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J. Biol. Chem., Vol. 275, Issue 35, 26683-26689, September 1, 2000

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Ability of Egr1 to Activate Tyrosine Hydroxylase Transcription in PC12 Cells

CROSS-TALK WITH AP-1 FACTORS^*

Nikolaos A. Papanikolaou and Esther L. Sabban

From the Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595

Received for publication, January 3, 2000, and in revised form, May 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL METHODS RESULTS DISCUSSION REFERENCES

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 µM 12-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL METHODS RESULTS DISCUSSION REFERENCES

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 (1). 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 (2). Recently, polymorphisms in the TH gene have been associated with a prevalent form of hypertension and with manic depression (3, 4). 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 (5, 6). 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 (7-11). 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. 12 and 13). 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 (14-18). 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 (16, 19-21). In addition, TPA increases the expression of c-fos and c-jun mRNAs and proteins in PC12 cells and also the binding of the AP-1 transcription factor complex to the TH AP-1 site (22). Interestingly, the transcription of egr1 in PC12 cells is also activated by both NGF (23) and by TPA (24) 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 (25). We now examine in PC12 cells if this element is functional and whether Egr1 can regulate TH transcription.

	EXPERIMENTAL METHODS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL METHODS RESULTS DISCUSSION REFERENCES

Materials

Plasmids-- 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) (26) 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 unique NarI restriction site of pCMVEgr1 at nucleotide 768, thus leading to termination after serine 170 (27). The pCMVgal plasmid was purchased from Invitrogen.

Oligonucleotides-- 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).

Antibodies-- 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/EBP was a rabbit polyclonal antiserum, which is not cross-reactive with C/EBP, C/EBP, or C/EBP, raised against an epitope at the C terminus of rat C/EBP.

Methods

Cell Culture-- PC12 rat pheochromocytoma cells (28) 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 (29). 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.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSAs)-- Extracts were prepared as described previously (30). 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 mM phenylmethylsulfonyl 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 M KCl, 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 (25). 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 (25).

Immunoblot Analysis of Whole Cell Extracts-- For immunoblot analysis, PC12 cells were lysed by three freeze-thaw cycles with 10 mM HEPES, pH 7.5, 90 mM KCl, 1 mM magnesium 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 mM Tris-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 (SuperSignal^TM, 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.

Site-directed Mutagenesis-- Mutagenesis of the Egr1/Sp1 site in the rat TH promoter region was performed with the QuickChange^TM 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.

Transfection of PC12 Cells-- 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 pCMV-gal vector were mixed with Superfect Reagent^TM 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.

Reporter Gene Assays-- For reporter assays, lysates from transfected cells were prepared and CAT activity was measured using a scintillation assay as described previously (17). The -galactosidase activity was determined (31). Cell lysates (10 µg) were added to 150 µl of 2× -galactosidase buffer (200 nM sodium phosphate, pH 7.3, 2 mM MgCl₂, 100 mM mercaptoethanol, 1.33 mg/ml o-nitrophenyl -D-galactopyranoside). Volumes were adjusted to 300 µl, and samples were incubated 2 h at 37 °C. The reaction was stopped with 0.5 ml of 1 M Na₂CO₃ solution, and absorbance was measured at 420 nm. The CAT and -galactosidase activities for each sample were normalized for equal amounts of protein in the cell lysates. Statistical significance was determined by analysis of variance. All experiments were performed at least twice.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL METHODS RESULTS DISCUSSION REFERENCES

Phorbol Ester Treatment Induces Egr1 and Reduces Sp1 Binding to the TH Promoter-- Electrophoretic mobility shift assays were carried out with a radiolabeled oligonucleotide encompassing the Sp1/Egr1 motif of the TH promoter (Fig. 1A) and nuclear extracts from control and PC12 cells treated with TPA (Fig. 1, B and C). Extracts from control cells formed two complexes (I and III) that were competed with excess unlabeled oligonucleotide (Fig. 1B, lane 2). The addition of an Sp1 consensus oligonucleotide also competed with complex formation, especially for complex I (Fig. 1B, lane 6).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Treatment with TPA alters the binding to TH Sp1/Egr1 motif. A, diagrammatic representation of the proximal rat TH promoter. The presence of putative and functional elements involved in regulation of TH transcription are shown. The later are shaded. The sequence of the 35-bp oligonucleotide (THSp1/Egr1, 138 to 104) containing the overlapping Sp1/Egr1 region and used in EMSA is indicated. B, EMSA analysis of complexes formed with the -32P-labeled THSp1/Egr1 oligonucleotide and extracts from untreated PC12 cells (lanes 1, 2, 5, and 6) or cells treated for 120 min with 2 µM TPA (lanes 3 and 4). The THSp1/Egr1 oligonucleotide (self, lanes 2 and 4) or consensus Sp1 oligonucleotide (lane 6) at a 100-fold excess were used as competitors. C, EMSA analysis of complexes at different times of TPA treatment. The -32P-labeled THSp1/Egr1 oligonucleotide was incubated with nuclear extracts from untreated PC12 cells (lanes 1 and 2), cells treated with 2 µM TPA for 60 min (lanes 3-5) or 120 min (lanes 6-8), or without any addition (lanes 1, 3, and 6), or with 100-fold excess Sp1 consensus oligonucleotide (lanes 4 and 7), or with Egr1-specific antisera (lanes 2, 5, and 8) as described under "Methods."

With nuclear extracts from PC12 cells treated with 2 µM TPA for 60 or 120 min, complex I was absent, and the intensity of complex III was reduced. Importantly, a new specific complex was formed (complex II). The formation of this complex was unaffected by competition with excess Sp1 consensus oligonucleotide (Fig. 1C, lanes 4 and 7). However, anti-Egr1-specific antisera prevented the formation of complex II and generated several supershifted complexes (lanes 5 and 8). These results indicate that complex II, which appears with TPA treatment, contains immnunoreactive Egr1 protein. Thus, TPA treatment induces an Egr1-containing complex.

With TPA treatment, the reduction in the Sp1-containing complex (mostly complex I) and the formation of the Egr1-specific complex (II) could be mediated by a number of possible mechanisms. First, the TPA-induced Egr1 might have greater affinity for this motif and thus effectively compete with Sp1 for binding. Second, the binding affinity of Sp1 might be reduced upon treatment of PC12 cells with TPA. Third, Sp1 levels might be reduced. This could result from increased degradation or reduced synthesis.

To distinguish among these possibilities, we examined the steady-state levels of Sp1 and Egr1 with immunoblots at various time intervals, up to 6 h of TPA treatment (Fig. 2, A and B). The results revealed that immunoreactive Sp1 protein is greatly reduced after 30 and 60 min of TPA. After 4 h, the levels were similar to those in untreated cells. Concomitantly, Egr1, which is undetectable in control extracts, was maximally induced at 60 min and subsequently declined to undetectable levels by 4-6 h. The same membranes were reprobed with antisera to actin (Fig. 2A) or to C/EBP (Fig. 2B), confirming equal loading of protein samples. Electrophoretic mobility shift assays demonstrated that complex II, containing Egr1, was prominent at 60 min of TPA treatment and gradually declined thereafter. Complex I, containing Sp1, reappeared after 4 h of TPA (Fig. 2C).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Changes in Sp1 and Egr1 with TPA treatment. The PC12 cells were treated with 2 µM TPA for the indicated times. A and B, cell lysates, recombinant Sp1 (rSp1), and molecular weight markers (MW) were fractionated on 6% SDS polyacylamide gels, transferred to nitrocellulose, and probed with anti-Sp1 or actin antibodies (A) or anti-Egr1 or C/EBP antibodies (B). C, electrophoretic mobility shift assays of nuclear extracts from the same cells as in B.

Egr1 Expression Induces CAT Reporter Activity Driven by the TH Promoter-- These results and the previously observed induction of Egr1 binding in adrenal medullary extracts from rats exposed to immobilization stress (25) suggested that Egr1 might be able to regulate TH transcription in response to physiological signals. To directly study the ability of Egr1 to activate the TH promoter, we transiently transfected PC12 cells with the reporter plasmids p5'THCAT(272/+27) and pCMVgal and the effector plasmid pCMVEgr1. The pCMVEgr1 contains the 3.1-kilobase pair Egr1 cDNA, which encodes the full-length human Egr1 protein, under the control of the cytomegalovirus (CMV) promoter (27). The pCMVgal plasmid served as an internal control for transfection efficiency. CAT activity was measured after 24, 48, and 72 h (Fig. 3A). Cells transfected with pCMVEgr1 displayed a 4-fold increase in CAT reporter activity 72 h post-transfection (Fig. 3A, lane 5). In contrast, when cotransfected with pCMVETTL, a plasmid expressing a truncated Egr1 protein lacking the N-terminal activation domain, no induction of CAT was observed at any time point (Fig. 3A, lanes 2, 4, and 6). Treatment with TPA tended to further increase the Egr1-mediated activation of the TH promoter construct at 48 and 72 h but not at 24 h post-transfection (Fig. 3B).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Egr1-triggered activation of CAT reporter activity under the control of the proximal rat TH promoter. A, PC12 cells were co-transfected with the p5'THCAT(272/+27) reporter and pCMVEgr1 effector plasmids (lanes 1, 3, and 5) or p5'THCAT(272/+27) and pCMVETTL (lanes 2, 4, and 6). B, p5'THCAT(272/+27) reporter and pCMVEgr1 effector plasmid were transfected for the indicated times and incubated in the absence (lanes 1, 3, and 5) or presence of 2 µM TPA for 3 h before harvesting (lanes 2, 4, and 6). C, p5'THCAT(108/+27) reporter plasmid was cotransfected with either the pCMVEgr1 or pCMVETTL effector plasmid and pCMVgal vector as described under "Methods." The CAT and -galactosidase (Gal) activities were measured at the indicated times after transfection. Results, mean ± S.E. (n = 4) of relative CAT/-galactosidase, are expressed relative to controls (with no effector plasmid) taken as 1.0. *, p  0.01 compared with controls.

To identify the region in the TH promoter required for the Egr1-mediated induction of CAT activity, the mutant reporter construct p5'THCAT(108/+27) was used (Fig. 3C). This construct, in which CAT is under the control of the first 108 nucleotides of the rat TH promoter, was cotransfected with the pCMVEgr1 and pCMVETTL expression vectors, and CAT reporter gene activity was measured. No appreciable increase in CAT activity was observed with this construct in the absence (Fig. 3C) or presence of TPA (not shown). These results indicate that the sequence up to 108 on the rat TH proximal promoter is not sufficient for the Egr1-mediated induction of CAT reporter activity.

To examine if the increased CAT reporter activity is mediated by the putative Egr1 motif, the four cytosines (underlined) at 119 to 116 on the Sp1/Egr1 sequence (5'-CACCCCCGCCT-3') were mutated to four thymines in the p5'THCAT(272/+27) reporter construct. Mutation of this site had little effect on basal activity but abolished induction of CAT reporter activity triggered by cotransfection with pCMVEgr1 (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Demonstration of the requirement for both the Sp1/Egr1 and AP-1 motifs for the Egr1-triggered activation of CAT reporter activity. PC12 cells were co-transfected without effector plasmid or with pCMVETTL or pCMVEgr1 effector plasmids and pCMVgal vector (to normalize for transfection efficiency) together with one of the following reporter constructs: p5'THCAT(272/+27) plasmid intact (wild type) or with mutation of Sp1/Egr1 or AP-1 motifs or the double mutant. The ratio of CAT and -galactosidase (Gal) activities in equal amounts of protein in homogenates were measured at 72 h post-transfection. Results are mean ± S.E. relative to controls taken as 1.0. *, p  0.01 compared with controls.

Since phorbol ester-mediated induction of TH has previously been reported to require the AP-1 sequence motif (19), we also used a construct with a mutant TH AP-1 site. As reported by some previous studies (26), mutation of this motif tended to reduce basal activity. Surprisingly, mutating the AP-1 motif alone or in combination with a mutant Egr-1 motif completely abolished the ability of the Egr1 expression vector to up-regulate the activity of the CAT reporter gene (Fig. 4). With these constructs, the reporter activity attained was comparable with that obtained with the full-length (pCMVEgr1) and truncated (pCMVETTL) Egr1 effector plasmids. These results suggest that the activation of TH transcription by Egr1 requires both the Sp1/Egr1 and AP-1 motifs.

An Oligonucleotide That Contains the TH AP-1/E-box Can Compete against the TH Sp1/Egr1 Oligonucleotide for Nuclear Protein Binding-- The mutation of either the AP-1 or the Sp1/Egr1 motifs abolished CAT induction in response to expressed Egr1, suggesting that they may compete for similar factors. We therefore examined whether the TH AP-1/E-box element could interfere with complex formation with the Sp1/Egr1 oligonucleotide in EMSA with PC12 nuclear extracts. Nuclear extracts from control or TPA-treated PC12 cells were incubated with the labeled THSp1/Egr1 oligonucleotide alone or with a 32-bp oligonucleotide corresponding to the TH AP-1/E-box. Surprisingly, the TH AP-1/E-box oligonucleotide efficiently competed and prevented formation of complexes I and II and to some extent of complex III (Fig. 5, lanes 3, 6, and 9). A shorter oligonucleotide containing only the AP-1 motif was not as effective a competitor (Fig. 5, lane 15). By comparison, the excess unlabeled TH Sp1/Egr1 oligonucleotide competed for all complexes with the three types of extracts (Fig. 5, lanes 2, 5, and 8), while a consensus Egr1, but not the consensus Sp1 oligonucleotide, competed for complex II (lane 12 versus lane 11). On the other hand, oligonucleotides representing the C/EBP consensus or AP-2 from the dopamine -hydroxylase promoter did not compete (lanes 13 and 14).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Interaction between the Sp1/Egr1 and AP-1/E-box motifs of the TH promoter. Oligonucleotide competition analysis of complexes formed in EMSA with the TH Sp1/Egr1 oligonucleotide. The labeled THSp1/Egr1 oligonucleotide was incubated with nuclear extracts from control (lanes 1-3) or PC12 cells treated with 2 µM TPA for 60 min (lanes 4-6 and 10-15) or 120 min (lanes 7-9). Competition with a 100-fold excess of the same oligonucleotide (lanes 2, 5, and 8) or with the same molar excess of the 32-bp THAP-1/E-box oligonucleotide (lanes 3, 6, and 9), 20-bp TH AP-1 (lane 15), consensus oligonucleotides for Sp1 (lane 11), Egr1 (lane 12), C/EBP (lane 14), or the AP-2 motif of the dopamine -hydroxylase promoter (lane 13) are shown.

TPA-induced Complex Formation at the TH AP-1/E-box Enhancer Sequence-- We also tested the effect of TPA on the binding of AP-1 factors in EMSA with the same nuclear extracts, using as a probe the 32-bp AP-1/E-box sequence oligonucleotide of the rat TH promoter. This sequence spans the region between 216 and 185 on the promoter (Fig. 1A) and is 85 nucleotides upstream from the Sp1/Egr1 motif. TPA treatment increased the binding of AP-1 factors to this sequence. Maximal complex formation was observed between 60 min and 4 h (Fig. 6, lanes 3-5). Since the Egr1 binding activity is also maximal within this time interval (see Fig. 2B) and because mutation of the AP-1/E-box region abrogates the Egr1-mediated activation of CAT under the control of the TH promoter (see Fig. 4), we performed supershift EMSAs with antisera raised against Egr1 to test for the presence of Egr1 in the complex formed at the AP-1/E-box sequence. There was no detectable Egr1 protein either in control (Fig. 6, lane 2), 60 min-treated (lane 5), or 4 h-treated PC12 cells (lane 8). We also used antisera raised against the transcription factor C/EBP, a protein frequently activated by cellular stress (47). No immunoreactive C/EBP protein was detectable in any of the extracts (lanes 3, 6, and 9).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Changes in AP-1 complex formation with TPA treatment. PC12 cells were treated with 2 µM TPA for the indicated times, and nuclear extracts were incubated with the labeled 32-bp TH AP-1/E-box oligonucleotide in the absence (lanes 1-6 (left panel) and lanes 1, 4, and 7 (right panel)) or presence of antisera to Egr1 (lanes 2, 5, and 8 in the right panel) or C/EBP (lanes 3, 6, and 9 in the right panel), as described under "Methods." The arrow points to the AP-1 complex.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL METHODS RESULTS DISCUSSION REFERENCES

Elevation of TH Transcription by Egr1-- The findings of this study indicate that the transcription factor Egr1 (also known as Zif268, NGFI-A, or Krox24; reviewed in Ref. 33) is likely to be involved in the regulation of TH gene expression. This is the first study to show that Egr1 can activate the transcription of the TH gene. The introduction of Egr1 into PC12 cells from an expression vector was sufficient in activating a CAT reporter gene under the control of the proximal rat TH promoter. Moreover, our findings reveal cross-talk between factors interacting with the AP-1 and Egr1 motifs.

Egr1 binding to the overlapping Sp1/Egr1 motif of the TH promoter was observed within 60 min of treatment of PC12 cells with TPA. egr1, like c-fos, was initially identified as an early response gene whose mRNA increased within minutes of treatment of PC12 cells with NGF (34). This response, which is not blocked by protein synthesis inhibition, is a component of the early or immediate early response that is presumed to have a key role in orchestrating a second wave of gene expression that underlies long term effects of these factors on cell growth and differentiation (35). Induction of Egr1 binding activity is reportedly also stimulated by serum, phorbol esters, or okadaic acid, a specific inhibitor of phosphatases 1 and 2 in a number of cell types (36-38). In PC12 cells, the induction of Egr1 by NGF is mediated by a regulatory region on the Egr1 promoter, which is also responsive to phorbol esters (23).

When the reporter plasmid p5'THCAT(272/+27) was co-transfected with the Egr1 expression vector, a nearly 4-fold induction of CAT activity was observed, which tended to increase further in the presence of TPA. The involvement of the Egr1 protein in the transcriptional induction of CAT via the TH promoter is also supported by the fact that the pCMVETTL effector plasmid, which encodes a truncated Egr1 protein, is unable to support activation via the reporter plasmid p5'THCAT(272/+27). The requirement for the Sp1/Egr1 motif in the activation of CAT reporter activity by the TH promoter was demonstrated by site-directed mutagenesis. The site-directed mutations introduced at the Sp1/Egr1 site (CCCC replaced by TTTT) had no effect on basal CAT reporter activity but eliminated the induction by the co-transfected Egr1 expression vector.

It remains unclear why activation of the CAT reporter occurs 72 h post-transfection with the Egr1 expression vector. One possibility is that the Egr1 protein itself is expressed within this time course. An alternative explanation is that the effect is indirect and that the Egr1 protein first induces other transcription factors, which subsequently activate TH. A third possibility is that another factor(s) is co-required with Egr1 and that the observed lag in the response of the reporter gene may be the result of this factor(s) being synthesized within 72 h. Nevertheless, the results with the mutated Egr1 motif suggest that this sequence is directly involved in activating TH transcription. Perhaps high levels of Egr1 expression are needed before it can activate CAT reporter activity, because in these transfected cells Sp1 should still be present and presumably bound to the Sp1/Egr1 site, in contrast to the situation in TPA-treated PC12 cells.

Sp1 binding is no longer evident in extracts from PC12 cells treated with TPA for 60 and 120 min. Our immunoblot results indicate that the treatment of PC12 with TPA results in a marked reduction of Sp1 protein within 30 min and up to 60 min. By 4 h, and perhaps earlier, Sp1 levels approached those of the control. This is corroborated by the EMSA results, which show that Sp1 binding activity returns to control levels after 4-6 h of TPA treatment. In contrast, the levels of Sp1 remained virtually unchanged in the adrenal medulla of rats subjected to either single or repeated stress,² although Egr1 was also induced in the later (25). In the TPA-treated cells, the Egr1 protein is undetectable in control extracts. It appears 30 min after TPA application and is highest at 60 min. After 4 h, when Sp1 reappears, Egr1 levels are greatly reduced. The mechanisms by which TPA rapidly down-regulates Sp1 and up-regulates Egr1 in PC12 cells remain to be determined. It is attractive to speculate that stimulation of the protein kinase C pathway by TPA may lead to phosphorylation of Sp1. In turn, this could lead to its targeting for degradation. It is likely that transcriptional mechanisms account for the rapid and transient elevation of Egr1 in the TPA-treated cells, since its promoter was shown to contain a TPA response element (24).

Interestingly, in addition to the findings of the present study on TH, another enzyme of the catecholamine biosynthetic pathway, phenylethanolamine N-methyltransferase, which catalyzes the conversion of norepinephrine to epinephrine, is also activated by Egr1 expression (24, 40). TPA activates the phenylethanolamine N-methyltransferase promoter in the PC12-derived, RS1 cell line (24). The co-transfection of Egr1 expression vector results in the activation of luciferase reporter plasmids under the control of the phenylethanolamine N-methyltransferase promoter. This effect is mediated by two overlapping consensus Sp1/Egr1 sequences located at 45 and 165 nucleotides in the phenylethanolamine N-methyltransferase promoter (41).

Induction of Egr1 may be important for the in vivo regulation of catecholamine biosynthesis. The appearance of an Egr1-containing complex with the TH Sp1/Egr1 motif was also observed in adrenomedullary nuclear extracts from rats exposed to immobilization stress (25). In the adrenomedullary nuclear extracts, the binding profiles specific for the TH Sp1/Egr1 motif were also altered by exposure to immobilization stress (25). Complexes similar in mobility to complexes I and III, observed here, were attained in mobility shift assays with nuclear extracts from control animals. The presence of Sp1, especially in the lower mobility complex (complex I), was confirmed by competition with Sp1 consensus oligonucleotide and with specific antisera. With nuclear extracts from the adrenal medulla of rats exposed to a single immobilization stress, a new complex comparable with complex II, containing Egr1, was formed. This complex was also evident, although somewhat less pronounced with extracts from rats exposed to repeated immobilization stress (25). These results suggest that expression of the egr1 gene in the adrenal medulla is stimulated by stress. These findings are consistent with results obtained by Wong and co-workers (32) on the induction of the stress-elicited elevation of adrenomedullary Egr1 mRNA levels. The induction of adrenomedullary phenylethanolamine N-methyltransferase mRNA levels is also rapidly elevated by immobilization stress (32, 39) and may also be mediated by induction of Egr1. Therefore, Egr1 may participate in coordinate mechanisms for the regulation for these catecholamine biosynthetic enzymes.

Cross-talk between Factors Interacting with Sp/Egr1 and AP-1 Motifs-- Previous studies on the regulation of TH by TPA have concentrated on the AP-1 promoter element (19, 21). Notably, deletion or mutation of the TH AP-1 element in a CAT reporter construct prevents its induction by TPA or NGF, indicating that it is an essential site (16, 19, 21).

Mutation of the Sp1/Egr1 motif was not the only mutation that prevented induction of TH promoter-directed expression of CAT activity by the Egr1 expression vector. Surprisingly, mutation of the AP-1 also prevented the Egr1-mediated induction of the CAT reporter. Previous reports have indicated that the AP-1 region is required for the binding of AP-1 proteins induced by TPA in PC12 cells (19).

It is plausible that Egr1 may act in concert with other stress-activated immediate early genes, like c-fos, in the initial stages of TH transcriptional activation. The AP-1 and Egr1/Sp1 sites might be cooperating in the transmission of the extracellular signals by sharing transcription factors required for complex binding at both sites or by cooperating with each other. The interaction between these motifs has not been previously investigated for TH gene promoter function. However, our study suggests that they are both required for full induction of CAT in PC12 cells by coexpressed Egr1 protein.

Further support for the interaction of the AP-1 region and Sp1/Egr1 motifs is provided by the competition EMSA results with the AP-1/E-box oligonucleotide. The ability of this oligonucleotide to compete for complex formation with the THSp1/Egr1 oligonucleotide suggests that it interacts with proteins necessary for complex formation.

The results of this work suggest a model for functional interactions between the Sp1/Egr1 and AP-1/E-box motifs in the induction of TH transcription by Egr1 (Fig. 7). In the unstimulated state, the Sp1 transcription factor binds the promoter. Sp1 and AP-1 factors may participate in its basal expression along with factors at the CRE site, which are clearly important for basal expression (42). Although the present study and that by Yoon and Chikaraishi (26) found that mutation of the Sp1 motif did not alter basal reporter expression under control of the proximal promoter, recent studies by Yang et al. (43), in which a much longer promoter construct was used, indicated that mutation of the Sp1/Egr1 greatly reduced basal expression. After stimulation of PC12 cells with TPA, the steady-state levels of Sp1 protein are decreased (perhaps by protein kinase C-stimulated degradative down-regulation), while Egr1 is induced. AP-1 binding and c-Fos expression are also induced (19). It is conceivable that Fos and Jun family members may also participate in the cross-talk.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Proposed model for cross-talk between factors at the Sp1/Egr1 and AP-1/E-box promoter motifs. In the unstimulated state, Sp1-containing complexes bind the overlapping Sp1/Egr1 motif, and Jun family members bind the AP-1 motif. With TPA treatment, the main complex at the Sp1/Egr1 motif contains Egr1, and the composition of the AP-1 factors is altered with induction of c-Fos and perhaps induction and/or phosphorylation of other AP-1 family members. The interaction between factors at these sites may include co-sharing of components of coactivator complexes.

The mechanism by which these factors cross-talk remains to be determined. c-Jun may be a candidate in mediating this cross-talk. In this regard, interactions between c-Jun and Sp1 to form a superactivator of the p21 gene have recently been reported and are based on physical interactions between these two transcription factors (44). Other interactions between Egr1/Sp1 sites and AP-1 motifs have been reported for elements that are in physical proximity (45). Recently, large cofactor complexes have been identified for Sp1-mediated promoter activation to function in conjunction with TATA binding protein-associated factors in HeLa cells (46). These complexes contain subunits that are unique to it as well as polypeptides that are shared with other cofactor complexes and may form a bridge between these two motifs. It remains to be determined whether the cross-talk revealed in this study between the Egr1/Sp1 and AP-1 motifs involves sharing of specific cofactor complexes bridging these two sites.

	ACKNOWLEDGEMENTS

We thank Dr. Bistra Nankova (New York Medical College) for useful discussions and suggestions, Dr. Dona Wong (Harvard Medical School) for useful discussions and the Egr expression vectors, and Dr. Dona Chikaraishi (Duke University Medical Center) for the reporter constructs.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant NS28869.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595. Tel.: 914-594-4068; Fax: 914-594-4058; E-mail: sabban@nymc.edu.

Published, JBC Papers in Press, June 6, 2000, DOI 10.1074/jbc.M000049200

² N. A. Papanikolaou and E. L. Sabban, unpublished results.

	ABBREVIATIONS

The abbreviations used are: TH, tyrosine hydroxylase; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; HIF, hypoxia-inducible factor-1; NGF, neural growth factor; bp, base pair(s); C/EBP, CCAAT/enhancer-binding protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; DTT, dithiothreitol; CMV, cytomegalovirus.

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