Cytokine Suppression of Dopamine-β-hydroxylase by Extracellular Signal-regulated Kinase-dependent and -independent Pathways
2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês
10.1074/jbc.m212480200
ISSN1083-351X
AutoresSuzan Dziennis, Beth A. Habecker,
Tópico(s)Nuclear Receptors and Signaling
ResumoCholinergic differentiation factors (CDFs) suppress noradrenergic properties and induce cholinergic properties in sympathetic neurons. The CDFs leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) bind to a LIFR·gp130 receptor complex to activate Jak/signal transducers and activators of transcription and Ras/mitogen-activated protein kinases signaling pathways. Little is known about how these differentiation factors suppress noradrenergic properties. We used sympathetic neurons and SK-N-BE(2)M17 neuroblastoma cells to investigate CDF down-regulation of the norepinephrine synthetic enzyme dopamine-β-hydroxylase (DBH). LIF and CNTF activated extracellular signal-regulated kinases (ERKs) 1 and 2 but not p38 or Jun N-terminal kinases in both cell types. Preventing ERK activation with PD98059 blocked CNTF suppression of DBH protein in sympathetic neurons but did not prevent the loss of DBH mRNA. CNTF decreased transcription of a DBH promoter-luciferase reporter construct in SK-N-BE(2)M17 cells, and this was also ERK-independent. Cytokine inhibition of DBH promoter activity did not require a silencer element but was prevented by overexpression of the transcriptional activator Phox2a. Inhibiting ERK activation increased basal DBH transcription in SK-N-BE(2)M17 cells, and DBH mRNA in sympathetic neurons. Transfection of Phox2a into PD98059-treated M17 cells resulted in a synergistic increase in DBH promoter activity compared with Phox2a or PD98059 alone. These data suggest that CDFs down-regulate DBH protein via an ERK-dependent pathway but inhibit DBH gene expression through an ERK-independent pathway. They further suggest that ERK activity inhibits basal DBH gene expression. Cholinergic differentiation factors (CDFs) suppress noradrenergic properties and induce cholinergic properties in sympathetic neurons. The CDFs leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) bind to a LIFR·gp130 receptor complex to activate Jak/signal transducers and activators of transcription and Ras/mitogen-activated protein kinases signaling pathways. Little is known about how these differentiation factors suppress noradrenergic properties. We used sympathetic neurons and SK-N-BE(2)M17 neuroblastoma cells to investigate CDF down-regulation of the norepinephrine synthetic enzyme dopamine-β-hydroxylase (DBH). LIF and CNTF activated extracellular signal-regulated kinases (ERKs) 1 and 2 but not p38 or Jun N-terminal kinases in both cell types. Preventing ERK activation with PD98059 blocked CNTF suppression of DBH protein in sympathetic neurons but did not prevent the loss of DBH mRNA. CNTF decreased transcription of a DBH promoter-luciferase reporter construct in SK-N-BE(2)M17 cells, and this was also ERK-independent. Cytokine inhibition of DBH promoter activity did not require a silencer element but was prevented by overexpression of the transcriptional activator Phox2a. Inhibiting ERK activation increased basal DBH transcription in SK-N-BE(2)M17 cells, and DBH mRNA in sympathetic neurons. Transfection of Phox2a into PD98059-treated M17 cells resulted in a synergistic increase in DBH promoter activity compared with Phox2a or PD98059 alone. These data suggest that CDFs down-regulate DBH protein via an ERK-dependent pathway but inhibit DBH gene expression through an ERK-independent pathway. They further suggest that ERK activity inhibits basal DBH gene expression. cholinergic differentiation factors ciliary neurotrophic factor dopamine-β-hydroxylase extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase MAPK/ERK kinase interleukin-6 leukemia inhibitory factor LIF receptor Janus tyrosine kinases c-Jun N-terminal kinases nerve growth factor signal transducers and activators of transcription vasoactive intestinal peptide stress-activated protein kinase green fluorescent protein cytomegalovirus phosphate-buffered saline reverse transcription glyceraldehyde-3-phosphate dehydrogenase Cholinergic differentiation factors (CDFs)1 suppress noradrenergic properties and induce cholinergic and peptidergic properties in sympathetic neurons. This occurs during development in response to target-derived differentiation factors (1Landis S.C. Prog. Brain Res. 1994; 100: 19-23Crossref PubMed Scopus (21) Google Scholar) and occurs following nerve injury due to the release of inflammatory cytokines (2Zigmond R.E. Hyatt-Sachs H. Mohney R.P. Schreiber R.C. Shadiack A.M. Sun Y. Vaccariello S.A. Perspect. Dev. Neurobiol. 1996; 4: 75-90PubMed Google Scholar). The developmental and injury-induced cholinergic differentiation factors that have been characterized to date are related to the inflammatory cytokine interleukin-6 (IL-6). The interleukin-6 cytokine family includes leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1, oncostatin M, and a sweat gland-derived differentiation factor. All interleukin-6 family members share the common signaling receptor gp130. Interleukin-6 uses a gp130 homodimer, whereas all other family members activate a gp130·LIFR heterodimer (3Baumann H. Ziegler S.F. Mosley B. Morella K.K. Pajovic S. Gearing D.P. J. Biol. 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Several IL-6 family members require an additional receptor subunit for cytokine binding, but signal transduction occurs through the gp130·LIFR complexes. Binding of these cytokines to their receptors induces dimerization of receptor subunits and activation of Janus tyrosine kinases (Jak1/Jak2 and Tyk2), which are constitutively associated with the receptors (9Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Science. 1994; 263: 92-95Crossref PubMed Scopus (849) Google Scholar, 10Lutticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Science. 1994; 263: 89-92Crossref PubMed Scopus (713) Google Scholar, 11Taga T. Kishimoto T. Curr. Opin. Immunol. 1995; 7: 17-23Crossref PubMed Scopus (92) Google Scholar). Ligand binding activates at least two major signaling cascades: a Jak/STAT pathway (12Akira S. Nishio Y. Inoue M. Wang X.J. Wei S. Matsusaka T. Yoshida K. 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Mitogen-activated protein kinases are serine/threonine kinases that exist in modules containing a three-kinase cascade. There are a least three classes of MAPKs activated in separate modules: ERKs, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), and p38s (18Pearson G. Robinson F. Beers G.T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3562) Google Scholar). Each of these types of kinases can be activated by IL-6-related cytokines in different cell types. The induction of cholinergic and peptidergic properties in sympathetic neurons requires STAT activation and can be modified by stimulation of the Ras/MAPK pathway (19Symes A. Gearan T. Eby J. Fink J.S. J. Biol. Chem. 1997; 272: 9648-9654Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In contrast to our knowledge of the signaling mechanisms that induce peptidergic properties, little is known about the mechanisms that suppress noradrenergic function. To identify the signaling pathways important for the loss of norepinephrine, we investigated the regulation of dopamine-β-hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine. DBH is suppressed by cholinergic differentiation factors both in vivo (20Landis S.C. Siegel R.E. Schwab M. Dev. Biol. 1988; 126: 129-140Crossref PubMed Scopus (99) Google Scholar) and in vitro (21Cervini R. Berrard S. Bejanin S. Mallet J. Neuroreport. 1994; 5: 1346-1348PubMed Google Scholar). Inasmuch as STATs typically induce transcription, we hypothesized that the suppression of DBH occurred primarily through a Ras/MAPK pathway. We investigated the role of MAPKs in cytokine-induced down-regulation of DBH mRNA and protein in sympathetic neurons. In addition, we examined cytokine regulation of DBH promoter activity in SK-N-BE(2)M17 neuroblastoma cells. In both cell types we found that ERK1 and -2 were activated by LIF and CNTF, whereas p38 and JNKs were not. Surprisingly, preventing ERK activation blocked suppression of DBH protein but not the decrease in DBH mRNA or promoter activity. The chronic absence of ERK activity elevated basal DBH transcription and mRNA, suggesting that ERK inhibits basal DBH transcription but does not mediate the cytokine suppression of DBH mRNA. Cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Sprague-Dawley rats were from Simonsen Laboratories (Gilroy, CA) or Charles River (Cambridge, MA). SK-N-BE(2)M17 human neuroblastoma cells were a generous gift from Dr. Kwang-Soo Kim (McLean Hospital/Harvard Medical School, Waltham, MA). Biochemicals and hormones were purchased from Sigma Chemical Co. (St. Louis, MO) except as noted. Dispase was obtained from Roche Applied Science (Indianapolis, IN), collagenase type II from Worthington Biochemicals (Freehold, NJ), and nerve growth factor (NGF) from Austral Biologicals (San Ramon, CA). CNTF and LIF were purchased from R&D Systems (Minneapolis, MN) and PeproTech (Rocky Hill, NJ). BioCoat plates and coverslips were from BD Biosciences (Bedford, MA). PD98059, U0126, antibodies specific for phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), and phospho-ERK 1/2 (Thr202/Tyr204), and the C6 and 293 cell extracts were purchased from Cell Signaling (Beverly, MA). Anti-DBH was from Chemicon, (Temecula, CA), and anti-ERK 2 was from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies conjugated to horseradish peroxidase were purchased from Cappel (Durham, NC), and Alexa Fluor secondaries were from Molecular Probes (Eugene, OR). Chemiluminescence reagents (Super Signal Dura) and a protein assay kit were from Pierce (Rockford, IL). Kodak X-Omat film was purchased from PerkinElmer Life Sciences (Boston, MA). Protease inhibitor mixture tablets were purchased from Roche Applied Science (Mannheim, Germany), nitrocellulose membranes were from Schleicher & Schuell (Dassel, Germany), maxiprep kits were from Qiagen (Valencia, CA), and Cells-to-cDNA II was from Ambion (Austin, TX). The LightCycler-FastStart SYBR Green I PCR amplification kit was from Roche Applied Science (Indianapolis, IN). The rat 394DBH-Luc and 232DBH-Luc promoter constructs, the Arix/Phox2a expression construct, and the pGL3 backbone luciferase vector were generous gifts from Dr. Elaine Lewis (Oregon Health & Sciences University, Portland, OR). The pRL-nullrenilla luciferase construct and the Dual-Luciferase Reporter Assay system were purchased from Promega. The pCMV-GFP plasmid was a generous gift from Dr. Rich Maurer (OHSU). The CyRE:VIP-Luc vasoactive intestinal peptide (VIP) promoter construct was a generous gift from Dr. Aviva Symes (Uniformed Services University of the Health Sciences, Bethesda, MD). Primary cultures of superior cervical ganglia were prepared as described previously (22Hawrot E. Patterson P.H. Methods Enzymol. 1979; 58: 574-584Crossref PubMed Scopus (238) Google Scholar, 23Rao M.S. Landis S.C. Patterson P.H. Dev. Biol. 1990; 139: 65-74Crossref PubMed Scopus (51) Google Scholar). Cells were preplated for 1–2 h to deplete non-neuronal cells and plated onto 96-well BioCoat plates or poly-l-lysine/laminin-coated plates at a density of 1000–2000 cells per well. Neurons were cultured in L-15 complete supplemented with 50 ng/ml NGF, 5% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate. Prior to any treatments, neurons were maintained for 2 days in the antimitotic agents fluorodeoxyuridine and uridine (10 μm each) to further deplete non-neuronal cells. SK-N-BE(2)M17 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and maintained in 5% CO2. For acute phosphorylation experiments, M17 cells were plated at 5 × 105 cells per well in six-well plates. For chronic (5-day) cytokine treatments, M17 cells were plated at 2–2.5 × 105 cells per well in six-well plates. A time course identified 15 min as the peak activity for LIF-activated phospho-ERK 1/2. Therefore, cytokines were added at the indicated concentration for 15 min. Sympathetic neurons were treated with PD98059 or U0126 (24English J.M. Cobb M.H. Trends Pharmacol. Sci. 2002; 23: 40-45Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar) for 30 min prior to addition of cytokine. Cells were placed on ice and collected in ERK lysis buffer (1% Igepal, 20 mm Tris-HCl (pH 8.0), 137 mm NaCl, 10% glycerol, 2 mm EDTA, 10 mm NaF, with 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 mm vanadate). 6× loading buffer (12% SDS, 60% glycerol, 360 mm Tris, pH 6.8, and 0.06% bromphenol blue with 600 mm dithiothreitol) was added to a total volume of 70 μl before samples were heated at 95 °C for 5 min and separated on a 10% acrylamide gel. M17 cells were treated as above with cytokine and lysed directly in SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromphenol blue). Extracts were sonicated for 10 s on ice to reduce viscosity. Equal volumes of protein extracts were heated at 95 °C for 5 min and separated on a 10% acrylamide gel. Membranes were blocked in TBST (100 mm NaCl, 10 mm Tris, pH 7.5, and 0.1% Tween 20) containing 5% nonfat milk, and incubated at 4 °C overnight with antibodies against phospho-p38, phospho-JNK, or phospho-ERK 1/2, diluted 1:1000 in TBST containing 5% bovine serum albumin. After washing, membranes were incubated for 1 h at room temperature with appropriate secondary antibodies (diluted 1:5000 in TBST/5% nonfat milk) and washed with TBST, and immunoreactive bands were visualized by chemiluminescence. Blots were stripped for 1 h at room temperature in stripping solution (62.5 mm Tris, pH 6.8, 2% SDS, and 0.7% (v/v) β-mercaptoethanol), followed by extensive washing in TBST. Phospho-p38 and phospho-JNK blots were re-assayed for phospho-ERK, while phospho-ERK blots were reincubated with a total ERK1/2 or ERK2 antibody (diluted 1:1000) in TBST/5% bovine serum albumin at 4 °C overnight, incubated with appropriate secondary antibodies, and immunoreactive bands visualized by chemiluminescence. Sympathetic neurons were treated 5–8 days with 50 or 100 ng/ml CNTF, with or without PD98059, as indicated in the figure legends. PD98059 was added 30 min prior to adding CNTF. PD98059 was added every other day, and media were replaced every 3–4 days. Cells were rinsed with PBS, and protein extracts were collected on ice in ERK lysis buffer. 5–10 μg of protein was diluted 1:2 with sample buffer, heated at 95 °C for 5 min, sized-fractionated on 10% SDS-PAGE gels, and transferred to membranes. Membranes were blocked in 5% nonfat dried milk/TBST, incubated overnight at 4 °C with rabbit anti-DBH (diluted 1:1,000 in TBST/5% milk), washed, and incubated 1 h at room temperature with goat anti-rabbit horseradish peroxidase (diluted 1:10,000 in TBST/5% milk), and immunoreactive bands were visualized by chemiluminescence. Band intensity was recorded by a −40 °C charge-coupled device camera, or Kodak X-Omat film, and analyzed using LabWorks software (UVP, Upland, CA). To quantify DBH protein levels, the band density obtained for DBH was normalized to the protein in that sample. An arbitrary value of 1 was assigned to the DBH value obtained in untreated control cultures. Total and mean band densities gave similar results. DNA used for transfection was purified using the Qiagen Maxiprep kit. M17 cells were plated at a density of 2–2.5 × 105 cells per well in six-well plates and immediately treated with cytokines and MEK inhibitors as described. 48 h after treatment/plating, media were removed, and cells were transfected by the CaPO4 method as previously described (25Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6498) Google Scholar, 26Shaskus J. Greco D. Asnani L.P. Lewis E.J. J. Biol. Chem. 1992; 267: 18821-18830Abstract Full Text PDF PubMed Google Scholar). Each cell was transfected with 1 μg of 394DBH-Luc, 1 μg of 232DBH-Luc, 1 μg of CyRE:VIP-Luc, or 1 μg of pGL3 basic, with 100 ng of pRL-null as a control for transfection efficiency, and 1 μg of pCMV-GFP DNA to bring the total to ∼2 μg. After a 4-h incubation with DNA, cells were shocked with 10% glycerol/PBS, washed with PBS, and put back into culture media containing cytokines and MEK inhibitors. Firefly luciferase activity from DBH-Luc or CyRE:VIP-Luc and Renilla luciferase activity from the pRL-null internal control were determined 48 h after transfection using the Dual-Luciferase Reporter Assay system. Firefly luciferase activities were normalized to the Renillaluciferase values. Neurons were plated onto 12-mm round Biocoat coverslips in 24-well plates. After 2 days NGF was decreased from 50 ng/ml to 10 ng/ml and cells were treated with cytokines as described in the figure legend. Cells were fixed in 4% paraformaldehyde, 4% sucrose, and 29.5 mm sodium phosphate monobasic, pH 7.4. Cells were washed in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were washed in PBS and blocked with 10% goat serum in PBS for 30 min and double-labeled with rabbit anti-phospho-ERK 1/2 and mouse anti-ERK 2 diluted 1:100 in blocking solution. Proteins were visualized with goat anti-rabbit Alexa 488 and goat anti-mouse Alexa 568 diluted 1:300 and examined by fluorescence microscopy. Digital images were treated identically in Photoshop 6.0, so that the differences in immunofluorescence seen in the figure reflect actual changes within the cell. RNA and reverse transcription (RT) reactions were generated from individual wells of sympathetic neurons using the Cells-to-cDNA II kit according to the manufacturer's protocol. An RT-negative control was included for each set of cells to control for genomic DNA contamination. PCR was performed with the LightCycler-FastStart SYBR Green I PCR amplification kit in the Roche Applied Science LightCycler. For the PCR amplification, 2 μl of RT reactions was used in a total volume of 20 μl. Controls lacking template were included to determine the level of primer dimer formation and/or contamination. Each 20-μl reaction included 2.0 mmMgCl2 for GAPDH or 3 mm MgCl2 for DBH, 0.5 μm of each primer, and 2 μl of DNA Master. The intron-spanning rat GAPDH primers generate a 238-bp fragment (27Comer A.M. Gibbons H.M. Qi J. Kawai Y. Win J. Lipski J. Brain Res. Brain Res. Protocol. 1999; 4: 367-377Crossref PubMed Scopus (27) Google Scholar): 5′-CCTGCACCACCAACTGCTTAGC and 3′-GCCAGTGAGCTTCCCGTTCAGC. The mouse DBH primers generate a 211-bp fragment: 5′-AAGGTGGTTACTGTGCTCGC and 3′-CACACATCTCCTCCAAGATTCC. GAPDH PCR parameters: denaturing at 94 °C for 10 min followed by 50 cycles of 94 °C for 0 s, 55 °C for 5 s, and 72 °C for 20 s. DBH PCR parameters: denaturing at 94 °C for 10 min followed by 50 cycles of 94 °C for 0 s, 60 °C for 5 s, and 72 °C for 15 s. The temperature transition rate was 20 °C/s. One fluorescence reading was taken after each cycle at the end of the 72 °C elongation time. Fluorescence was plotted as a function of cycle number, to determine when reactions were in the linear phase of amplification. To confirm that only specific PCR products were generated, a melt analysis was carried out to determine the specific Tm for each amplification product. Standard curves for DBH and GAPDH were generated by performing individual cells-to-cDNA reactions on known amounts of cells, ranging from 500 to 4000. A slope was generated from the standard curve PCR amplifications and unknown samples were compared with the known standard values. Values for DBH were normalized to GAPDH from the same sample. Analysis of variance was carried out using GraphPad Prism 3.0. The Dunnett post hoc test was used to compare treatments to the control, and Tukey's post hoc test to compare all conditions. LIF and CNTF activate Ras in cultured sympathetic neurons (28Schwarzschild M.A. Dauer W.T. Lewis S.E. Hamill L.K. Fink J.S. Hyman S.E. J. Neurochem. 1994; 63: 1246-1254Crossref PubMed Scopus (17) Google Scholar), and these cytokines can cause the activation of ERK, p38, and/or JNK MAPKs in different cell types (29Li W.Q. Dehnade F. Zafarullah M. J. Immunol. 2001; 166: 3491-3498Crossref PubMed Scopus (154) Google Scholar, 30Faris M. Ensoli B. Stahl N. Yancopoulos G. Nguyen A. Wang S. Nel A.E. AIDS. 1996; 10: 369-378Crossref PubMed Scopus (35) Google Scholar). It is not known which MAPKs are activated by LIF and CNTF in sympathetic neurons. In the absence of NGF, CNTF activates ERK1 and -2 in these cells (31Virdee K. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 2159-2169Crossref PubMed Scopus (64) Google Scholar), but NGF stimulates sustained activation of ERK1/2 (32Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4239) Google Scholar), raising the possibility that CNTF has little affect on ERK activity in the presence of NGF. Because suppression of DBH occurs over several days and the neurons must be maintained in NGF, we first tested whether LIF or CNTF stimulated phosphorylation of ERK in addition to that caused by NGF. A time course revealed that cytokines induced transient ERK phosphorylation that peaked between 10 and 15 min (data not shown). Treatment with either cytokine induced phosphorylation of ERK1/2 as determined by immunoblot analysis (Fig. 1). Similar results were obtained using serum-free Opti-MEM, but subsequent experiments were carried out in the presence of serum. To determine if these cytokines also activated p38 or JNKs in sympathetic neurons, cells were stimulated for 15 min with LIF (10 ng/ml) or 1 h with anisomycin (10 μg/ml), a cell-permeable activator of p38 and JNK, as a positive control. LIF did not stimulate phosphorylation of either p38 (Fig. 1 B) or JNK (Fig.1 C). The lack of p38 or JNK phosphorylation was not due to a lack of cytokine activity, because all blots were stripped and reblotted with a phospho-ERK antibody, confirming that LIF had induced ERK phosphorylation. A total ERK 2 antibody was used on the same blots to confirm equivalent protein loading and transfer. Total ERK was used to control for protein loading, because total p38 levels were very low in these cells (data not shown and Ref. 33Eilers A. Whitfield J. Babij C. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 1713-1724Crossref PubMed Google Scholar). Immunoblot analysis revealed that LIF and CNTF stimulated phosphorylation of ERK. To determine if phospho-ERK remained in the cytoplasm or translocated to the nucleus, neurons were treated for varying times with CNTF, and total and phospho-ERK were visualized by double-label immunofluorescence. CNTF-induced ERK phosphorylation was visible in both the neurites and in the cell soma within 15 min. Although some nuclear phospho-ERK was visible, widespread translocation of phospho-ERK from the cytoplasm to the nucleus was not observed up to 2 h after treatment (Fig. 2). To test whether ERKs were required for the LIF- or CNTF-mediated down-regulation of DBH protein, the MEK inhibitor PD98059 was used to prevent phosphorylation and activation of ERK1/2. 20 μmPD98059 was sufficient to inhibit the phosphorylation of ERK induced by exposure to 100 ng/ml CNTF (Fig.3 A). Neurons were treated for 5–8 days with 100 ng/ml LIF or CNTF, with or without 20 μm PD98059. DBH levels were assessed by immunoblot analysis and normalized to total protein. Addition of PD98059 elevated basal levels of DBH and blocked the suppression of DBH by cytokines (Fig. 3, B and C). LIF and CNTF also decrease DBH mRNA in sympathetic neurons (21Cervini R. Berrard S. Bejanin S. Mallet J. Neuroreport. 1994; 5: 1346-1348PubMed Google Scholar), raising the possibility that these cytokines inhibit transcription of the DBH gene. To determine if this occurred, reporter assays were carried out with the DBH promoter in SK-N-BE(2)M17 neuroblastoma cells (M17 cells). These cells express DBH mRNA (34Kim C.H. Kim H.S. Cubells J.F. Kim K.S. J. Biol. Chem. 1999; 274: 6507-6518Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) as well as the receptor subunits required for LIF and CNTF signaling, 2S. Dziennis and B. Habecker, unpublished observations. suggesting that they are a suitable model for these studies. LIF stimulated phosphorylation of ERK1/2 but caused no detectable phosphorylation of p38 or JNKs in M17 cells (Fig. 4), indicating that LIF activated the same MAPK pathways in these cells as in sympathetic neurons. To determine if LIF or CNTF inhibited DBH transcription, M17 cells were treated for 2 days with cytokines and then transfected with a reporter construct containing the proximal 394 bp of the rat DBH promoter driving firefly luciferase (394DBH-Luc) (26Shaskus J. Greco D. Asnani L.P. Lewis E.J. J. Biol. Chem. 1992; 267: 18821-18830Abstract Full Text PDF PubMed Google Scholar). Treatment of cells with either LIF or CNTF resulted in decreased DBH promoter activity (Fig.5 A). As a positive control for cytokine treatments and cell viability, cells were transfected with a cytokine-responsive VIP promoter construct (CyRE:VIP-Luc) (35Symes A.J. Rao M.S. Lewis S.E. Landis S.C. Hyman S.E. Fink J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 572-576Crossref PubMed Scopus (58) Google Scholar). VIP promoter activity was stimulated by the same treatments that inhibited DBH promoter activity (Fig. 5 B), indicating that cytokines did not disrupt cell viability or cause nonspecific suppression of transcription.Figure 5Cytokines decrease DBH transcription.M17 cells were grown in control medium or in medium containing 10 ng/ml LIF. After a two-day pretreatment, cells were transfected with 1 μg of 394DBH-Luc or the backbone vector pGL3-Luc (A) or 1 μg of CyRE:VIP-Luc (B) and 100 ng of pRL-null as an internal control. Luciferase activities were determined 48 h after transfection, and firefly luciferase was normalized toRenilla luciferase. Data are expressed as -fold induction and are the mean ± S.E. of triplicate samples. Experiments were repeated at least three times with similar results. A, DBH promoter (394DBH-Luc) activity was decreased by LIF and CNTF pretreatment, but the vector alone control was unchanged. B, the cytokine response element from the VIP promoter (CyRE:VIP-Luc) was stimulated by LIF and CNTF.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cytokines could suppress DBH transcription by inducing a repressor or by decreasing a transcriptional activator. The 394-bp rat DBH promoter construct used in this study contains a silencer region and several positive regulatory elements. The silencer region binds a suppressor protein, and induction of this repressor is one way that differentiation factors could inhibit DBH gene expression (26Shaskus J. Greco D. Asnani L.P. Lewis E.J. J. Biol. Chem. 1992; 267: 18821-18830Abstract Full Text PDF PubMed Google Scholar, 36Ishiguro H. Kim K.S. Joh T.H. Brain Res. Mol. Brain Res. 1995; 34: 251-261Crossref PubMed Scopus (28) Google Scholar, 37Afar R. Silverman R. Aguanno A. Albert V.R. Brain Res. Mol. Brain Res. 1996; 36: 79-92Crossref PubMed Scopus (26) Google Scholar). To test whether induction or activation of a repressor protein was required for LIF down-regulation of DBH transcription, M17 cells were pretreated with LIF and then transfected with a 232-bp DBH-Luc construct lacking the silencer region. LIF decreased DBH promoter activity to the same extent in the presence or absence of the suppressor binding site (Fig.6 A). This suggests that cytokines inhibit DBH transcription through suppression of a transcriptional activator rather than induction of a transcriptional repressor. The homeodomain proteins Phox2a/Arix and Phox2b/NBPhox are required for basal expression o
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