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• W2030732769 abstract "Phosphorylation (P-) of cAMP-response element-binding protein (CREB) by protein kinase A or mitogen-activated protein kinases was implicated in mediating the increased tyrosine hydroxylase (TH) gene expression after prolonged exposure to nicotine in vivo and in cell culture. We examined the time course and signaling pathways for phosphorylation of CREB and possible involvement of ATF-2. Treatment of PC12 cells with 200 μm nicotine triggered rapid but transient elevation of P-CREB followed by a second sustained rise after 2-5 h of continuous nicotine. In contrast, ERK1/2 was only phosphorylated with short term nicotine exposure. MEK inhibitor U0126 abolished nicotine-induced rise in P-ERK1/2, but not P-CREB, nor did it inhibit nicotine-evoked elevation in TH promoter activity, indicating that ERK1/2 was not needed for induction of TH gene expression by nicotine. In contrast, protein kinase A inhibitor H-89 or Ca2+/calmodulin-activated protein kinase inhibitor KN-93 reduced the nicotine-triggered rise in P-CREB and TH promoter activity. There was a delayed elevation of P-ATF-2 after 1 h of nicotine treatment, accompanied by increased ATF-2 protein. Upstream kinase JNK, but not p38, was phosphorylated especially after 5 min to 2 h of nicotine exposure. To examine the requirement for CREB and ATF-2, cells were transfected with dominant negative forms of ATF-2 or CREB. Both reduced the basal TH promoter activity and the response to nicotine. Knockdown of ATF-2 or CREB with siRNA did not alter basal TH promoter activity or mRNA but greatly attenuated the response to nicotine. The results suggest that both ATF-2 and CREB mediate activation of TH gene transcription by nicotine. Phosphorylation (P-) of cAMP-response element-binding protein (CREB) by protein kinase A or mitogen-activated protein kinases was implicated in mediating the increased tyrosine hydroxylase (TH) gene expression after prolonged exposure to nicotine in vivo and in cell culture. We examined the time course and signaling pathways for phosphorylation of CREB and possible involvement of ATF-2. Treatment of PC12 cells with 200 μm nicotine triggered rapid but transient elevation of P-CREB followed by a second sustained rise after 2-5 h of continuous nicotine. In contrast, ERK1/2 was only phosphorylated with short term nicotine exposure. MEK inhibitor U0126 abolished nicotine-induced rise in P-ERK1/2, but not P-CREB, nor did it inhibit nicotine-evoked elevation in TH promoter activity, indicating that ERK1/2 was not needed for induction of TH gene expression by nicotine. In contrast, protein kinase A inhibitor H-89 or Ca2+/calmodulin-activated protein kinase inhibitor KN-93 reduced the nicotine-triggered rise in P-CREB and TH promoter activity. There was a delayed elevation of P-ATF-2 after 1 h of nicotine treatment, accompanied by increased ATF-2 protein. Upstream kinase JNK, but not p38, was phosphorylated especially after 5 min to 2 h of nicotine exposure. To examine the requirement for CREB and ATF-2, cells were transfected with dominant negative forms of ATF-2 or CREB. Both reduced the basal TH promoter activity and the response to nicotine. Knockdown of ATF-2 or CREB with siRNA did not alter basal TH promoter activity or mRNA but greatly attenuated the response to nicotine. The results suggest that both ATF-2 and CREB mediate activation of TH gene transcription by nicotine. Administration of nicotine in vivo and in cell culture triggers increased expression of a number of genes related to neurosecretion including the catecholamine biosynthetic enzymes. These changes in gene expression may be involved in neurochemical and cardiovascular effects of smoking. Nicotine is a potent sympathomimetic agent, and its administration, in doses similar to those obtained in smoking, increases heart rate and systolic and diastolic blood pressure in humans and many animal species (for review, see Ref. 1Benowitz N.L. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 597-613Crossref PubMed Scopus (516) Google Scholar). These cardiovascular effects are largely attributed to the direct stimulation of release of catecholamines, epinephrine and norepinephrine, from the adrenal medulla and peripheral sympathetic nerve endings (2Haass M. Kubler W. Cardiovasc. Drugs Ther. 1997; 10: 657-665Crossref PubMed Scopus (212) Google Scholar, 3Cryer P.E. Haymond M.W. Santiago J.V. Shah S.D. N. Engl. J. Med. 1976; 295: 573-577Crossref PubMed Scopus (937) Google Scholar, 4Kilaru S. Frangos S.G. Chen A.H. Gortler D. Dhadwal A.K. Araim O. Sumpio B.E. J. Am. Coll. Surg. 2001; 193: 538-546Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In this regard a polymorphism in the gene for tyrosine hydroxylase (TH), 2The abbreviations used are: TH, tyrosine hydroxylase; CREB, cAMP-response element-binding protein; CRE/CaRE, cAMP/calcium response element; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; JNK, Jun N-terminal kinase; P-, phosphorylated; PKA, protein kinase A; CaM, calmodulin; CaMK, Ca2+/calmodulin-activated protein kinase; MAP, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; si-, small interfering; d/n, dominant-negative; RT, reverse transcription; nAChR, nicotinic acetylcholine receptor. the rate-limiting enzyme in catecholamine biosynthesis, has been associated with tobacco use (5Anney R.J. Olsson C.A. Lotfi-Miri M. Patton G.C. Williamson R. Pharmacogenetics. 2004; 14: 73-81Crossref PubMed Scopus (38) Google Scholar). Transcriptional mechanisms are at least partially responsible for the nicotine triggered changes in TH gene expression. The response of TH to nicotine has been proposed to be mediated by CREB. Nicotine treatment both in vivo and in vitro induces phosphorylation of CREB (6Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar, 7Nakayama H. Numakawa T. Ikeuchi T. Hatanaka H. J. Neurochem. 2001; 79: 489-498Crossref PubMed Scopus (129) Google Scholar, 8Brunzell D.H. Russell D.S. Picciotto M.R. J. Neurochem. 2003; 84: 1431-1441Crossref PubMed Scopus (167) Google Scholar). The cAMP/calcium response element (CRE/CaRE) in the TH promoter is required for its transcriptional activation by nicotine in PC12 cells (6Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar). CREB is activated through the phosphorylation at its regulatory site on serine residue 133 (Ser-133) in response to cellular stimulations that result in increased [Ca2+]i and/or cAMP levels (9Montminy M.R. Gonzalez G.A. Yamamoto K.K. Trends Neurosci. 1990; 13: 184-188Abstract Full Text PDF PubMed Scopus (382) Google Scholar, 10Bito H. Deisseroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar, 11Kornhauser J.M. Cowan C.W. Shaywitz A.J. Dolmetsch R.E. Griffith E.C. Hu L.S. Haddad C. Xia Z. Greenberg M.E. Neuron. 2002; 34: 221-233Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Phosphorylated CREB (P-CREB) bound to the CRE/CaRE on the promoter of a target gene facilitates its transcription, an event called stimulus-transcription coupling (10Bito H. Deisseroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar, 12Silva A.J. Kogan J.H. Frankland P.W. Kida S. Annu. Rev. Neurosci. 1998; 21: 127-148Crossref PubMed Scopus (1220) Google Scholar, 13Montminy M.R. Bilezikjian L.M. Nature. 1987; 328: 175-178Crossref PubMed Scopus (968) Google Scholar). The phosphorylation of CREB on Ser-133 can be mediated by several kinases including protein kinase A (PKA), protein kinase C, CaM kinase II/IV, or MAP kinases (14Johannessen M. Delghandi M.P. Moens U. Cell. Signal. 2004; 16: 1211-1227Crossref PubMed Scopus (530) Google Scholar). The involvement of PKA was implicated by the finding that nicotine failed to increase TH mRNA levels in PKA-deficient PC12 cells or in the presence of adenylyl cyclase inhibitors (6Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar, 15Gueorguiev V.D. Zeman R.J. Hiremagalur B. Menezes A. Sabban E.L. Am. J. Physiol. 1999; 276: C54-C65Crossref PubMed Google Scholar). In addition, the phosphorylation of CREB in response to nicotine has been proposed to be mediated by MAP kinases (7Nakayama H. Numakawa T. Ikeuchi T. Hatanaka H. J. Neurochem. 2001; 79: 489-498Crossref PubMed Scopus (129) Google Scholar, 16Tang K. Wu H. Mahata S.K. O'Connor D.T. Mol. Pharmacol. 1998; 54: 59-69Crossref PubMed Scopus (60) Google Scholar). However, the temporal characteristics of nicotine-triggered CREB phosphorylation are not well established. The effects of nicotine on phosphorylation of CREB were generally examined during short term treatments for up to 1 h (6Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar, 7Nakayama H. Numakawa T. Ikeuchi T. Hatanaka H. J. Neurochem. 2001; 79: 489-498Crossref PubMed Scopus (129) Google Scholar) but not during the long term exposure. CRE-mediated gene expression was found to correlate more with sustained, rather than transient, P-CREB elevation (10Bito H. Deisseroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar). Although the long term response of P-CREB to nicotine was not yet examined, our previous findings suggest that long-rather than short term elevation of [Ca2+]i is more pertinent for the activation of TH gene transcription. The addition of the calcium chelator BAPTA to PC12 cells even 2 h after the addition of nicotine still prevented the elevation in TH mRNA levels. In addition to CREB, a number of other transcription factors such as CREM, ATF-1, and ATF-2 can also bind the CRE/CaRE element (17Foulkes N.S. Borrelli E. Sassone-Corsi P. Cell. 1991; 64: 739-749Abstract Full Text PDF PubMed Scopus (534) Google Scholar, 18Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Crossref PubMed Scopus (1797) Google Scholar). Their role in the regulation of TH gene expression in the response to nicotine has not been well studied. Use of various chimeras of transcription factors with Gal4 indicated that in conjunction with CREBbinding protein, they can mediate CRE-dependent transcriptional activation of the TH promoter (19Lim J. Yang C. Hong S.J. Kim K.S. Mol. Cell. Biochem. 2000; 212: 51-60Crossref PubMed Google Scholar, 20Lewis-Tuffin L.J. Quinn P.G. Chikaraishi D.M. Mol. Cell. Neurosci. 2004; 25: 536-547Crossref PubMed Scopus (90) Google Scholar). ATF-1 was only marginally effective in increasing TH promoter activity, suggesting that it does not play a major role in the regulation of TH gene expression. ATF-2 has been implicated in transcriptional regulation of TH during neural development (21Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Its expression in human brain is neuronal, and cell bodies of catecholamine-synthesizing neurons, such as the substantia nigra and locus coeruleus, are among the regions with especially high levels of ATF-2 expression (22Pearson A.G. Curtis M.A. Waldvogel H.J. Faull R.L. Dragunow M. Neuroscience. 2005; 133: 437-451Crossref PubMed Scopus (45) Google Scholar). Therefore, ATF-2 may play a role in the regulation of TH gene expression. To elucidate the molecular mechanisms and transcription factors mediating the induction of TH transcription by nicotine, we examined the temporal changes in phosphorylation of CREB and ATF-2 and the signaling pathways involved as well as their role in CRE/CaRE-mediated TH promoter. Our results suggest that both CREB and ATF-2 are required for nicotine-triggered elevation of TH transcription. Materials—Dulbecco's modified Eagle's medium (DMEM), streptomycin, and penicillin were obtained from Invitrogen. Tissue culture dishes were from Falcon (Lincoln Park, NJ). Fetal bovine serum and donor horse serum were from Gemini Bio-Products (Woodland, CA). Anti-phospho-ERK1/2 (Th-202/Tyr-204), anti-ERK1/2, anti-phospho-ATF-2, anti-ATF-2, anti-Jun N-terminal kinase (JNK), and anti-phospho-JNK antibodies were from Cell Signaling Technology (Beverley, MA). Anti-phospho-CREB, (Ser-133) and anti-CREB antibodies were from Upstate Group Inc. (Waltham, MA). The MEK inhibitor U0126 was from Promega Corp. (Madison, WI). Protein kinase A inhibitor H-89 was from Calbiochem, and CaMKII inhibitor KN-93 was from Alexis Corp. (San Diego, CA). The molecular mass markers and Laemmli sample buffer were supplied by Bio-Rad. The Super Signal West Pico chemiluminescent kit, anti-rabbit, and anti-mouse IgG were obtained from Pierce. SuperFect™ and TransMessenger™ transfection reagents were from Qiagen Inc. (Valencia, CA). Cy™3-labeled glyceraldehyde-3-phosphate dehydrogenase siRNA, CREB, and ATF-2 siRNAs were obtained from Ambion (Austin, TX). Dominant-negative (d/n) CREB expression plasmid pCMV-KCREB, containing mutations in DNA binding domain, was obtained from BD Biosciences; d/n ATF-2 was a generous gift from Dr. Phyllis LuValle (University of Florida, Gainesville, FL). All other reagents were purchased from Sigma. Cell Culture—PC12 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 5% heat-inactivated donor horse serum, 50 μg/ml streptomycin, and 50 IU/ml penicillin in a humidified atmosphere at 37 °C and 7% CO2 as described previously (15Gueorguiev V.D. Zeman R.J. Hiremagalur B. Menezes A. Sabban E.L. Am. J. Physiol. 1999; 276: C54-C65Crossref PubMed Google Scholar). Cells were maintained at a medium density (∼3 × 105/cm2). For nicotine treatment, nicotine ditartrate dissolved in sterile water was added to obtain the desired final concentration. Western Blot Analysis—Cells were collected in 0.5 ml of lysis buffer containing 125 mm Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromphenol blue, and 2% β-mercaptoethanol. Proteins from total cell lysates were resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes (Trans-Blot Transfer Medium, Bio-Rad). Equal loading was ensured by Ponceau S staining. Membranes were blocked in phospho-buffered saline, 0.05% Triton X-100 containing 5% skim milk powder and were then probed overnight with specific primary antibodies (1:1000 phospho-CREB, 1:2000 phospho-ERK. or 1:1000 phospho-ATF-2). Antibodies were detected with the corresponding horseradish peroxidase-linked secondary antibodies. Blots were developed using SuperSignal West Pico chemiluminescent substrate (Pierce) detection reagents. Membranes were stripped with stripping buffer (2% SDS, 100 mm Tris-HCl, 0.1% β-mercaptoethanol) for 45 min at 60 °C and reprobed with the corresponding antibodies to total CREB, ERK1/2, ATF-2, or β-actin for loading control. The membranes were then exposed to x-ray films for various time intervals. The images were captured with a GS-800 calibrated densitometer (Bio-Rad), and the ratio was quantified by densitometric analyses within the linear range of each captured signal. Plasmids and Transfections—The preparation of the constructs with the first 272 nucleotides of the rat TH promoter driving the expression of a firefly luciferase gene (p5′TH-Luc (-272/+27)) has been previously described (23Nakashima A. Ota A. Sabban E.L. Brain Res. Mol. Brain Res. 2003; 112: 61-69Crossref PubMed Scopus (51) Google Scholar). The corresponding plasmids were mixed with SuperFect™ in a ratio of 5 μg to 10 μl according to the manufacturer's instructions (Qiagen), and then the mixtures were added to PC12 cells grown on 6-well plates in 0.6 ml of serum-free DMEM. The cells were incubated with the transfection mixtures for 3 h at 37 °Cinan incubator with 5% CO2. The transfection mixtures were removed, and the cells were washed twice with phospho-buffered saline, which was then replaced with 1 ml of DMEM containing 10% horse serum and 5% fetal bovine serum, and the cells were incubated for an additional 24 h at 37 °C in humidified air containing 7% CO2 and then treated with nicotine for another 16 h. The cells were harvested in 1 ml of phospho-buffered saline and collected by centrifugation. Firefly luciferase activity was determined using luciferase reporter assay system from Promega. The PC12 cells were lysed with 40 μl of passive lysis buffer, and 4 μl of the lysate was added to 20 μl of the luciferase substrate. Luminescence was measured immediately with a Luminometer, model TD-20/20 (Turner, Sunnyvale, CA). Luciferase activity was normalized to a protein concentration of the samples determined with the Bio-Rad protein assay system using the Bradford method. At least three or four cell culture plates were used for each treatment. All experiments were performed at least twice. siRNA—We tested at least thee different annealing double-stranded siRNAs for CREB or ATF-2 genes to find the siRNA that has the strongest effect in reducing CREB or ATF-2 protein levels. The sense strand for siATF-2 was 5′-CCUUCUGUUGUAGAAACAAtt-3′, and the anti-sense strand was 5′-UUGUUUCUACAACAGAAGGtt-3′. The sense strand for siCREB was 5′-GGAGUCUGUGGAUAGUGUAtt-3′, and antisense strand was 5′-UACACUAUCCACAGACUCCtg-3′. Down-regulation of total CREB or ATF-2 proteins was evaluated by Western blot using specific antibodies as mentioned above. We followed the guidelines (Qiagen) for co-transfection of adherent cells with siRNA and plasmid DNA using TransMessenger™ transfection reagent. First we diluted 2.4 μl of Enhancer R in the appropriate volume of buffer EC-R followed by 0.1 μg of siRNA and 0.2 μg of plasmid DNA. After incubation at room temperature for 5 min, 2.5 μl of TransMessenger transfection reagent was added to the siRNA-plasmid DNA-Enhancer R mixture. The mixture was incubated for 10 min and then added to PC12 cells grown on 24-well plates in 0.1 ml of serum-free DMEM. The cells were incubated with the transfection mixtures for 4 h at 37°C in an incubator with 5% CO2. The transfection mixtures were removed, the cells were washed twice with phospho-buffered saline, which was then replaced with 0.4 ml of DMEM containing 10% horse serum and 5% fetal bovine serum, and the cells were incubated for an additional 48 h at 37 °C and 7% CO2 and then treated with nicotine for another 16 h. Isolation of RNA and RT-PCR—The levels of TH mRNA were determined by real-time RT-PCR as described before (24Cheng S.Y. Glazkova D. Serova L. Sabban E.L. Pharmacol. Biochem. Behav. 2005; 82: 559-568Crossref PubMed Scopus (15) Google Scholar, 25Serova L.I. Maharjan S. Huang A. Sun D. Kaley G. Sabban E.L. Brain Res. 2004; 1015: 1-8Crossref PubMed Scopus (77) Google Scholar). Total RNA was isolated from quadruplicate cell cultures by using RNA-STAT 60 (Tel-Test, Friendswoods, TX), and the concentration was quantified by using Ribo-Green fluorescent dye (Molecular Probes, Eugene, OR). RNA (300 ng) was reverse-transcribed with avian myeloblastosis virus reverse transcriptase (Sigma) and 1 μm specific reverse transcription primer for the TH gene, 5′-TCAGGCTCCTCTGACAG-3′,in5 μl of RT mixture. RT was carried out at 42 °C for 1 h followed by 10 min on 85 °C. Quantitative real-time PCR was performed using Light Cycler System with SYBR Green buffer (Roche Applied Science) with the following primers: forward primer, 5′-GTGAACCAATTCCCCATG-3′; reverse primer, 5′-CAGTACACCGTGGAGAG-3′. The denaturation program (95 °C for 7 min) was followed by a four-segment amplification and quantification program repeated 37 times (95 °C for 3s; 59 °C for 3 s; 72 °C for 18 s; 85 °C for 0 s for a single fluorescence measurement) and a melting curve program (70 °C to 99 °C) and, finally, a cooling program down to 40 °C. The threshold cycle was determined using the Fit Points method to provide optimal standard curve values (0.98-1.0). For quantification assays, a standard curve was used, produced by amplification of several 10-fold dilutions of linearized plasmids constructs containing TH cDNA. The results of real-time PCR were normalized to the amount of total RNA used in the PCR reaction. Statistical Analysis—Statistical significance was determined by Student's t test for experiments with two groups or by performing an analysis of variance followed by the Fisher least significant difference test for experiments with more than two groups. A level of p < 0.05 was considered statistically significant. Temporal Effect of Nicotine on Phosphorylation of ERK1/2 and Long Term Activation of CREB—Our previous experiments indicated the importance of prolonged elevation of calcium for nicotine-induced changes in TH gene expression (15Gueorguiev V.D. Zeman R.J. Hiremagalur B. Menezes A. Sabban E.L. Am. J. Physiol. 1999; 276: C54-C65Crossref PubMed Google Scholar). Nicotine led to sustained elevation of [Ca2+]i for up to 6 h of constant exposure. Therefore, we examined the effect of prolonged exposure to 200 μm nicotine, a concentration leading to maximal induction of TH mRNA (6Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar). Results shown in Fig. 1A reveal a sharp transient increase in phosphorylation of ERK1/2 by 5 min followed by a decline to basal levels with no change in the level of phosphorylation afterward. In contrast, although the level of P-CREB was elevated after 5 and 30 min of nicotine treatment and declined to near basal levels by 1 h of treatment, P-CREB increased again at the 2-h time point and rose even further for up to 5 h of exposure to nicotine. Densitometry (Fig. 1B) indicated that there was about a 5-fold increase in P-CREB with transient as well as prolonged treatment with nicotine. The results suggest that long term elevation of P-CREB occurs without concomitant activation of ERK1/2. Exposure to lower concentrations of nicotine (10 and 50 μm) for up to 1 h also led to transient but weaker phosphorylation of CREB and ERK1/2 with similar kinetics (data not shown). Phosphorylation of ERK1/2 Is Not Required for the Nicotine-triggered Regulation of TH Promoter Activity—The phosphorylation of ERK1/2 was found to be very transient, whereas phosphorylation of CREB was sustained with prolonged treatment in PC12 cells. Previous studies implicated the involvement of MAP kinases in the short term nicotine-triggered elevation in P-CREB and TH gene expression (7Nakayama H. Numakawa T. Ikeuchi T. Hatanaka H. J. Neurochem. 2001; 79: 489-498Crossref PubMed Scopus (129) Google Scholar, 8Brunzell D.H. Russell D.S. Picciotto M.R. J. Neurochem. 2003; 84: 1431-1441Crossref PubMed Scopus (167) Google Scholar); therefore, we examined whether phosphorylation of ERK1/2 is also necessary for the nicotine-induced phosphorylation of CREB. Pretreatment for 15 min with 10 μm MEK inhibitor U0126 prevented the phosphorylation of ERK1/2 triggered by 200 μm nicotine as expected (Fig. 2A). However, under the same conditions this inhibitor did not prevent the phosphorylation of CREB in nicotine-treated cells. Because the phosphorylation of CREB is implicated in the nicotine-triggered activation of TH transcription, we investigated if nicotine can induce elevation of TH promoter-driven reporter activity in the presence of MEK inhibitor U0126. Cells were transfected with reporter constructs in which luciferase activity was controlled by the first 272 nucleotides of the rat TH promoter (p5′TH/Luc(-272/+27)). This promoter region contains several regulatory elements including an AP-1 like motif and the Egr1/Sp1 site as well as a perfect consensus CRE/CaRE (TGACGTCA, at -45 to -38), which is necessary for the response to nicotine (6Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar, 26Yang C. Kim H.S. Seo H. Kim K.S. J. Neurochem. 1998; 71: 1358-1368Crossref PubMed Scopus (48) Google Scholar, 27Fung B.P. Yoon S.O. Chikaraishi D.M. J. Neurochem. 1992; 58: 2044-2052Crossref PubMed Scopus (87) Google Scholar). As shown in Fig. 2B, after incubation with 200 μm nicotine, there was a 2.5-fold increase in TH promoter-driven luciferase activity. In the presence of 10 μm MEK inhibitor U0126, nicotine still was able to elevate TH promoter-driven luciferase activity. These results indicate that the MAP kinase pathway is not involved in the regulation of TH promoter activity in response to nicotine. PKA Inhibitor H-89 and CaMK Inhibitor KN-93 Attenuated the Phosphorylation of CREB and TH Promoter Activity—Because ERK1/2 is not involved in CREB and TH promoter activation and we have previously shown the importance of PKA in the regulation of TH gene expression (6Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar, 15Gueorguiev V.D. Zeman R.J. Hiremagalur B. Menezes A. Sabban E.L. Am. J. Physiol. 1999; 276: C54-C65Crossref PubMed Google Scholar), we examined the requirements for PKA and CaM kinase. PC12 cells were pretreated for 30 min with 10 μm H-89, a specific inhibitor for PKA, or 10 μm KN-93, a selective inhibitor of CaMKII. Inhibition of PKA decreased the level of P-CREB after3hof treatment with nicotine. The effect of CaMKII inhibitor was even more pronounced. There was no elevation of the level of P-CREB on the 5′ and no detectable P-CREB after3hofnicotine treatment (Fig. 3A). To further elucidate the role of PKA and CaMKII on TH transcription, we transfected PC12 cells with the TH promoter construct. The cells were pretreated with H-89 or KN-93 inhibitors for 30 min before nicotine was added, and respective luciferase activity was measured. Nicotine induced the TH promoter activity more than 2.5-fold compared with the untreated controls. However, both inhibitors prevented the effect of nicotine on TH promoter activity (Fig. 3B). Basal transcription levels were not changed with both inhibitors, suggesting that only the induction is affected. Nicotine Triggered Long Term Phosphorylation of ATF-2 as Well as JNK—Next, we examined the effect of nicotine on phosphorylation of ATF-2. For these experiments PC12 cells were treated for different time periods with 200 μm nicotine, and the levels of phosphorylation of ATF-2 were examined by Western blots using antibodies to P-ATF-2 (Th-71) and to total ATF-2 (Fig. 4). Short term incubation with nicotine (5-30 min) did not change the levels of P-ATF-2. However, both phosphorylation of ATF-2 and the protein levels were induced after 1 h of continual incubation with nicotine. They continued to increase and remained significantly elevated for up to 5 h (longest time examined) (Fig. 4A). Densitometry was used to estimate the level of induction, and the results are shown in Fig. 4B. The elevation of P-ATF-2 accompanied the rise in ATF-2 protein levels. Stress-activated kinases, such as p38 (28Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar) and JNK (29Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1339) Google Scholar), are involved in the stimulation of ATF-2. Therefore, we used Western blotting to determine the level of phosphorylation of JNK with antibody against the P-JNK. We found an increase in the level of P-JNK, reaching maximal levels at 5 min and remaining high for 2 h, then slowly decreasing to the basal level at 5 h (Fig. 5A). The level of total JNK was not changed at any of the time points. Our experiments showed no change in the level of P-p38 (data not shown). Data from the densitometry of the changes in the levels of P-JNK and total JNK protein are shown (Fig. 5B). Attenuation of the Nicotine-triggered Activation of TH Promoter by Dominant Negative CREB or ATF-2—To analyze whether ATF-2 and CREB are directly involved in the regulation of TH gene expression in PC12 cells, the cells were co-transfected with p5′TH-Luc(-272/+27) and either expression plasmid for d/n ATF-2 or d/n CREB. Untreated controls and nicotine-treated cells were co-transfected with p5′TH-Luc(-272/+27) reporter construct and empty expression vector (pcDNA3). The results showed a marked decrease in TH promoter activity in cell cultures transfected with either of these dominant negative constructs (Fig. 6). The addition of nicotine increased TH promoter driven luciferase activity in control cells but did not elicit any significant change in the cells expressing d/n-ATF-2 or d/n CREB. Thus, both d/n-ATF-2 and d/n-CREB reduced the basal expression and prevented the response to nicotine. These results suggest that ATF-2 and CREB transcription factors are likely required for the nicotine-triggered regulation of TH gene promoter in PC12 cells. Knockdown of CREB or ATF-2 Inhibits the Basal and Nicotine-stimulated TH Promoter Activity and the Level of TH mRNA—To further study the requirement for CREB and ATF-2, we used siRNAs to selectively knock down these genes. First we used fluorescent dye-labeled siRNA to determine the efficiency of siRNA uptake by transfection. PC12 cells were transfected with Cy™3-labeled glyceraldehyde-3-phosphate dehydrogenase siRNA. One day after transfection, 90% of the cells were labeled, demonstrating successful uptake of the siRNA (Fig. 7A). Additional preliminary experiments were performed to evaluate the ability of CREB or ATF-2 siRNAs to inhibit the levels of targeted proteins and to select the appropriate siRNA concentration and incubation time. PC12 cells were transfected with CREB or ATF-2 siRNAs or scrambled RNA, and levels of CREB or ATF-2 proteins were determined by Western blo" @default.
• W2030732769 created "2016-06-24" @default.
• W2030732769 creator A5006293529 @default.
• W2030732769 creator A5031670347 @default.
• W2030732769 creator A5048913695 @default.
• W2030732769 date "2006-04-01" @default.
• W2030732769 modified "2023-10-16" @default.
• W2030732769 title "Prolonged Activation of cAMP-response Element-binding Protein and ATF-2 Needed for Nicotine-triggered Elevation of Tyrosine Hydroxylase Gene Transcription in PC12 Cells" @default.
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