGACACGGGCGAGC-3′ (forward) and 5′-TGCGCAGCTCAGGGGTGGGC-3′ (reverse), human Nab2, 5′-GACCCTGCAGCCCAGACTC-3′ (forward) and 5′-CCAGGCAGTGGTGATAGCTTC-3′ (reverse) and human GAPDH, 5′-AATTCCATGGCACCGTCAAG-3′ (forward) and 5′-GCCTGCTTCACCACCTTCTT-3′ (reverse). To check for linear amplification each PCR was performed with 1 μl of undiluted cDNA and serial dilutions of 1:10, 1:100, and 1:1000. Linear amplification for each PCR was achieved with 1 μl of 1:10 diluted cDNA. Therefore, each PCR was performed using 1 μl of 1:10 diluted cDNA in a 20-μl PCR with the use of Taq polymerase and buffer Y from Peqlab (Erlangen, Germany). Products were separated by electrophoresis through a 1.2% agarose gel. To control for the integrity and uniformity of the sample preparations, GAPDH mRNA was amplified. Densitometric analysis was performed using SigmaGel software (SPSS science). Nuclear Extract Preparation—Mel 888 cells were either left unstimulated or stimulated for 3 h with PMA. Nuclear extracts were prepared from 5 × 107 cells using the high salt extraction as described (10Leung S. McCracken S. Ghysdael J. Miyamoto N.G. Oncogene. 1993; 8: 989-997PubMed Google Scholar). EMSAs—Double-stranded oligonucleotides were end-labeled with [γ-32P]dATP (Hartmann Analytic, Braunschweig, Germany) using T4 polynucleotide kinase (New England Biolabs). Nuclear extracts (12 μg of protein) were incubated for 40 min on ice with 32P-labeled oligonucleotide in a final volume of 15 μl of binding buffer containing 10 mm Tris-HCl (pH 7.5), 1 mm dithiothreitol, 1 mm EDTA, 50 mm NaCl, 5% glycerol, and 2 μg of poly(dI-dC). For oligonucleotide competitions and antibody supershift/blocking experiments, the gel shift mixture was preincubated on ice for 30 min with 40 ng of oligonucleotide competitor or 2 μg of antibody. Preincubation with Egr-1 antibody was performed at room temperature. DNA-protein complexes were separated on a 4% nondenaturing polyacrylamide gel containing 0.5× Tris borate EDTA at 125V for 3 h at room temperature. Gels were dried and exposed to x-ray film with intensifying screens at –80 °C. Antibodies against Egr-1 (sc-110 X) and Sp1 (sc-59 X) as well as oligonucleotides for consensus and mutant DNA-binding domains of Egr-1 (sc-2529/sc-2530) and Sp1 (sc-2502/sc-2503) were purchased from Santa Cruz (Santa Cruz, CA) Rabbit control serum (X903) was obtained from DAKO (Hamburg, Germany). Isolation of the Nab2 Genomic Sequence—The full-length Nab2 gene was isolated from a human leukocyte genomic library screened with a 463-bp probe from the 5′-region of the cDNA. Two phage clones, m1 and m4, were obtained. A 7.4-kb BamHI fragment (4B7) from m1 and a 2.1-kb EcoRI fragment (1E10) from m4 were subcloned and sequenced. Clone 4B7 was found to contain the entire Nab2 gene as well as an 1845-bp region 5′ to the first ATG (Fig. 1). As previously reported the Nab2 gene consists of 7 exons (3Svaren J. Sevetson B.R. Apel E.D. Zimonjic D.B. Popescu N.C. Milbrandt J. Mol. Cell Biol. 1996; 16: 3545-3553Crossref PubMed Scopus (322) Google Scholar). All 6 introns fulfill the GT-AG rule. Comparison of the exon sequence with the sequence of the drop8 variant cDNA (GenBank AJ011081), which was isolated from a human melanoma cell line, showed that this is a splice variant lacking exon 6 (Fig. 1). Identification of Nab2 Promoter Region—The Nab2 expression is driven by a promoter with high GC content, which contains no typical core promoter elements such as TATA box, initiator sequence, or CAAT box. In silico analysis indicated a CpG island at bp –876 to +82, a putative promoter region at bp –705 to –82 relative to translation start site and several putative Egr, Sp1, NFκB, and AP2 transcription factor binding sites as well as a cAMP-responsive element (Fig. 2). To identify promoter elements that control Nab2 transcription, the 5′-region of Nab2 was used to drive a promoterless firefly luciferase reporter plasmid. This construct as well as serially 5′-truncated fragments were assessed for activity by transient transfection in Nab2 expressing Colo 320DM and Mel 888 cells (Fig. 2). The activity of the luciferase reporter maluc-679 was comparable with that of maluc-872 and a construct containing the 5′-full-length genomic DNA of Nab2 (data not shown), whereas the activity of maluc-558, lacking two Egr sites, one Sp1, and one AP2 binding site, was reduced by 20–30%. These findings indicate that maluc-679 includes the complete Nab2 promoter. The removal of an additional 90 bp, including one Egr and one Egr/Sp1 binding site, up to nucleotide –468 reduced the activity by another 20%, whereas further deletion up to bp –379 had no influence on the luciferase activity. This indicates that the Egr/Sp1 binding site at bp –422 has no important function in basal expression. Further 5′-truncation of the Nab2 promoter to nucleotide –263 resulted in a loss of luciferase activity of 72% in Mel 888 and 60% in Colo 320DM, compared with maluc-379, suggesting the presence of a major regulatory element in the promoter region between bp –379 to –263. This region contains multiple overlapping Egr/Sp1 sites and one AP2 binding site (see Fig. 6A). The activity of maluc-195, lacking one Egr/Sp1 binding site and a cAMP-responsive element compared with maluc-263, declined by a further 60%. Further truncation of an additional 121 nucleotides, comprising one Egr binding site, up to bp –74 essentially abolished transcriptional activity.FIGURE 6Members of the Egr family bind to the Nab2 promoter. A, detailed representation of the Nab2 oligonucleotides 1, 2, and 3, spanning the region between bp –329 and –260 of the Nab2 promoter, used for EMSAs. Positions relative to translation start site (+1) in the Nab2 promoter are indicated. Dotted lines represent putatuve Sp1 binding sites, solid lines represent putative Egr binding sites, and an AP2 binding site is noted. B, binding of Egr family members to the Nab2 oligonucleotide 3 is induced by stimulation with PMA, whereas binding of Sp1 is not influenced. Nuclear extracts (12 μg of protein) of PMA stimulated or unstimulated Mel 888 cells were incubated with the 32P-labeled Nab2 oligonucleotide 3 together with the indicated Egr or Sp1 competitor oligonucleotides as described under “Experimental Procedures.” Nab2 oligo, no competitor added; Cons, consensus; Mut, mutated. The observed complexes are indicated with roman numerals. C, members of the Egr family bind to the region between bp –329 and –260 of the Nab2 promoter after PMA treatment. Nuclear extracts (12 μg of protein) of Mel 888 cells stimulated for 3 h with PMA were incubated with the indicated 32P-labeled Nab2 oligonucleotides in the presence or absence of an Egr consensus sequence oligonucleotide (Egr cons) as described under “Experimental Procedures.” The observed complexes are indicated with roman numerals.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These results indicate that the Nab2 promoter is located from bp –679 to –74 relative to translation start site, corresponding precisely to the putative promoter region predicted by in silico analysis software. This region includes four Egr, three overlapping Egr/Sp1 binding sites, one Sp1, one AP2 site, a cAMP-responsive element, and one cluster of multiple Egr/Sp1 binding sites. Analysis of the deletion constructs indicate a major regulatory element at –329 to –260 bp, which contains multiple Egr/Sp1 sites and a single AP2 site (see Fig. 6A). The correlation of the activity with the number of Sp1 sites in the deletion constructs points to a role for the constitutively expressed transcription factor Sp1 in basal promoter activity. The Nab2 Promoter Is Activated by Egr-1—The Nab2 promoter contains several putative Egr or overlapping Egr/Sp1 binding sites (Fig. 2), suggesting that members of the Egr transcription factor family are major regulators of Nab2 expression. Egr-1 has been shown to induce Nab2 mRNA expression when transfected into murine neuroblastoma cells (11Ehrengruber M.U. Muhlebach S.G. Sohrman S. Leutenegger C.M. Lester H.A. Davidson N. Gene (Amst.). 2000; 258: 63-69Crossref PubMed Scopus (59) Google Scholar) and was identified as a Egr-1 target gene by Affymetrix microarray (12Fu M. Zhu X. Zhang J. Liang J. Lin Y. Zhao L. Ehrengruber M.U. Chen Y.E. Gene (Amst.). 2003; 315: 33-41Crossref PubMed Scopus (139) Google Scholar). As can be seen in Fig. 3A transfection of Egr-1 leads to Nab2 gene expression in human colon carcinoma cells. Colo 320DM cells were transfected with human Egr-1 or control vector and RT-PCR was performed 48 h later. Nab2 mRNA was increased after Egr-1 transfection, but remained unaffected after transfection of the control protein Phlda-1. To determine whether Egr-1 influences the Nab2 promoter activity, human Egr-1 was transfected together with the human Nab2 promoter reporter construct. Sixty ng of the reporter construct maluc-872, which contains the complete Nab2 promoter, were transfected into Colo 320DM, LoVo, Mel 888, and Mel Ei cells together with 40 ng of Egr-1 or Phlda-1 as control. In all cell lines, Egr-1 increased Nab2 promoter activity compared with Phlda-1 (Fig. 3B). Induction was higher in melanoma cell lines (4–5-fold) than in colon carcinoma cell lines (2–3-fold). Additional experiments indicated that activation of the Nab2 promoter by Egr-1 is dose-dependent, reaching a plateau at 20 ng of Egr-1 (Fig. 3C). Transfection of Phlda-1 did not influence the Nab2 promoter activity. These results indicate that Egr-1 induces the expression of its own corepressor by stimulating Nab2 promoter activity. Depletion of Egr-1 Reduces the Inducibility of Nab2 in Response to Phorbol Ester—To examine the significance of Egr-1 in the regulation of Nab2 expression two distinct siRNAs specific for Egr-1 (Egr-1-1 and Egr-1-2) and a negative control siRNA, with no similarity to human gene sequences, were transfected into HeLa L cells. After 48 h the cells were stimulated with PMA, and RT-PCR analysis of Egr-1 and Nab2 expression was performed at 20 min and 1 h. As can be seen from Fig. 4 this cell line expresses both Egr-1 and Nab2 under normal growth conditions. Transfection of control siRNA did not influence the typical mRNA expression patterns of either Egr-1 or Nab2. A 3-fold Egr-1 induction in response to PMA was observed after 20 min, which increased to 4-fold after 1 h, whereas a 2-fold induction of Nab2 expression was first seen 1 h after PMA treatment. Transfection of two siRNAs directed against Egr-1 (Egr-1-1 and Egr-1-2) resulted in a decrease of basal Egr-1 mRNA expression of 40% (Egr-1-2) and 70% (Egr-1-1), respectively (Fig. 4B). Although PMA stimulation induced Egr-1 expression after 20 min in Egr-1 siRNA-treated cells, the Egr-1 mRNA levels were reduced by 60% in Egr-1-1 and 70% in Egr-1-2-transfected cells compared with control cells. In contrast to control siRNA-treated cells, no further increase in Egr-1 expression was detectable after 1 h. Depletion of Egr-1 influenced Nab2 expression as well. Transfection of Egr-1 siRNAs reduced basal Nab2 mRNA levels by 65% (Egr-1-2) and 80% (Egr-1-1) (Fig. 4C). Stimulation with PMA increased Nab2 mRNA levels at 1 h, but this was reduced by 70% compared with cells transfected with control siRNA. Transfection of each of the two siRNAs directed against Egr-1 inhibited not only Egr-1 expression but also resulted in a decrease of Nab2 expression, confirming that endogenous Egr-1 has a significant influence on the regulation of Nab2 expression. Identification of Elements Mediating the Nab2 Induction—Nab2 is a delayed early response gene and can be induced by exposure to growth factors and mitogens as well as by stimulation with phorbol esters such as PMA. To identify elements in the Nab2 promoter mediating induction by phorbol esters the 5′-truncated fragments were transfected into Colo 320DM and Mel 888 cells and cultured in the presence or absence of PMA for 24 h (Fig. 5A). In Colo 320DM cells truncation of the Nab2 complete promoter to bp –379 relative to translation start site had no influence on luciferase activity in response to PMA. Further deletion up to bp –263 reduced luciferase activity by 80%. A 4–5-fold stimulation in response to PMA is still observed with maluc-263, and this is reduced to 2–3-fold in maluc-195. These results indicate an important element between bp –379 and –263, which is necessary for maximum activation of the Nab2 promoter in response to PMA in Colo 320DM cells, as well as responsive elements between bp –263 and –74. The region between bp –379 and –263 has already been shown to be important for basal promoter activity and contains a complex cluster of binding sites, including several Egr/Sp1 motifs (Fig. 6A). An additional Egr/Sp1 binding site, a cAMP-responsive element, and a single Egr binding site are located between bp –263 and –74. The importance of the region between bp –379 and –263 in mediating PMA induction is also observed in Mel 888 cells. However, in these cells additional elements located further upstream contribute significantly to PMA induction. In Mel 888 cells luciferase activity of maluc-872, containing the full Nab2 promoter, was 2.5-fold higher than that of maluc-379 and maluc-468 after PMA treatment. This region contains additional four Egr binding sites, one Egr/Sp1 site, one AP2, and a single NFκB binding motif. This implies that a cluster of multiple Egr/Sp1 binding sites and one AP2 binding site located at bp –329 to –260, present in maluc-379 but not in maluc-263, is the major regulatory element in the Nab2 promoter for induction of transcription after PMA stimulation. Identification of Egr-1-responsive Elements in the Nab2 Promoter— The expression of transcription factors of the Egr family but not Sp1 (2Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Gineitis D. Treisman R. J. Biol. Chem. 2001; 276: 24531-24539Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14Raychowdhury R. Schaefer G. Wang T.C. Hoecker M. Gastroenterology. 2001; 120: A305Abstract Full Text PDF PubMed Google Scholar) is induced after stimulation with PMA. To investigate whether Egr-1 activates the Nab2 promoter deletion reporter constructs in the same manner as PMA, the 5′-truncated fragments were assessed for activity in Colo 320DM and Mel 888 cells after transfection with 40 ng of human Egr-1 or Phlda-1 DNA as control (Fig. 5B). In Colo 320DM cells the pattern of induction of the Nab2 promoter by Egr-1 was nearly identical to that after PMA stimulation, although the activation after PMA treatment was higher than after Egr-1 transfection. In addition the construct maluc-195 responds to PMA, whereas it does not respond to Egr-1 transfection in Colo 320DM cells. These results indicate the involvement of additional transcription factors in response to PMA in this cell line. Corresponding to the results obtained after PMA stimulation, the cluster of multiple Egr/Sp1 sites and one AP2 binding motif, located between bp –379 and –263, was necessary for maximum Egr-1-mediated activation of the Nab2 promoter in Colo 320DM cells. These results indicated that in this cell line the same promoter regulatory elements that respond to PMA are also responsive to Egr-1, suggesting that Egr-1 binding sites mediate the PMA stimulation. In Mel 888 cells both the pattern and strength of induction after Egr-1 transfection coincides with that observed after PMA stimulation. The region between bp –379 and –260 is also of major importance in response to E" @default.
• W2029537608 created "2016-06-24" @default.
• W2029537608 creator A5038400103 @default.
• W2029537608 creator A5063708946 @default.
• W2029537608 creator A5080013197 @default.
• W2029537608 date "2005-12-01" @default.
• W2029537608 modified "2023-09-30" @default.
• W2029537608 title "Egr-1 Induces the Expression of Its Corepressor Nab2 by Activation of the Nab2 Promoter Thereby Establishing a Negative Feedback Loop" @default.
• W2029537608 cites W1228798347 @default.
• W2029537608 cites W1544307004 @default.
• W2029537608 cites W1984580777 @default.
• W2029537608 cites W1995529555 @default.
• W2029537608 cites W2000045473 @default.
• W2029537608 cites W2001352795 @default.
• W2029537608 cites W2001381510 @default.
• W2029537608 cites W2006345574 @default.
• W2029537608 cites W2012886530 @default.
• W2029537608 cites W2030248303 @default.
• W2029537608 cites W2042153179 @default.
• W2029537608 cites W2044570331 @default.
• W2029537608 cites W2044643825 @default.
• W2029537608 cites W2054498037 @default.
• W2029537608 cites W2056714530 @default.
• W2029537608 cites W2064879068 @default.
• W2029537608 cites W2067754245 @default.
• W2029537608 cites W2081662078 @default.
• W2029537608 cites W2087367550 @default.
• W2029537608 cites W2109356886 @default.
• W2029537608 cites W2123995229 @default.
• W2029537608 cites W2150901931 @default.
• W2029537608 cites W2151325576 @default.
• W2029537608 cites W2157972566 @default.
• W2029537608 cites W2158841551 @default.
• W2029537608 cites W2169110289 @default.
• W2029537608 cites W2188854244 @default.
• W2029537608 cites W4250733479 @default.
• W2029537608 doi "https://doi.org/10.1074/jbc.m511079200" @default.
• W2029537608 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16260776" @default.
• W2029537608 hasPublicationYear "2005" @default.
• W2029537608 type Work @default.
• W2029537608 sameAs 2029537608 @default.
• W2029537608 citedByCount "87" @default.
• W2029537608 countsByYear W20295376082012 @default.
• W2029537608 countsByYear W20295376082013 @default.
• W2029537608 countsByYear W20295376082014 @default.
• W2029537608 countsByYear W20295376082015 @default.
• W2029537608 countsByYear W20295376082017 @default.
• W2029537608 countsByYear W20295376082018 @default.
• W2029537608 countsByYear W20295376082019 @default.
• W2029537608 countsByYear W20295376082020 @default.
• W2029537608 countsByYear W20295376082021 @default.
• W2029537608 countsByYear W20295376082022 @default.
• W2029537608 countsByYear W20295376082023 @default.
• W2029537608 crossrefType "journal-article" @default.
• W2029537608 hasAuthorship W2029537608A5038400103 @default.
• W2029537608 hasAuthorship W2029537608A5063708946 @default.
• W2029537608 hasAuthorship W2029537608A5080013197 @default.
• W2029537608 hasBestOaLocation W20295376081 @default.
• W2029537608 hasConcept C104317684 @default.
• W2029537608 hasConcept C114614502 @default.
• W2029537608 hasConcept C146777309 @default.
• W2029537608 hasConcept C150194340 @default.
• W2029537608 hasConcept C158448853 @default.
• W2029537608 hasConcept C184670325 @default.
• W2029537608 hasConcept C33923547 @default.
• W2029537608 hasConcept C502942594 @default.
• W2029537608 hasConcept C54355233 @default.
• W2029537608 hasConcept C86803240 @default.
• W2029537608 hasConceptScore W2029537608C104317684 @default.
• W2029537608 hasConceptScore W2029537608C114614502 @default.
• W2029537608 hasConceptScore W2029537608C146777309 @default.
• W2029537608 hasConceptScore W2029537608C150194340 @default.
• W2029537608 hasConceptScore W2029537608C158448853 @default.
• W2029537608 hasConceptScore W2029537608C184670325 @default.
• W2029537608 hasConceptScore W2029537608C33923547 @default.
• W2029537608 hasConceptScore W2029537608C502942594 @default.
• W2029537608 hasConceptScore W2029537608C54355233 @default.
• W2029537608 hasConceptScore W2029537608C86803240 @default.
• W2029537608 hasIssue "52" @default.
• W2029537608 hasLocation W20295376081 @default.
• W2029537608 hasOpenAccess W2029537608 @default.
• W2029537608 hasPrimaryLocation W20295376081 @default.
• W2029537608 hasRelatedWork W1207770767 @default.
• W2029537608 hasRelatedWork W1971981721 @default.
• W2029537608 hasRelatedWork W2003755002 @default.
• W2029537608 hasRelatedWork W2116188114 @default.
• W2029537608 hasRelatedWork W2230458223 @default.
• W2029537608 hasRelatedWork W2346548024 @default.
• W2029537608 hasRelatedWork W2554098092 @default.
• W2029537608 hasRelatedWork W2565274046 @default.
• W2029537608 hasRelatedWork W4213416581 @default.
• W2029537608 hasRelatedWork W4323295151 @default.
• W2029537608 hasVolume "280" @default.
• W2029537608 isParatext "false" @default.
• W2029537608 isRetracted "false" @default.
• W2029537608 magId "2029537608" @default.
• W2029537608 workType "article" @default.