Ability of Egr1 to Activate Tyrosine Hydroxylase Transcription in PC12 Cells

We have recently identified an Egr1 motif that overlaps with the Sp1 element in the tyrosine hydroxylase (TH) promoter. Here we examine whether this motif has a functional role in the regulation of TH transcription in PC12 cells. In nuclear extracts from control PC12 cells, an oligonucleotide containing the TH Sp1/Egr1 motif binds Sp1-containing complexes. Treatment of PC12 cells with phorbol ester (2 μm12-O-tetradecanoylphorbol-13-acetate (TPA)) gives rise to a new Egr1-containing complex. TPA treatment reduces the steady-state levels of the Sp1 protein and leads to the appearance of immunoreactive Egr1 protein within 30–60 min. Expression of the Egr1 protein in PC12 cells stimulates the chloramphenicol acetyltransferase reporter gene placed under the control of the first 272 nucleotides of the rat TH promoter. Site-directed mutagenesis of either the Sp1/Egr1 motif or of an upstream AP-1 motif or both abolishes the Egr1-mediated induction of chloramphenicol acetyltransferase activity. An oligonucleotide encompassing the AP-1/E-box sequence of the rat TH promoter competes in electrophoretic mobility shift assays for binding of nuclear extracts from control and TPA-treated cells to an oligonucleotide containing the Sp1/Egr1 element, indicating that these two enhancers may interact. The results show that Egr1 can activate TH transcription and reveals cross-talk between Sp1/Egr1 and AP-1 factors. We have recently identified an Egr1 motif that overlaps with the Sp1 element in the tyrosine hydroxylase (TH) promoter. Here we examine whether this motif has a functional role in the regulation of TH transcription in PC12 cells. In nuclear extracts from control PC12 cells, an oligonucleotide containing the TH Sp1/Egr1 motif binds Sp1-containing complexes. Treatment of PC12 cells with phorbol ester (2 μm12-O-tetradecanoylphorbol-13-acetate (TPA)) gives rise to a new Egr1-containing complex. TPA treatment reduces the steady-state levels of the Sp1 protein and leads to the appearance of immunoreactive Egr1 protein within 30–60 min. Expression of the Egr1 protein in PC12 cells stimulates the chloramphenicol acetyltransferase reporter gene placed under the control of the first 272 nucleotides of the rat TH promoter. Site-directed mutagenesis of either the Sp1/Egr1 motif or of an upstream AP-1 motif or both abolishes the Egr1-mediated induction of chloramphenicol acetyltransferase activity. An oligonucleotide encompassing the AP-1/E-box sequence of the rat TH promoter competes in electrophoretic mobility shift assays for binding of nuclear extracts from control and TPA-treated cells to an oligonucleotide containing the Sp1/Egr1 element, indicating that these two enhancers may interact. The results show that Egr1 can activate TH transcription and reveals cross-talk between Sp1/Egr1 and AP-1 factors. tyrosine hydroxylase chloramphenicol acetyltransferase electrophoretic mobility shift assay hypoxia-inducible factor-1 neural growth factor base pair(s) CCAAT/enhancer-binding protein 12-O-tetradecanoylphorbol-13-acetate dithiothreitol cytomegalovirus The first and major rate-limiting regulatory step in the biosynthesis of the catecholamines (dopamine, norepinephrine, and epinephrine) is catalyzed by tyrosine hydroxylase (TH)1 (1Nagatsu T. Levitt M. Udenfriend S. J. Biol. Chem. 1964; 239: 2910-2917Abstract Full Text PDF PubMed Google Scholar). TH is expressed in catecholaminergic cells of the central and peripheral nervous systems and in the adrenal medulla. The catecholamines are important in the maintenance of internal homeostasis. Aberrations in catecholamine neurotransmission are thought to underlie several prevalent diseases including neuropsychiatric disorders, such as schizophrenia and depression and cardiovascular disorders, such as hypertension. Dysregulation of catecholamine biosynthesis and long term changes in TH activity are some of the mechanisms implicated in the etiology of these disorders (2Mallet J. Trends Neurosci. 1996; 19: 191-196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Recently, polymorphisms in the TH gene have been associated with a prevalent form of hypertension and with manic depression (3Wei J. Ramchand C.N. Hemmings G.P. Life Sci. 1997; 61: 1341-1347Crossref PubMed Scopus (37) Google Scholar, 4Sharma P. Hingorani A. Jia H. Ashby M. Hopper R. Clayton D. Brown M.J. Hypertension. 1998; 32: 676-682Crossref PubMed Scopus (75) Google Scholar). Moreover, Parkinson's disease is characterized by degeneration of the dopaminergic nigro-striatal pathways. Its symptoms may be ameliorated by gene therapy with TH expression vectors (5Corti O. Sanchez-Capelo A. Colin P. Hanoun N. Hamon M. Mallet J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12120-12125Crossref PubMed Scopus (79) Google Scholar, 6Imaoka T. Date I. Ohmoto T. Nagatsu T. Hum. Gene Ther. 1998; 9: 1093-1102Crossref PubMed Scopus (54) Google Scholar). Therefore, an understanding of the intricate physiological mechanisms that regulate TH gene expression is crucial. Physiological and pharmacological stimuli that are associated with long term stimulation of catecholaminergic cells in vivo increase TH gene expression. For example, TH transcription, mRNA levels, and immunoreactive protein are increased in the adrenal medulla of rats exposed to a variety of stressors or with pharmacological treatments such as administration of reserpine or nicotine (7Nankova B. Kvetnansky R. McMahon A. Viskupic E. Hiremagalur B. Frankle G. Fukuhara K. Kopin I.J. Sabban E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5937-5941Crossref PubMed Scopus (120) Google Scholar, 8Kvetnansky R. Sabban E.L. Ann. N. Y. Acad. Sci. 1998; 851: 342-356Crossref PubMed Scopus (45) Google Scholar, 9Faucon Biguet N. Buda M. Lamouroux A. Samolyyk D. Mallet J. EMBO J. 1986; 5: 287-291Crossref PubMed Scopus (184) Google Scholar, 10Wessel T.C. Joh T.H. Mol. Brain Res. 1992; 15: 349-360Crossref PubMed Scopus (55) Google Scholar, 11Fossom L.L.H. Sterling C.R. Tank A.W. Mol. Pharmacol. 1992; 42: 898-908PubMed Google Scholar). In cell cultures of adrenomedullary origin, increased cAMP or calcium, growth factors, phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), and glucocorticoids increase TH mRNA levels, transcription, and/or promoter activity (reviewed in Refs. 12Sabban L.E. Semin. Cell Dev. Biol. 1997; 8: 101-111Crossref PubMed Scopus (40) Google Scholar and 13Kumer S.C. Vrana K.E. J. Neurochem. 1996; 67: 443-462Crossref PubMed Scopus (620) Google Scholar). A number of studies have revealed that alterations in transcription are a primary regulatory mechanism mediating long term changes in TH gene expression. The TH promoter contains several motifs that are homologous to known cis-acting regulatory elements including the hypoxia-inducible factor-1 (HIF) element, AP-1, AP-2, E-box, octamer/heptamer, Sp1, and a cAMP/calcium response element. The relative positions of the AP-1, Sp1, and cAMP/calcium response elements are strictly conserved in the rat, mouse, and human TH genes. The perfect consensus cAMP/calcium response element sequence (−45 to −38) is required for activation of TH transcription by cAMP, nicotine, elevated calcium, and FGF-2 but not by phorbol esters (14Kim K.S. Tinti C. Song B. Cubells J.F. Joh T.H. J. Neurochem. 1994; 63: 834-842Crossref PubMed Scopus (59) Google Scholar, 15Lewis E.J. Harrington C.A. Chikaraishi D.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3550-3554Crossref PubMed Scopus (299) Google Scholar, 16Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar, 17Osaka H. Sabban E.L. Mol. Brain Res. 1997; 49: 222-228Crossref PubMed Scopus (12) Google Scholar, 18Osaka H. Sabban E.L. Soc. Neurosci. 1994; 20: 125.3Google Scholar). In contrast, the AP-1 motif (TGATTCA at −204 to −198), which differs from the consensus sequence (5′-TGACTCA-3′) by a single nucleotide (underlined), appears to be required for TPA- and NGF-induced transcription of TH in PC12 cells (16Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar, 19Icard-Liepkalns C. Biguet N.F. Vyas S. Robert J.J. Sassone-Corsi P. Mallet J. J. Neurosci. Res. 1992; 32: 290-298Crossref PubMed Scopus (105) Google Scholar, 20Leonard D.G.B. Ziff E.B. Green L.A. Mol. Cell. Biol. 1987; 7: 3156-3167Crossref PubMed Scopus (222) Google Scholar, 21Gizang-Ginsberg E. Ziff E.B. Genes Dev. 1990; 4: 477-491Crossref PubMed Scopus (224) Google Scholar). In addition, TPA increases the expression of c-fos and c-junmRNAs and proteins in PC12 cells and also the binding of the AP-1 transcription factor complex to the TH AP-1 site (22Gizang-Ginsberg E. Ziff E.B. Mol. Endocrinol. 1994; 8: 249-262Crossref PubMed Scopus (59) Google Scholar). Interestingly, the transcription of egr1 in PC12 cells is also activated by both NGF (23DeFranco C. Damon D.H. Endoh M. Wagner J.A. Mol. Endocrinol. 1993; 7: 365-379PubMed Google Scholar) and by TPA (24Morita K. Ebert S.N. Wong D.L. J. Biol. Chem. 1995; 270: 11161-11167Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) with typical immediate early kinetics. Egr1 might be a new candidate in the regulation of TH transcription. We have recently demonstrated that the rat TH promoter contains an Egr1 motif that overlaps with the Sp1 motif. Egr1 binding, which is absent in control extracts, is prominent in the adrenal medulla of rats exposed to immobilization stress (25Papanikolaou N.A. Sabban E.L. J. Neurochem. 1999; 73: 433-436Crossref PubMed Scopus (34) Google Scholar). We now examine in PC12 cells if this element is functional and whether Egr1 can regulate TH transcription. The parental wild type plasmid p5′THCAT(−272/+27) containing the first 272 nucleotides of the rat TH promoter, the deletion plasmid p5′THCAT(−108/+27) and the mutant AP-1 (GTGATTCA to TCTCGAGC at −205 to −198) plasmid p5′THCATmAP-1(−272/+27) (26Yoon S.O. Chikaraishi D.M. Neuron. 1992; 9: 55-67Abstract Full Text PDF PubMed Scopus (138) Google Scholar) were generously provided by Dr. Dona Chikaraishi (Duke University Medical Center). The derivative plasmids containing the mutant Sp1/Egr1 site and the double Sp1/Egr1-AP-1 mutant were generated from the p5′THmAP-1CAT(−272/+27), as described under “Site-directed Mutagenesis.” The plasmids pCMVEgr1 and pCMVETTL, which express a full-length and a truncated Egr1 protein, respectively, were a gift from Dr. Dona Wong (Harvard Medical School). In pCMVETTL, a linker with stop codons in all three reading frames is inserted into the uniqueNarI restriction site of pCMVEgr1 at nucleotide 768, thus leading to termination after serine 170 (27Gupta M.P. Gupta M. Zak R. Sukhatme V.P. J. Biol. Chem. 1991; 266: 12813-12816Abstract Full Text PDF PubMed Google Scholar). The pCMVβgal plasmid was purchased from Invitrogen. For EMSA, the following oligonucleotides and their complementary strands were synthesized by Life Technologies, Inc.: 1) THSp1/Egr1 (35 bp 5′-GCCCTCGCTCCATGCCCACCCCCGCCTCCCTCAGG-3′ (−138 to −104); 2) THmSp1 (5′-GCCCTCGCTCCATGCCCACCCTTGCCTCCCTCAGG-3′ (−138 to −104) with two thymines at positions −117 and −116 replacing two cytosines; 3) THAP-1/E-box (5′-CGGGCTGAGGGTGATTCAGAGGCAGGTGCCTG-3′ (−216 to −185), containing both the AP-1/E-box regions; 4) THAP-1, 5′-GGCTGAGGGTGATTCAGAGG-3′ (−215 to −195). The double-stranded, consensus Sp1 (5′-ATTCGATCGGGGCGGGGGGAGC-3′) and C/EBP (5′-TGCAGATTGCGCAATCTGCA-3′) oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All antibodies were from Santa Cruz Biotechnology. Anti-Egr1 was a rabbit polyclonal antibody raised against an epitope corresponding to a C-terminal peptide of human Egr1 (p82). Anti-Sp1 was a goat polyclonal antibody raised against a peptide within the internal domain of rat Sp1 protein. It recognizes both p95 and p106 Sp1 proteins but does not cross-react with Sp2, Sp3, or Sp4. The anti-actin antiserum was a goat polyclonal against the carboxyl terminus of human actin. Anti-C/EBPβ was a rabbit polyclonal antiserum, which is not cross-reactive with C/EBPα, C/EBPδ, or C/EBPε, raised against an epitope at the C terminus of rat C/EBPβ. PC12 rat pheochromocytoma cells (28Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4853) Google Scholar) were originally obtained from Drs. Lloyd Greene (Columbia University) and Daniel O'Connor (University of California, San Diego). The cells were grown to medium density in culture dishes (Falcon) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% heat-inactivated fetal bovine serum and 5% horse serum (Gemini BioProducts), as well as 100 μg/ml streptomycin/penicillin at 37 °C, 7% CO2, as described previously (29Menezes A. Zeman R. Sabban E.L. J. Neurochem. 1996; 67: 138-146PubMed Google Scholar). The cells were treated with 2 μm (final concentration) of TPA (Research Biochemicals Inc.) for up to 6 h. They were washed twice with 1 ml of ice-cold phosphate-buffered saline, harvested, and used to prepare nuclear extracts. Extracts were prepared as described previously (30Nankova B. Devlin D. Kvetnansky R. Kopin I.J. Sabban E.L. J. Neurochem. 1993; 61: 776-779Crossref PubMed Scopus (39) Google Scholar). Briefly, the cells were suspended in three packed cell volumes of hypotonic buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT with protease inhibitors (0.05 mmphenylmethylsulfonyl fluoride, 1 mm each of pepstatin, leupeptin, and aprotinin) and allowed to swell for 10 min on ice. The cells were homogenized, transferred to new tubes, and centrifuged for 30 min at 10,000 rpm. The released nuclei were suspended in half the packed cell volume of low salt buffer (20 mm HEPES, pH 7.9, 20 mm KCl, 1.5 mm MgCl2, 0.1 mm EDTA, 25% glycerol, 0.2 mm DTT, and the mixture of protease inhibitors), followed by the dropwise addition of high salt buffer (20 mm HEPES, pH 7.9, 0.6 mKCl, 1.5 mm MgCl2, 25% glycerol, 0.2 mm DTT, and the mixture of protease inhibitors). The nuclear suspensions were extracted for 30 min at 4 °C with gentle agitation, and the suspension was centrifuged for 30 min at 14,000 rpm. The supernatants (nuclear extracts) were stored at −80 °C in aliquots. Protein concentrations were determined with the Bradford assay method (Bio-Rad). EMSA was performed as described before (25Papanikolaou N.A. Sabban E.L. J. Neurochem. 1999; 73: 433-436Crossref PubMed Scopus (34) Google Scholar). Prior to the addition of labeled DNA probe, 4 μg of PC12 nuclear extracts were incubated for 20 min on ice in 20 μl of reaction buffer containing 10 mm HEPES, pH 7.5, 2.5 mm MgCl2, 50 mm NaCl, 0.5 mm DTT, 4% glycerol, 1 μg of double-stranded poly(dI-dC), and 1 μg of BSA. Radiolabeled probe was added (0.5 ng, 40,000 cpm/assay), and the incubation was continued for another 20 min at room temperature. In competition experiments, the nuclear extracts were preincubated with the indicated molar excess of unlabeled, double-stranded oligonucleotides for 20 min on ice. In supershift experiments, the extracts were preincubated with antibodies for 60 min on ice. Protein-DNA complexes were analyzed on nondenaturing polyacrylamide gels as before (25Papanikolaou N.A. Sabban E.L. J. Neurochem. 1999; 73: 433-436Crossref PubMed Scopus (34) Google Scholar). For immunoblot analysis, PC12 cells were lysed by three freeze-thaw cycles with 10 mm HEPES, pH 7.5, 90 mm KCl, 1 mmmagnesium acetate, 1 mm DTT, 5% glycerol, and 0.5% Nonidet P-40 plus protease inhibitors (5 μm each of phenylmethylsulfonyl fluoride, pepstatin, leupeptin, and aprotinin). Cell debris was removed by centrifugation. The amounts of total protein present in the supernatants were determined. Equal amounts of protein were fractionated in a 6% SDS-polyacrylamide gel. The proteins were transferred to supported nitrocellulose membranes (Bio-Rad). After transfer, the membranes were briefly rinsed with 10 mmTris-Cl, pH 7.5, 150 mm NaCl, 0.1% Tween 20 (1× TBST). The membranes were blocked overnight in 6% (w/v) nonfat milk (Carnation) in TBST and washed three times at room temperature with TBST. Subsequently, the membranes were incubated in primary antibody. After three washes with TBST, the membranes were incubated with appropriate secondary antibodies. They were washed three times, and the bands of interest were detected with chemiluminescence (SuperSignalTM, Pierce). For reprobing, the membranes were stripped at 55 °C for 30 min in buffer (62.5 mm Tris-Cl, pH 5.6, 150 mm NaCl) and washed three times for 5 min each time with 1× TBST at room temperature. They were then blocked with 5% 1× TBST for 45 min and reincubated with antibodies as described above. Mutagenesis of the Egr1/Sp1 site in the rat TH promoter region was performed with the QuickChangeTM polymerase chain reaction-based method (Stratagene, CA), using two primers (5′-CTGAGGGAGGCTTTTGTGGGC-3′ and 5′-GCCCACAAAAGCCTCCCTCAG-3′), which replaced four cytosines with four thymines (underlined) at −119 to −116. Following transformation, amplification, selection, and screening, the mutations were verified with enzymatic sequencing. For transfections, the plasmids (3 μg each of the reporter (p5′THCAT constructs) and Egr1 (pCMVEgr1 or pCMVETTL) effector plasmids as well as 0.5 μg of the pCMVβ-gal vector were mixed with Superfect ReagentTM according to the manufacturer's instructions (Qiagen) and added to PC12 cells grown in quadruplicate on 60-mm dishes (Falcon) in 1 ml of Dulbecco's modified Eagle's medium. The cells were incubated with the transfection mixtures for 3 h at 37 °C. The transfection mixtures were removed, and the cells were washed twice with phosphate-buffered saline, which was then replaced with 2 ml of complete Dulbecco's modified Eagle's medium, and the cells were incubated for 24, 48, and 72 h. The cells were harvested in 1 ml of phosphate-buffered saline and collected by centrifugation. For reporter assays, lysates from transfected cells were prepared and CAT activity was measured using a scintillation assay as described previously (17Osaka H. Sabban E.L. Mol. Brain Res. 1997; 49: 222-228Crossref PubMed Scopus (12) Google Scholar). The β-galactosidase activity was determined (31Maniatis T. Whittemore L.A. Du W. Fan C.M. Keller A. Palonbella V. Thanos D. McKnight S.L. Yamamoto K. Transcriptional Regulation, Part 2. Cold Spring Harbor Cold Spring Scholar). Cell lysates (10 were added to 150 μl of β-galactosidase buffer pH 2 mm MgCl2, 100 mm were to and were incubated 2 h at 37 °C. The reaction was with 0.5 ml of 1 and was measured at The CAT and β-galactosidase for each were for amounts of protein in the cell was determined by of All were performed at mobility shift assays were with a oligonucleotide encompassing the Sp1/Egr1 motif of the TH promoter 1 and nuclear extracts from control and PC12 cells treated with TPA and Extracts from control cells two complexes and that were with excess oligonucleotide 1 The addition of an Sp1 consensus oligonucleotide also with complex for complex 1 nuclear extracts from PC12 cells treated with 2 for 60 or complex was and the of complex was a new complex was The of this complex was by competition with excess Sp1 consensus oligonucleotide 4 and the of complex and generated several complexes 5 and results that complex which appears with TPA contains Egr1 protein. TPA treatment an Egr1-containing complex. TPA the in the Sp1-containing complex complex and the of the complex be by a number of the Egr1 might have for this motif and thus with Sp1 for the binding of Sp1 might be treatment of PC12 cells with Sp1 levels might be from increased or these we the steady-state levels of Sp1 and Egr1 with at time up to 6 h of TPA treatment and The results revealed that immunoreactive Sp1 protein is after 30 and 60 min of After 4 the levels were to in cells. which is in control extracts, was at 60 min and to levels by h. The membranes were with to 2 or to of protein mobility shift assays demonstrated that complex containing was prominent at 60 min of TPA treatment and containing Sp1, after 4 h of TPA 2 results and the previously induction of Egr1 binding in adrenal extracts from rats exposed to immobilization stress (25Papanikolaou N.A. Sabban E.L. J. Neurochem. 1999; 73: 433-436Crossref PubMed Scopus (34) Google Scholar) that Egr1 might be to regulate TH transcription in response to physiological the of Egr1 to activate the TH promoter, we transfected PC12 cells with the reporter plasmids p5′THCAT(−272/+27) and pCMVβgal and the effector plasmid The pCMVEgr1 contains the Egr1 which the full-length human Egr1 protein, under the control of the cytomegalovirus promoter (27Gupta M.P. Gupta M. Zak R. Sukhatme V.P. J. Biol. Chem. 1991; 266: 12813-12816Abstract Full Text PDF PubMed Google Scholar). The pCMVβgal plasmid as an internal control for transfection CAT activity was measured after 24, 48, and 72 h transfected with pCMVEgr1 a increase in CAT reporter activity 72 h 3 In contrast, with pCMVETTL, a plasmid a truncated Egr1 protein the activation induction of CAT was at time 3 and Treatment with TPA to increase the Egr1-mediated activation of the TH promoter at and 72 h but not at h the region in the TH promoter required for the Egr1-mediated induction of CAT the mutant reporter p5′THCAT(−108/+27) was used 3 in which CAT is under the control of the first nucleotides of the rat TH promoter, was with the pCMVEgr1 and expression and CAT reporter gene activity was increase in CAT activity was with this in the 3 or of TPA results that the sequence up to on the rat TH promoter is not for the Egr1-mediated induction of CAT reporter activity. examine if the increased CAT reporter activity is by the Egr1 the four cytosines (underlined) at −119 to −116 on the Sp1/Egr1 sequence were to four thymines in the p5′THCAT(−272/+27) reporter of this site on activity but induction of CAT reporter activity by with pCMVEgr1 phorbol induction of TH has previously been to the AP-1 sequence motif C. Biguet N.F. Vyas S. Robert J.J. Sassone-Corsi P. Mallet J. J. Neurosci. Res. 1992; 32: 290-298Crossref PubMed Scopus (105) Google we also used a with a mutant TH AP-1 by some studies (26Yoon S.O. Chikaraishi D.M. Neuron. 1992; 9: 55-67Abstract Full Text PDF PubMed Scopus (138) Google of this motif to activity. the AP-1 motif or in with a mutant motif the of the Egr1 expression vector to the activity of the CAT reporter gene these the reporter activity was with that obtained with the full-length and truncated Egr1 effector results that the activation of TH transcription by Egr1 both the Sp1/Egr1 and AP-1 The of either the AP-1 or the Sp1/Egr1 motifs CAT induction in response to expressed that may for factors. We whether the TH AP-1/E-box element with complex with the Sp1/Egr1 oligonucleotide in EMSA with PC12 nuclear extracts. extracts from control or TPA-treated PC12 cells were incubated with the labeled THSp1/Egr1 oligonucleotide or with a oligonucleotide corresponding to the TH the TH AP-1/E-box oligonucleotide and of complexes and and to some of complex and A oligonucleotide containing the AP-1 motif was not as a the excess TH Sp1/Egr1 oligonucleotide for all complexes with the three of extracts and a consensus but not the consensus Sp1 for complex the oligonucleotides the consensus or from the promoter not and We also the of TPA on the binding of AP-1 in EMSA with the nuclear extracts, using as a probe the AP-1/E-box sequence oligonucleotide of the rat TH promoter. sequence the region between and on the promoter and is nucleotides upstream from the Sp1/Egr1 motif. TPA treatment increased the binding of AP-1 to this complex was between 60 min and 4 h the Egr1 binding activity is also within this time 2 and of the AP-1/E-box region the Egr1-mediated activation of CAT under the control of the TH promoter we performed supershift with raised against Egr1 to for the of Egr1 in the complex at the AP-1/E-box was Egr1 protein either in control 60 or 4 PC12 cells We also used raised against the transcription factor a protein activated by stress V. R. Cell. 1990; 63: Full Text PDF PubMed Scopus Google Scholar). immunoreactive protein was in of the extracts The of this that the transcription factor Egr1 known as or in J. Trends Neurosci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) is to be in the regulation of TH gene expression. is the first to show that Egr1 can activate the transcription of the TH The of Egr1 into PC12 cells from an expression vector was in a CAT reporter gene under the control of the rat TH promoter. Moreover, cross-talk between with the AP-1 and Egr1 Egr1 binding to the Sp1/Egr1 motif of the TH promoter was within 60 min of treatment of PC12 cells with was identified as an early response gene mRNA increased within of treatment of PC12 cells with NGF J. 1987; PubMed Scopus Google Scholar). which is not blocked by protein is a of the early or immediate early response that is to have a role in a of gene expression that long term of these on cell growth and Ann. N. Y. Acad. Sci. PubMed Scopus Google Scholar). of Egr1 binding activity is also by phorbol or a of 1 and 2 in a number of cell R. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, R. M. H. S. D. Pharmacol. 1992; PubMed Scopus Google Scholar, C. M. M. Wagner J.A. Res. 1993; PubMed Scopus Google Scholar). In PC12 cells, the induction of Egr1 by NGF is