Cytokine Suppression of Dopamine-beta -hydroxylase by Extracellular Signal-regulated Kinase-dependent and -independent Pathways*

Suzan Dziennis and Beth A. HabeckerDagger

From the Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239

Received for publication, December 9, 2002, and in revised form, January 31, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1) and occurs following nerve injury due to the release of inflammatory cytokines (2). 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 (3-8). 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 (9-11). Ligand binding activates at least two major signaling cascades: a Jak/STAT pathway (12-16) and a Ras/(mitogen-activated protein kinase) MAPK pathway (17). Upon phosphorylation by Jaks, STATs dimerize and translocate to the nucleus where they bind to cytokine-responsive elements to induce gene transcription. 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 (18). 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 (19).

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-beta -hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine. DBH is suppressed by cholinergic differentiation factors both in vivo (20) and in vitro (21). 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 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-null renilla 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).

Cell Lines and Cell Culture-- Primary cultures of superior cervical ganglia were prepared as described previously (22, 23). 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.

MAPK Phosphorylation-- 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 (24) 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% beta -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) beta -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.

DBH Immunoblot Analysis-- 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.

Transient Transfection and Reporter Assays-- 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 (25, 26). 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 Renilla luciferase values.

Immunohistochemistry-- 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.

Real-time PCR-- 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 mM MgCl2 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 (27): 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.

Statistics-- 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LIF and CNTF activate Ras in cultured sympathetic neurons (28), and these cytokines can cause the activation of ERK, p38, and/or JNK MAPKs in different cell types (29, 30). 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 (31), but NGF stimulates sustained activation of ERK1/2 (32), 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.


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Fig. 1.   Cytokine activation MAPKs in sympathetic neurons. A, sympathetic neurons were maintained in 50 ng/ml NGF in serum-containing (control) or serum-free (Opti-MEM) medium and were stimulated with 10 ng/ml CNTF for 15 min. Phospho ERK1/2 (p-ERK 1/2) were detected by Western blot analysis using an anti-phospho ERK 1/2 antibody. The blot was then stripped and incubated with a total ERK 1/2 antibody (ERK 1/2). B and C, neurons grown in serum and 50 ng/ml NGF were stimulated for 15 min with 10 ng/ml LIF, or for 30 min with 10 µg/ml anisomycin. Phospho-p38 (p-p38) and phospho-JNKs (p-JNK) were detected by immunoblot analysis. Commercially prepared extracts from control (C-6) and anisomycin-treated (C-6+Anis.) C-6 cells were included as negative and positive controls for phospho-p38. Commercially prepared extracts from control (293) and ultraviolet light-treated (293-UV) 293 cells were included as negative and positive controls for phospho-JNK. Blots were then stripped and incubated with a phospho-ERK 1/2 antibody (middle panels). Blots were stripped again and incubated with total ERK 1/2 or ERK2 as a loading control (bottom panels).

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. 1B) or JNK (Fig. 1C). 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. 33).

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).


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Fig. 2.   ERK localization after CNTF stimulation. Sympathetic neurons were maintained in 10 ng/ml NGF and treated with 100 ng/ml CNTF for 15-120 min. Cells were fixed, permeabilized, and double-labeled for ERK 2 (left panel) and phospho-ERK1/2 (right panel). ERK 2 was detected using goat-anti-mouse Alexa 568, and phospho-ERK was detected using goat-anti-rabbit Alexa 488. Proteins were visualized by fluorescence microscopy. ERK appears to be located primarily in the cytoplasm and neurites before and after stimulation with CNTF.

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 µM PD98059 was sufficient to inhibit the phosphorylation of ERK induced by exposure to 100 ng/ml CNTF (Fig. 3A). 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).


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Fig. 3.   Cytokine suppression of DBH protein requires ERK. A, inhibition of ERK activation. Neurons were pretreated for 30 min with 20 µM PD98059 (PD) and then stimulated for 10 min with 50 or 100 ng/ml CNTF. Phospho-ERK1/2 (p-ERK 1/2) was assayed by immunoblot. The membrane was stripped and re-blotted for total ERK 2 protein. B, immunoblot analysis of DBH. Sympathetic neurons were treated with 100 ng/ml CNTF or LIF, with or without 20 µM PD98059, for 5-8 days. 5-µg samples were assayed for DBH by immunoblot analysis. Panel B shows a representative blot. C, DBH blots, including the one shown in panel B, were quantified as described under "Experimental Procedures." The mean ± S.E. of DBH immunoreactivity (normalized for protein loading) from four independent experiments is graphed as a percentage of control.

LIF and CNTF also decrease DBH mRNA in sympathetic neurons (21), 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 (34) as well as the receptor subunits required for LIF and CNTF signaling,2 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) (26). Treatment of cells with either LIF or CNTF resulted in decreased DBH promoter activity (Fig. 5A). As a positive control for cytokine treatments and cell viability, cells were transfected with a cytokine-responsive VIP promoter construct (CyRE:VIP-Luc) (35). VIP promoter activity was stimulated by the same treatments that inhibited DBH promoter activity (Fig. 5B), indicating that cytokines did not disrupt cell viability or cause nonspecific suppression of transcription.


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Fig. 4.   Cytokine activation of MAPKs in M17 cells. M17 cells were treated with for 15 min with 10 ng/ml LIF or for 60 min with 10 µg/ml anisomycin (Anis.). Equal volumes of cell lysates were analyzed by immunoblot analysis for either phospho-p38 (p-p38; A) or phospho-p46/p54 JNK (p-JNK; B). Membranes were stripped and then blotted for phospho-ERK1/2 (p-ERK 1/2). Membranes were stripped a second time and blotted with an ERK 2 antibody to control for total protein.


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Fig. 5.   Cytokines 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 to Renilla 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.

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 (26, 36, 37). 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. 6A). 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 of DBH (38), and three Phox binding sites are present in the 232- and 394-bp DBH promoter constructs. To determine if the addition of Phox2a could overcome cytokine suppression of DBH transcription, cells were pretreated with LIF for 2 days and then transfected with a Phox2a expression construct and the 394DBH-Luc reporter. LIF decreased DBH transcription (p < 0.05), and exogenous Phox2a restored promoter activity to control levels (Fig. 6B).


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Fig. 6.   Cytokines regulate DBH transcription through activators rather than repressors. M17 cells were grown in control medium or in medium containing 10 ng/ml LIF. After a 2-day pretreatment, cells were transfected with 1 µg of 394DBH-Luc, which contains the silencer region, or 1 µg of 232DBH-Luc, which lacks the repressor binding site (A), and 100 ng of pRL-null as an internal control or 1 µg of 394DBH-Luc and 100 ng of pRL-null as an internal control, with or without 1 µg of Phox2a (B). Luciferase activities were determined 48 h after transfection, and firefly luciferase was normalized to Renilla luciferase. Data are expressed as -fold induction and are the mean ± S.E. of triplicate samples. Each experiment was repeated three times with similar results. A, LIF decreased DBH promoter activity even in the absence of the repressor binding site (p < 0.05). B, LIF decreased DBH promoter activity (p < 0.05), but Phox2a addition restored transcription to control levels.

To determine if ERK activation played a role in the cytokine inhibition of DBH transcription, transient transfection assays were carried out with 394DBH-LUC in M17 cells with or without MEK inhibitors. Inhibition of ERK activity with UO126 or PD98059 (data not shown) increased basal activation of the DBH promoter in M17 cells (Fig. 7). The decrease in transcriptional activity between cells treated with MEK inhibitor alone and MEK inhibitor plus CNTF was similar to the decrease in transcription between untreated and CNTF-treated cells. This suggests that ERK decreases basal expression of DBH mRNA but is not involved in the suppression of DBH transcription by LIF or CNTF.


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Fig. 7.   CNTF down-regulation of DBH transcription does not require ERK. A, 20 µM UO126 (UO) was added 30 min prior to stimulating M17 cells with LIF (10 ng/ml). Cell extracts were assayed for phospho-ERK1/2 (p-ERK 1/2) by immunoblot. Samples were re-blotted for ERK 2 as a loading control. B, M17 cells were treated with 10 ng/ml LIF, 20 µM UO126 (UO), or 20 µM U0126 plus 10 ng/ml LIF (LIF+UO) for 2 days and then transfected with 1 µg of 394DBH-Luc, 100 ng of pRL-null, and 1 µg of CMV-GFP to bring the total DNA concentration to 2.1 µg. Cultures were harvested 48 h later and assayed for luciferase activity. Firefly luciferase was normalized to Renilla luciferase and expressed as -fold induction with untreated cells set at 1. Data are the mean ± S.E. of triplicate samples. Similar results were obtained in three experiments.

Given the difference between the regulation of DBH protein in neurons and the regulation of DBH transcription in M17 cells, endogenous DBH mRNA was measured in sympathetic neurons using real-time PCR. CNTF decreased DBH mRNA levels as expected, and this was not altered by chronic MEK 1/2 inhibition with PD98059 (Fig. 8). Preventing ERK activation with PD98059 elevated basal DBH mRNA levels, consistent with the increased transcriptional activity observed with MEK inhibitors. These data indicate that ERKs were not required for the cytokine-mediated down-regulation of DBH mRNA but that ERK activity decreased the basal level of DBH gene expression.


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Fig. 8.   Cytokine suppression of DBH mRNA does not require ERK. Neurons were treated with 100 ng/ml CNTF, 100 ng/ml CNTF plus 20 µM PD98059 (CNTF+PD), or 20 µM PD98059 (PD) for 8 days. RNA extraction and reverse transcription were performed using the Cells to cDNA kit. DBH and GAPDH mRNAs were quantified using real-time PCR. Each sample was processed with an individual RT and PCR reaction. DBH values were normalized to GAPDH and expressed as a percentage of control. These data are the mean ± S.E. (n = 3) from a single experiment. This experiment was performed three times with each condition tested in triplicate.

In the absence of cytokines, chronic blockade of ERK activation with MEK inhibitors increased DBH transcription and mRNA. This suggests that under basal conditions ERK inhibits DBH transcription. The transcriptional activator Phox2a/Arix, which stimulates DBH transcription, is inhibited by phosphorylation (39). To test whether ERK1/2 decreased Phox2a activity under basal conditions, Phox2a/Arix was transfected, along with the 394DBH-Luc reporter, into M17 cells grown with or without PD98059 for 2 days to prevent ERK activation. 394DBH-Luc promoter activity was assayed 2 days after transfection. Addition of exogenous Phox2a into cells maintained in PD98059 caused a synergistic increase in 394DBH-Luc activity compared with Phox2a or PD98059 alone (Fig. 9). This suggests that, under basal conditions, ERK activity decreased DBH transcription by stimulating the phosphorylation of Phox2a, rendering it less active.


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Fig. 9.   Activation of the DBH promoter by Phox2a and PD98059 in M17 cells. M17 cells were maintained in control media or treated with 20 µM PD98059 (PD) for 2 days and then transiently transfected with 1 µg of 394DBH-Luc and 100 ng of pRL-null, with or without 1 µg of Phox2a. Total DNA was adjusted to ~2.1 µg using CMV-GFP. Cultures were harvested 48 h later and assayed for luciferase activity. Firefly luciferase was normalized to Renilla luciferase, and control values were standardized to 1. These data represent the mean ± S.E. of triplicate samples. This experiment was carried out twice with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cholinergic differentiation factors induce cholinergic and suppress noradrenergic properties in sympathetic neurons, and it is not clear how a single protein simultaneously elicits two such distinct changes in neurotransmitter phenotype. CDFs activate both the Jak/STAT and the Ras/MAPK signaling cascades, and one attractive hypothesis is that the STAT pathway induces cholinergic and peptidergic properties, whereas the Ras/MAPK pathway suppresses noradrenergic function. Consistent with this idea, STATs are required for activation of the choline acetyltransferase and VIP promoters (40-42), whereas Ras activation has little affect on induction of either of these genes in some systems (43). In other cell lines, STAT activity is potentiated by serine/threonine kinases, suggesting there is cross-talk between the Ras/MAPK and STAT pathways (19).

We investigated the down-regulation of DBH in sympathetic neurons to determine if activation of a MAPK pathway was crucial for suppression of noradrenergic function. The suppression of noradrenergic properties is well-characterized in sympathetic neurons, but the signaling pathways activated by LIF and CNTF have not been thoroughly investigated in these cells. We found that LIF and CNTF activated ERK1/2 in sympathetic neurons, consistent with the observation that LIF and CNTF activate ERK1/2 in these cells in the absence of NGF (31). In contrast, we saw no evidence for cytokine activation of p38 or JNK in sympathetic neurons. Although we carried out time courses for up to 1 h, we cannot exclude the possibility that these pathways were activated in sympathetic neurons over a longer time frame. However, it is unlikely that either of these pathways is critical for the regulation of neurotransmitter phenotype, because p38 levels are very low in these cells (33), and JNK activation in these cells is associated with apoptosis rather than differentiation (44).

Prolonged inhibition of ERK prevented the cytokine-induced down-regulation of DBH protein but had no effect on cytokine suppression of DBH mRNA (Fig. 10). The retention of high DBH protein levels despite decreased gene expression suggests that inhibiting ERK activity increases the half-life of the DBH enzyme. Estimates of the half-life of membrane-bound DBH range from 32 h (45) to 3-4 days (46). In our experiments, neurons were treated for 5-8 days with cholinergic differentiation factors. This was sufficient time to detect a loss of DBH protein in CDF-treated cells, despite the long protein half-life. MEK inhibitors completely prevented the decrease in DBH protein over this time frame, suggesting that the half-life of the enzyme is longer than 3 or 4 days in the absence of ERK activity. Thus, activation of ERK by cholinergic differentiation factors like LIF and CNTF appears to decrease the half-life of the DBH enzyme. ERK-dependent phosphorylation can target cells for ubiquitination and degradation by the proteasome (47), suggesting a potential mechanism for cytokine suppression of DBH protein. ERK-dependent changes in protein synthesis seem less likely than changes in protein stability, because DBH mRNA levels are very low in CDF-treated cells. We expect that, given enough time, DBH protein levels would eventually decrease in CDF-treated neurons, because DBH mRNA is reduced in these cells. It might take weeks, however, for this loss of protein to become evident, and additional experiments will be required to fully elucidate the mechanism by which ERK decreases DBH protein.


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Fig. 10.   Models for the regulation of DBH. A, CDF suppression of DBH. CDFs induce ERK phosphorylation, which acts through an unknown mechanism to decrease DBH protein levels. CDFs suppress DBH mRNA and transcription through an ERK-independent mechanism, likely by inhibiting a transcriptional activator. B, ERK inhibition increases basal DBH transcription. Under basal conditions there is some phosphorylation, and therefore activation, of ERK (phosphates represented by dark circles). This results in the phosphorylation of downstream effectors, which may include Phox2a and Phox2b. Phosphorylation of Phox decreases its ability to bind DNA and activate the DBH promoter. Preventing ERK phosphorylation with PD98059 or UO126 may result in decreased phosphorylation of Phox2a/b and, therefore, increased DBH transcription.

In contrast to the ERK-dependent changes in DBH enzyme expression, cytokine suppression of DBH mRNA in sympathetic neurons was independent of ERK activation. Similar results were obtained using DBH-Luciferase reporter constructs to examine transcriptional activation of the DBH promoter. Cytokines decreased activation of the 394DBH-Luc promoter in M17 cells, and blocking ERK activation did not alter this. The reduction in DBH mRNA was greater than the decrease in transcription, particularly in cells treated with both cytokines and MEK inhibitors. There are several possible explanations for this discrepancy. First, the half-life of DBH mRNA may be shorter than the half-life of the luciferase reporter, although the half-life of the luciferase reporter is just 3 h.3 Second, the reporter construct may not contain all of the sites that confer responsiveness to cytokines. Alternatively, it is possible that inhibition of transcription is just one way in which cytokines decrease DBH mRNA.

Cytokines could suppress DBH transcription by inducing a repressor or by decreasing a transcriptional activator. The observation that cells must be exposed to cytokines for 2 days prior to transfection of the reporter construct is consistent with the need to either synthesize a repressor protein or remove a transcriptional activator. The 394-bp DBH promoter construct used in this study contains a negative regulatory element and several positive regulatory elements. Our data with the 232-bp DBH promoter construct indicate that the silencer region, which binds a suppressor protein (26, 36, 37), is not required for the suppression of DBH transcription. This suggests that decreased expression or activity of a transcriptional activator mediates the loss of DBH gene expression.

Candidate transcriptional activators that bind to the 232-bp region of the DBH promoter include Phox2a/Arix and Phox2b/NBPhox, Fos, Jun, CREB, and AP-2 (48-51). CREB, Fos, and Jun regulate the expression of a wide variety of genes. In contrast, Phox2a and Phox2b are expressed only in neurons and are required for the expression of a much narrower set of genes, including DBH. Phox2a and Phox2b are crucial for inducing DBH expression during development (52-55), and suppression of these transcription factors is a way in which CDFs could decrease DBH expression selectively. Consistent with this possibility, overexpression of Phox2a restored DBH transcription to control levels in cells treated with LIF. This raises the possibility that cytokines decrease DBH transcription through suppression of Phox2a and/or Phox2b, but additional experiments are required to determine if cytokines regulate the expression of Phox2a or Phox2b.

Our data suggest that the decreases in DBH mRNA and transcription are mediated through one or more ERK-independent pathways. The SHP-2 phosphatase, which is involved in the activation of ERK1/2, can also recruit PI3 kinase, which stimulates downstream effectors, including the mammalian target of rapamycin (56). CNTF activation of the mammalian target of rapamycin, and subsequently p70S6 kinase, increases transcription from the VIP promoter due to serine phosphorylation of STAT3 (57). p70S6K can interact with other transcription factors as well (58) and may be involved in the suppression of DBH transcription. Thus, there are several ERK-independent mechanisms by which LIF and CNTF could decrease DBH gene expression.

Although ERK was not required for cytokine suppression of DBH mRNA or transcription, chronic inhibition of ERK increased basal DBH mRNA and transcription. Low levels of phospho-ERK were present in both sympathetic neurons and M17 neuroblastoma cells in our experiments, likely due to NGF and growth factors present in the serum. The addition of MEK inhibitors completely blocked ERK phosphorylation and resulted in the elevation of DBH mRNA and transcription in cells that were not treated with cytokines. This suggests that, under basal conditions, ERK activity inhibits DBH gene expression.

ERK activity may decrease DBH transcription by inhibiting the activity of a transcription factor. The Phox transcription factors are particularly good candidates for mediating an ERK-dependent decrease in basal DBH transcription. Phox2a is expressed in M17 cells (34), and both Phox2a and Phox2b are present in sympathetic neurons (54, 55). In contrast to many DNA-binding proteins, the Phox2 proteins are inactivated by phosphorylation. Unphosphorylated Phox2a/Arix strongly binds to and transactivates the DBH promoter, whereas the serine-phosphorylated protein binds the DBH promoter weakly and stimulates only modest activation (39). In our experiments, addition of Phox2a/Arix together with a MEK inhibitor caused a synergistic increase in DBH-Luc activity compared with Phox2a or MEK inhibitor alone. This provides indirect evidence that ERK decreases DBH transcription by stimulating the phosphorylation of Phox2a and/or Phox2b. This increase in DBH transcription likely accounts for the elevation of DBH mRNA seen in neurons maintained in MEK inhibitor.

Cholinergic differentiation factors such as LIF and CNTF suppress noradrenergic function by decreasing DBH protein, mRNA, and transcription. We hypothesized that LIF and CNTF suppressed DBH expression by an ERK-dependent pathway but found that cytokines regulate DBH protein levels and gene expression via distinct signaling pathways. DBH protein levels are regulated by ERK-dependent degradation or changes in translation that are independent of changes in gene expression. Although ERK is not involved in the suppression of DBH mRNA by LIF or CNTF, under basal conditions ERK appears to decrease DBH gene expression by inhibiting Phox2a activity. The mechanisms responsible for the down-regulation of DBH gene expression are currently being investigated.

    ACKNOWLEDGEMENTS

We thank Dr. Elaine Lewis for the DBH-Luc, Arix/Phox2a, and pGL3 constructs; Dr. Rich Maurer for the CMV-GFP plasmid; Dr. Aviva Symes for the CyRE:VIP-Luc construct; Dr. Kwang-Soo Kim for the SK-N-BE(2)M17 human neuroblastoma cells; and Dr. Elaine Lewis and Dr. Philip Stork for helpful discussions and critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL68231 and American Heart Association Grant 0151349Z (to B. A. H.) and by a Tartar Trust Fellowship (to S. D.).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.

Dagger To whom correspondence should be addressed: Dept. of Physiology/Pharmacology, L334, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-0497; Fax: 503-494-4352; E-mail: habecker@ohsu.edu.

Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M212480200

2 S. Dziennis and B. Habecker, unpublished observations.

3 Available at www.promega.com.

    ABBREVIATIONS

The abbreviations used are: CDFs, cholinergic differentiation factors; CNTF, ciliary neurotrophic factor; DBH, dopamine-beta -hydroxylase; ERK 1/2, extracellular signal-regulated kinases 1 and 2; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; IL-6, interleukin-6; LIF, leukemia inhibitory factor; LIFR, LIF receptor; Jak, Janus tyrosine kinases; JNK, c-Jun N-terminal kinases; NGF, nerve growth factor; STAT, signal transducers and activators of transcription; VIP, vasoactive intestinal peptide; SAPK, stress-activated protein kinase; GFP, green fluorescent protein; CMV, cytomegalovirus; PBS, phosphate-buffered saline; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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