Stimulation of Calcitonin Gene-Related Peptide Expression and Secretion from Rat Trigeminal Ganglion Neurons

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Articles by College of Natural and Applied Sciences Faculty

1-1-2006

Tumor necrosis factor-α stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons

Elizabeth J. Bowen Missouri State University

Thomas W. Schmidt

Christina S. Firm

Andrew F. Russo

Paul L. Durham Missouri State University

Follow this and additional works at: https://bearworks.missouristate.edu/articles-cnas

Recommended Citation Bowen, Elizabeth J., Thomas W. Schmidt, Christina S. Firm, Andrew F. Russo, and Paul L. Durham. "Tumor necrosis factor‐α stimulation of calcitonin gene‐related peptide expression and secretion from rat trigeminal ganglion neurons." Journal of neurochemistry 96, no. 1 (2006): 65-77.

This article or document was made available through BearWorks, the institutional repository of Missouri State University. The work contained in it may be protected by copyright and require permission of the copyright holder for reuse or redistribution. For more information, please contact [email protected]. Journal of Neurochemistry, 2006, 96, 65–77 doi:10.1111/j.1471-4159.2005.03524.x

Tumor necrosis factor-a stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons

Elizabeth J. Bowen,* Thomas W. Schmidt, Christina S. Firm,à Andrew F. Russo ,à and Paul L. Durham*

*Department of Biology, Missouri State University, Springﬁeld, Missouri, USA Department of Physiology and Biophysics and àGenetics Program, University of Iowa, Iowa City, Iowa, USA

Abstract activated the transcription factor NF-jB, as well as the Jun N- Expression of the neuropeptide calcitonin gene-related pep- terminal kinase (JNK) and p38 mitogen-activated protein tide (CGRP) in trigeminal ganglion is implicated in neurovas- (MAP) kinase pathways. The importance of TNF-a induction cular headaches and temporomandibular joint disorders. of MAP kinase pathways was demonstrated by inhibiting MAP Elevation of cytokines contributes to the pathology of these kinases with pharmacological reagents and gene transfer with diseases. However, a connection between cytokines and an adenoviral vector encoding MAP kinase phosphatase-1 CGRP gene expression in trigeminal ganglion nerves has not (MKP-1). We propose that selective and regulated inhibition of been established. We have focused on the effects of the MAP kinases in trigeminal neurons may be therapeutically cytokine tumor necrosis factor-a (TNF-a). TNFR1 receptors beneﬁcial for inﬂammatory disorders involving elevated CGRP were found on the majority of CGRP-containing rat trigeminal levels. ganglion neurons. Treatment of cultures with TNF-a stimula- Keywords: calcitonin gene-related peptide, cytokine, ted CGRP secretion. In addition, the intracellular signaling migraine, mitogen-activated protein kinase, trigeminal, tumor intermediate from the TNFR1 receptor, ceramide, caused a necrosis factor-a. similar increase in CGRP release. TNF-a caused a coordinate J. Neurochem. (2006) 96, 65–77. increase in CGRP promoter activity. TNF-a treatment

Release of the neuropeptide calcitonin gene-related peptide theme among these disorders is the apparent involvement (CGRP) from trigeminal ganglion nerve terminals is impli- of an inflammatory cascade of cytokines, including cated in the underlying pathology of several disorders tumor necrosis factor-a (TNF-a) (Covelli et al. 1992; involving the cerebrovasculature and craniofacial structures. Kopp 2001). CGRP is the most potent vasodilatory peptide known and it Reciprocal communication between the immune and is a major contributor to neurogenic inflammation and nervous systems has long been recognized (Hopkins and nociception (Brain et al. 1985; Williamson and Hargreaves Rothwell 1995; Rothwell and Hopkins 1995). Important 2001). Increased levels of CGRP have been reported in serum and saliva during migraine headache and cluster headache (Nicolodi and Del Bianco 1990; Goadsby and Received May 21, 2005; revised manuscript received August 22, 2005; accepted August 23, 2005. Edvinsson 1993, 1994; Fanciullacci et al. 1995). High levels Address correspondence and reprint requests to Paul L. Durham PhD, of CGRP have been found in the synovial fluid of arthritic Department of Biology, 225 Temple Hall, Missouri State University, temporomandibular joints in association with spontaneous Springfield, MO 65897, USA. E-mail: [email protected] pain, impairment of mandibular mobility, and occlusal signs Abbreviations used: CCD, charge coupled device; CGRP, calcitonin of destruction in patients with rheumatoid arthritis and gene-related peptide; CMV, cytomegalovirus; ERK, extracellular signal- regulated kinase; JNK, Jun N-terminal kinase; MAP, mitogen-activated inflammation (Appelgren et al. 1993, 1995; Sessle 2001). protein; MKP-1, MAP kinase phosphatase-1; NF-jB, nuclear factor-jB; Results from these studies demonstrate that elevated levels NGF, nerve growth factor; PBS, phosphate-buffered saline; TMD, tem- of CGRP correlate with inflammation and pain. A common poromandibular joint disorder; TNF-a, tumor necrosis factor-a.

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 65 66 E. J. Bowen et al. messengers between these systems are the multifunctional Materials and methods cytokine peptides that are released by both immune and non- immune cells. One of the best characterized is TNF-a, which Animals and cell culture has been implicated as a potential pathogenic mechanism for The animal care and procedures were conducted in accordance with a number of inflammatory diseases (Taylor et al. 2004). institutional and National Institutes of Health guidelines. Adult female Sprague–Dawley rats (Charles River Laboratories Inc., TNF-a levels have been shown to positively correlate with Wilmington, MA, USA) were housed in clean plastic cages on a acute and chronic joint inflammation, connective tissue 12-h light/dark cycle with unrestricted access to food and water. destruction, and pain in temporomandibular joint disorder Trigeminal ganglia primary cultures were established based on our (TMD) (Appelgren et al. 1993, 1995). For migraine, while published protocol (Durham and Russo 2003). Briefly, ganglia sampling difficulties and the complexity of migraine have led isolated from 3- to 4-day-old rats were incubated for 30 min at 37C to conflicting reports on which cytokines are elevated, among in L-15 (Leibovitz, Sigma, St Louis, MO, USA) media containing the most consistent findings is elevation of TNF-a (Kemper 10 mg/mL Dispase II (Invitrogen Life Technologies, Gaithersburg, et al. 2001; Perini et al. 2005). MD, USA). Following centrifugation at 250 g for 1 min, the cells Although the cellular mechanisms by which cytokines were dissociated in plating media by trituration using a 5-mL pipet, might increase CGRP synthesis and release during these and large debris removed using a Pasteur pipet. The remaining diseases are not well understood, cytokines are known to supernatant was transferred to a new tube and then spun at 250 g for 3 min. The resultant cell pellet was re-suspended in L-15 medium activate multiple signaling pathways, including mitogen- containing 10% fetal bovine serum, 50 mM glucose, 250 lM activated protein (MAP) kinase signaling pathways. It is now ascorbic acid, 8 lM glutathione, 2 mM glutamine, and 10 ng/mL known that at least four distinctly regulated groups of MAP mouse 2.5 S nerve growth factor (Alomone Laboratories, Jerusalem, kinases are present in mammalian cells: extracellular signal- Israel) at 37C at ambient CO2. Penicillin (100 units/mL), strepto- regulated kinases (ERK-1, -2), Jun N-terminal kinases mycin (100 lg/mL), and amphotericin B (2.5 lg/mL, Sigma) were (JNK1, -2, -3), p38 proteins (p38a,-b,-d), and ERK5, also added to the supplemented L15 media, which is referred to as which are in turn activated by specific MAP kinase kinases L15 complete medium. Dissociated cells were plated on either 6- (Schramek 2002). TNF-a has been reported to activate the well (promoter studies), 24-well (secretion studies) tissue culture ERK, JNK, and p38 pathways in various systems, including plates (BD Biosciences, Bedford, MA, USA), or 11-mm glass or neurons (Barbin et al. 2001; Schafers et al. 2003). However, plastic coverslips (immunostaining) coated with poly D-lysine the TNF-a-induced paths in trigeminal ganglion neurons (MW 30,000–70 000; Sigma). The cells from the equivalent of three ganglia were plated per 6-well plate, one per 24 well, and 0.5 have not been reported. per 11-mm coverslip. The medium was changed after 24 h We have focused on TNF-a induction of MAP kinases. and every other day thereafter. TNF-a and ceramide (C2 or CGRP gene transcription is regulated by MAP kinases at N-acetylsphingosine) were purchased from Sigma, while two sites: a distal 18-bp cell-specific enhancer and a SB 203580 hydrochloride and SP 600125 were purchased from proximal cAMP- and Ras-responsive region (deBustros Tocris Cookson (Ballwin, MO, USA). et al. 1986; Tverberg and Russo 1993; Thiagalingam et al. 1996; Lanigan and Russo 1997). At least in trigeminal CGRP secretion ganglion neurons, the stimulation by MAP kinases appears After 24 h in culture, cells were incubated in HEPES-buffered saline to be mediated predominantly through the cell-specific (HBS; 22.5 mM HEPES, 135 mM NaCl, 3.5 mM KCl, 1 mM MgCl2, enhancer (Durham and Russo 2003). A complex of the 2.5 mM CaCl2, 3.3 mM glucose, and 0.1% bovine serum albumin, basic helix-loop-helix zipper proteins USF-1 and -2, and pH 7.4) and the amount of CGRP released into the medium was determined using a CGRP-specific radioimmunoassay (Bachem/ the Foxa2 forkhead protein regulates the enhancer in a Peninsula Laboratories Inc., San Carlos, CA, USA) as previously neuronal-like cell line (Viney et al. 2004). Whether the described (Durham and Russo 1999). As a control, the basal same or similar proteins play a role in trigeminal neurons (unstimulated) rate of CGRP secreted into the media in 1 h is not known, but it is interesting that the USF proteins immediately prior to addition of the stimuli was determined, and 2+ can be regulated by elevated Ca (Tabuchi et al. 2002; these values were used to normalize for differences between dishes. Chen et al. 2003) and phosphorylated by p38 MAP kinase Cells were treated with TNF-a, the cell-permeable form of ceramide, (Galibert et al. 2001). and/or KCl for 1 h. For each experiment, one set of wells in the In this study, we used primary cultures of trigeminal 24-well plate was treated with 60 mM KCl to determine the ganglion neurons to investigate the mechanisms by which responsiveness of the cultures to depolarizing stimuli as described TNF-a regulates CGRP gene expression. We show that TNF- previously (Durham and Russo 1999). Cultures that were found to be a can stimulate CGRP secretion from trigeminal neurons and either unresponsive or exhibit a weak response to the depolarizing stimulus on CGRP release (< 2-fold) were not analyzed. As a control, that the TNFR1 signaling intermediate ceramide can also cells were treated with equivalent amounts of the appropriate vehicle increase CGRP release. Importantly, CGRP promoter activity (buffered saline for TNF-a, dimethylsulfoxide for ceramide). Neuro- was stimulated in response to TNF-a activation of MAP nal survival was assessed in cultures 24 h after the secretion studies by kinases. Finally, we describe the use of gene transfer to applying 1 mL of trypan blue diluted 1 : 10 in phosphate-buffered down-regulate TNF-a induction of the CGRP promoter.

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 TNF-a stimulation of CGRP expression 67 saline (PBS) for 3 min, after which the cultures were washed three previously (Tverberg and Russo 1993; Lanigan and Russo 1997). times with PBS and viewed by brightfield microscopy. The ratio of The 1250-bp rat CGRP promoter–luciferase reporter contains viable cells to total cells was determined for each secretion condition. sequences from the KpnI site ()1250) to the Sau3A site (+21) in Neuronal viability was shown to be > 90% for untreated cultures (88/ exon 1. Cultured cells were transiently transfected 1 h after 93 total cells) as well as for cultures treated with TNF-a (111/120) and plating with Lipofectamine 2000 (Invitrogen Life Technologies) ceramide (92/99). Each experimental condition was repeated in at following manufacturer’s instructions. Approximately 3–5 · least four independent experiments carried out in duplicate. The data 104 cells (3–4 ganglia) per well were transfected 1 h with were reported as mean ± SEM. Statistical analysis was performed 1–2 lg of CGRP–luciferase reporter plasmid or with 0.5 lgof using the Wilcoxon rank test. Results were considered significant c-Jun reporter plasmids (PathDetect, Stratagene, La Jolla, CA, when p was less than 0.05. USA). The amount of DNA was kept constant by addition of the empty expression vector CMV-5 (Durham and Russo 1998). The Immunocytochemistry DNA and Lipofectamine 2000 reagent (1 : 2 ratio) were incuba- Dissociated trigeminal ganglion cells were plated at a density of 500– ted together for 20 min in L15 media. The DNA : Lipofectamine 1000 cells on poly D-lysine coated 13-mm glass or plastic coverslips complex was then added to the trigeminal cultures maintained in and incubated in complete L-15 medium. Untreated 2-day-old the fully supplemented L15 complete medium [including serum, primary cultures or cultures treated with 50 ng/mL TNF-a, 0.6 M nerve growth factor (NGF), antibiotics, and amphotericin B] and sorbitol, or vehicle were briefly rinsed with PBS and incubated in 4% incubated for 24 h before assaying for reporter activity. Under paraformaldehyde for 30 min and then 0.2% trition X-100 in PBS for these transfection conditions using Lipofectamine 2000, > 90% of 15 min to fix and permeabilize the cells. Stimulation with sorbitol the non-neuronal cells are no longer viable 1 day after plating (30 min) was used as a positive control for p38, JNK, ERK, and NF- (Durham and Russo 2003). Based on cell counts, it is estimated jB activation. For the signaling pathway studies, the cultures were that < 3% of the neurons were transfected by this method. TNF-a subcultured in L-15 medium that contained 0.5% FBS for 24 h prior was added to transfected cells 2 h before harvesting. For the to addition of stimulatory agents. Cultures immunostained for the MAP kinase inhibitor studies, the JNK inhibitor SP 600125 or phosphorylated MAP kinase proteins were stimulated for 30 min, p38 inhibitor SB 203580 hydrochloride was added 30 min prior while those used to determine NF-jB activity were stimulated for to addition of TNF-a. Luciferase activity was measured using the 120 min. Fixed cells were incubated for 30 min in PBS containing Luciferase Assay System (Promega) and was reported as relative 5% donkey serum, then for 1 h with primary antibodies and for 1 h light units/20 lg protein or fold change normalized to vehicle with secondary antibodies. For TNFR1 and TNFR2 receptor control. Protein concentrations were determined by Bradford localization, cultures were stained with rabbit anti-TNFR1 or anti- assays (Bio-Rad Laboratories, Hercules, CA, USA). In all TNFR2 antibodies (diluted 1 : 50 in PBS, R & D Systems, experiments, transfection efficiencies were normalized to CMV– Minneapolis, MN, USA), as well as with rabbit a-CGRP antibodies b-galactosidase activity measured using Galacto-Light Plus rea- (Sigma, 1 : 1000). Cultures were also stained with antibodies gents (Applied Biosystems, Foster City, CA, USA). No difference directed against the phosphorylated (active) MAP kinase proteins in cell viability was observed following transfection with the p38 (Promega, Madison, WI, USA; 1 : 8000), JNK (Promega, different plasmids or treatments as determined by trypan blue 1 : 4000), and ERK (Promega, 1 : 8000), antibodies that recognize exclusion. The normalized luciferase activities are reported as the unphosphorylated and phosphorylated forms (total protein) of means with standard errors per 20 lg protein. Each experimental p38, JNK, or ERK (1 lg/mL, Santa Cruz Biotechnology, Santa Cruz, condition was repeated in at least three independent experiments CA, USA), and antibodies against the active form of NF-jB done in duplicate. Statistical analysis was performed using the (Chemicon, Temecula, CA, USA; 1 : 1000). Immunoreactive pro- Wilcoxon rank test. teins were detected following incubation with FITC-conjugated donkey anti-rabbit IgG, Texas red-conjugated donkey anti-rabbit Adenoviral infections and bioluminescence imaging IgG, or FITC-conjugated donkey anti-mouse IgG polyclonal The 1.25-kb rat CGRP promoter and firefly luciferase gene were antibodies (diluted 1 : 100 in PBS, Jackson Immuno-Research subcloned from pGL3-rCAP (Lanigan and Russo 1997) by Laboratories, West Grove, PA, USA). No staining was observed in digestion with KpnI and XbaI and inserted into shuttle plasmid the absence of primary antibodies. Cultures were co-stained with 4¢,6 pacAd5 K-N pA to generate Ad CGRP-Luc. Mouse MKP-1 was diamidino-2-phenylindole (DAPI) to identify nuclei (Vector Labor- subcloned from pSG5 MKP-1-myc (Durham and Russo 2000) by atories, Burlingame, CA, USA). The number of neuronal and non- EcoRI and BamHI digestion and inserted into shuttle plasmid neuronal cells that responded to TNF-a stimulation was determined pacAd5 CMV-K-N pA to generate Ad CMV-MKP-1. Unaltered by analysis of immunopositive cells from 10 random fields of cells pacAd5 CMV-K-N pA was used to generate the control virus Ad from three coverslips. The reported numbers are the average of the CMV-Empty. The Ad-NF-jB responsive luciferase reporter virus counts obtained by two laboratory members that were blinded to the has been described (Sanlioglu et al. 2001). Briefly, a KpnI and experimental design. Neuronal cells were identified based on their XbaI fragment of the pNF-jB-Luc plasmid (Clontech Inc., Palo unique cell morphology (round cell body of 30–50 lm). Alto, CA, USA) containing the luciferase gene driven by four tandem copies of the NF-jB consensus sequence fused to a TATA-like promoter from the herpes simplex virus-thymidine Transfection of trigeminal cultures kinase gene was inserted into a promoterless adenoviral shuttle The CGRP luciferase reporter plasmids and the cytomegalovirus plasmid (pAd5mcspA). The insertions were confirmed by sequen- (CMV) b-galactosidase reporter plasmid have been described cing. The University of Iowa Gene Transfer Vector Core provided

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 68 E. J. Bowen et al. shuttle plasmids and generated the replication-deficient type 5 medium. As shown in Fig. 1(c–e), robust TNFR1 receptor adenoviruses. The recombinant viruses have the transgenes immunoreactivity was observed primarily on the cell body of inserted into a deletion of the E1 region. Viral titers were small diameter neurons (30–40 lm) that also expressed CGRP. determined by plaque assays on 293 cells. The purified viruses Faint TNFR1 staining was detectable in a small percentage of were aliquoted in PBS containing 3% sucrose and stored at non-neuronal cells. Staining for TNFR2 revealed that this )80C. receptor was also abundantly expressed by most CGRP- For the infection of primary cultures, the dissociated cells were plated on 25-mm glass cover slips coated with EHS-laminin positive neuronal cells, and was less robust in non-neuronal (Sigma) and incubated for 24 h in L15 complete medium at 37C cells (Figs 1f–h), Our results are in agreement with the findings of Li et al. (2004) that neuronal cells express higher levels of and ambient CO2. The next day, the cultures were infected with 2 lL Ad CGRP-Luc (1 · 107 pfu/lL) virus in 0.5 mL L15 TNFR1 mRNA than non-neuronal cells. However, while the complete medium. For the NF-jB reporter infections, after the authors reported exclusive TNFR2 mRNA expression in first 24 h the medium was changed to neurobasal medium neuronal cells, our data indicated TNFR2 protein expression in (Invitrogen) without NGF for infection with 2 lL of Ad NF-jB- neurons as well as non-neuronal cells. luciferase reporter virus (4 · 107 pfu/lL) per well. The cultures with neurobasal medium were incubated in 5% CO2. After 4 h, 1.5 mL L15 complete medium was added and cultures were incubated overnight. The next day, the medium was removed and the cultures were infected with either 2 lL Ad CMV-MKP-1 (5 · 107 pfu/lL) or 2 lL Ad CMV-Empty (2 · 107 pfu/lL) virus in 0.5 mL L15 complete medium. After 4 h, 1.5 mL L15 complete medium was added and cultures were incubated overnight. The next day, the medium was removed and the cells were incubated in 2 mL L15 complete medium for 1–1.5 h. Luciferin (0.25 mM) (Xenogen, Alameda, CA, USA) was added to each well and pre- stimulus luciferase activity was measured using an IVIS 100 charged coupled device (CCD) camera with Igor pro-4.2 living image software (Xenogen). Cells were treated with 50 ng/mL mouse TNF-a or vehicle for 4 h at 37C and ambient CO2. Fresh luciferin (0.25 mM) was added to each well and post-stimulus luciferase activity was measured. All light measurements were recorded for 3 min and reported as photons/s/steridan/cm2. Data are reported as the fold change in luciferase activity between post- and pre-stimulus readings from the same cultures. For comparison between cultures, the post-/pre-stimulus fold changes from each culture were first calculated, and then normalized to the pre- stimulus value of the control cultures from each experiment. Conditions were performed in duplicate unless otherwise noted. Statistical analysis was performed using the Wilcoxon rank test.

Results

Presence of TNFR1 and TNFR2 receptors on cultured trigeminal ganglion neurons Primary trigeminal ganglia cultures were used to investigate the signaling mechanisms by which TNF-a regulates CGRP secretion and promoter activity. Under our culture conditions, > 90% of the neurons (67/72 cells) express detectable levels of Fig. 1 Expression of TNFR1 and TNFR2. Day-2 trigeminal ganglia CGRP-IR based on cell morphology (round cell body of cultures were stained for expression of CGRP (a,b) and co-stained for 30–50 lm) (Figs 1a and b). These data are in agreement with CGRP and TNFR1 (c–e) or TNFR2 (f–h). Phase microscopy was used to identify neuronal (large arrows) and non-neuronal cells (small our previously published ﬁndings (Durham and Russo 1999; arrows) (a, c, f). CGRP was expressed exclusively in neuronal cells Durham et al. 2004a). This is a higher percentage than (b). The same cultures were stained for CGRP (d) and TNFR1 (e) or reported in human and rat trigeminal ganglia in situ, where CGRP (d) and TNFR2 (h). Scale bar, 50 lm. TNFR1 and TNFR2 were 40% of the neurons are CGRP positive (Edvinsson et al. easily detected on the cell body of CGRP-positive trigeminal neurons. 1998). The basis for this bias is not known, but may reﬂect the TNFR1 and TNFR2 staining of some non-neuronal cells was inclusion of only one neurotrophin, NGF, in the culture observed.

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 TNF-a stimulation of CGRP expression 69

TNF-a stimulation of CGRP secretion from cultured TNF-a treatment. Taken together, these data are suggestive trigeminal ganglia neurons that TNF-a stimulation of CGRP gene expression involves To determine whether TNF-a could stimulate CGRP release TNFR1 activation and induction of the ceramide pathway. from the cultured neurons, cultures were treated with increasing concentrations of TNF-a. The soluble form of TNF-a stimulation of CGRP promoter activity TNF-a used in these studies is reported to fully activate the Having shown that TNF-a could stimulate CGRP secretion, TNFR1 receptor but have minimal effects on TNFR2 (Grell transient transfections of primary trigeminal ganglia cultures et al. 1995) TNF-a treatment at the higher concentrations (50 were used to determine whether TNF-a could also stimulate and 100 ng/mL) caused a 4-fold increase in CGRP CGRP synthesis via activation of the CGRP promoter. secretion when compared with unstimulated basal values Trigeminal ganglia cultures were transfected with a 1.25-kb (Fig. 2). For comparison, treatment of 1-day-old trigeminal fragment that contains the cell-specific enhancer and cAMP- ganglia cultures with a depolarizing stimulus, 60 mM KCl, and Ras-responsive elements of the rat CGRP gene. We have caused a 6-fold increase in CGRP secretion (Fig. 2). The previously shown that this promoter directs neuronal specific vehicle control did not stimulate CGRP release. expression and is regulated by MAP kinases in primary trigeminal neurons (Durham and Russo 2003; Durham et al. Involvement of ceramide in CGRP release 2004b). Treatment with 50 or 100 ng/mL TNF-a resulted in a The TNFR1 receptor stimulates neutral and acidic sphin- marked increase in CGRP promoter activity (> 3-fold) gomyelinase isoforms to produce the secondary messenger compared with vehicle-treated control values (Fig. 4a). ceramide (Adam-Klages et al. 1998). To investigate the role These data provide evidence that TNF-a can also stimulate of the TNFR1-induced secondary messenger ceramide on CGRP synthesis via activation of its promoter in trigeminal CGRP secretion, cultures were treated with increasing neurons. concentrations of the cell permeable form of ceramide (C2) for 1 h. As shown in Fig. 3, ceramide stimulated CGRP TNF-a activation of JNK and p38 MAP kinases release from trigeminal neurons. All concentrations of Based on our previous data demonstrating that activity of the ceramide tested in our studies significantly (p<0.05) CGRP promoter is stimulated by MAP kinases (Durham and increased CGRP release when compared with control or Russo 2003), we wanted to determine whether TNF-a could vehicle levels. The maximum fold-increase observed with ceramide (3–4-fold) was similar to that observed following

Fig. 2 TNF-a increases CGRP secretion from primary trigeminal neurons. CGRP secretion from untreated cultures (CON) or cultures Fig. 3 Stimulation of CGRP secretion by ceramide. The relative treated for 1 h with vehicle (VEH), 60 mM KCl, or increasing amounts amount of CGRP secreted from control cultures (CON) or cultures of TNF-a was determined. The mean basal rate of CGRP release was treated with vehicle (VEH) or increasing amounts of ceramide for 1 h. 132 ± 13 pg/h/well. The secretion rate for each condition was nor- The mean basal rate of CGRP release was 107 ± 9 pg/h/well. The malized to the basal rate for each well. The means and the SE from secretion rate for each condition was normalized to the basal rate for ﬁve independent experiments are shown. *p<0.05 when compared each well. The means and the SE from four independent experiments with control levels; p<0.01 when compared with control levels. are shown. *p<0.05 when compared with control levels.

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 70 E. J. Bowen et al. activate MAP kinase pathways in cultured trigeminal neurons. Two approaches were taken. The first was to use a reporter gene controlled by a Gal4-c-Jun fusion protein that is directly phosphorylated by MAP kinases. Treatment of trigeminal cultures with 50 ng/mL TNF-a increased c-Jun reporter activity > 3-fold (Fig. 4b). These data demonstrate that TNF-a can stimulate the JNK MAP kinase signaling pathway in the trigeminal cultures. The second approach to test whether TNF-a activated MAP kinases was to stain trigeminal cultures for the active forms of these signaling molecules. We used phospho- specific ERK, JNK, and p38 antibodies that recognize only the dual phosphorylated (activated) proteins. Trigemi- nal ganglia cultures were treated for 30 min with vehicle, 50 ng/mL TNF-a, or 0.6 M sorbitol (positive control). Sorbitol was used as a positive control as it has been reported to strongly stimulate ERK, JNK, and p38 kinase activity (Kayali et al. 2000). In the vehicle control cultures, cytoplasmic and nuclear levels of active MAP kinases were barely detectable (Figs 5a–c). However, treatment of cultures with TNF-a caused a large increase in the percentage of cells exhibiting nuclear staining for active JNK and p38 (Figs 5b and c, and Fig. 6). The observed levels were similar to those seen following treatment with sorbitol. In contrast, TNF-a did not cause an increase in active ERK. As a control, cultures treated with sorbitol had a marked increase in the number of neurons with nuclear localization of phosphorylated ERK. In addition, we stained untreated and treated trigeminal cultures with antibodies that recognize both the phosphorylated as well as unphosphorylated forms of ERK, JNK, and p38. However, the level of cytoplasmic or nuclear staining for ERK, JNK, and p38 total proteins in the TNF-a-treated cultures was not increased when compared with untreated cultures (data not shown). Taken together, these data demonstrate that TNF-a treatment Fig. 4 TNF-a stimulates CGRP promoter and c-Jun activity. The leads to increased phosphorylation and nuclear localization )1250-bp CGRP promoter fragment contains proximal cAMP- and of JNK and p38, but not ERK in trigeminal ganglion ras-responsive regions (gray box) and a distal enhancer that contains neurons. both cell-specific (open box) and non-cell-specific (striped box) elements. (a) Trigeminal cultures transfected with the CGRP–luciferase TNF-a activation of NF-jB reporter were either untreated (CON) or treated 2 h with 50 or The same two approaches were used to test whether TNF-a 100 ng/mL TNF-a and reporter activity measured. (b) c-Jun reporter treatment leads to activation of the NF-jB pathway in activity in cultures treated with 50 ng/mL TNF-a for 2 h. Mean lucif- trigeminal ganglion neurons. In the first approach, activated erase activity (normalized to b-galactosidase activities) per 20 lg nuclear NF-jB was determined by immunocytochemical protein with SEM from four experiments is shown. *p < 0.05 when staining. Cultures were treated for 120 min with sorbitol or compared with control levels. TNF-a prior to staining with a phospho-specific NF-jB antibody. Under unstimulated conditions, cytoplasmic and gene (Sanlioglu et al. 2001). An advantage of using a viral nuclear levels of active NF-jB were barely detectable delivery system is that we can detect luciferase activity using (Fig. 5d). Treatment with TNF-a increased the percentage a sensitive CCD camera and imaging system before and after of neuronal cells exhibiting nuclear staining for active NF- treatment in the same culture. This bioluminescent detection jB in neurons (Fig. 5d and Fig. 6). The observed levels were is possible as the viral vector delivers the reporter gene to similar to those seen following treatment with sorbitol over 10 times the number of neurons than chemical trans- (Fig. 6). In the second approach, we used an adenoviral fection (Durham et al. 2004b). The bioluminescence imaging vector containing an NF-jB responsive luciferase reporter system is advantageous to the traditional in vitro luciferase

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 TNF-a stimulation of CGRP expression 71

Fig. 5 TNF-a stimulation of JNK and p38 MAP kinases and NF-jB. active JNK and p38, but not ERK, was observed in the nucleus of Unstimulated trigeminal ganglia cultures (control, CON) or cultures TNF-a-treated cultures. Nuclear expression of NF-jB was also in- treated with TNF-a (TNF) were stained with antibodies directed creased in response to TNF-a. The same cultures were co-stained against the active (phosphorylated) forms of ERK (a), JNK (b), and with DAPI to identify nuclei (right panels). Cell bodies of neurons are p38 (c), as well as NF-jB (d) (left panels). Increased expression of indicated by arrows. assay as the ability to acquire promoter activity from the trigeminal cultures leads to activation of NF-jB signaling same population of cells before and after treatment eliminates pathways and responsive genes. the inherent prestimulus variability of primary cultures. Hence, fewer cultures and animals are required with this Requirement of MAP kinases for TNFa stimulation of approach. As a control, when the cultures are treated with CGRP promoter activity vehicle, there is little or no change in the light signal from the Because TNF-a treatment led to activation of both MAP cultures (Fig. 7a). When the cultures were treated with kinases and NF-jB, we then asked which pathway was 50 ng/mL TNFa for 4 h, there was a robust 4–5-fold increase involved in activation of the CGRP promoter. Given our in NF-jB-responsive luciferase activity (Figs 7b and c). previous ﬁndings that MAP kinases activate the promoter in These data provide evidence that TNFa treatment of trigeminal neurons (Durham and Russo 2003), we reasoned

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 72 E. J. Bowen et al.

Fig. 6 Percentage of trigeminal neurons expressing the active form of ERK, JNK, p38 or NF-jB in response to TNF-a treatment. The number of neuronal cells in which nuclear staining was detected following sorbitol (positive control, gray bars) or TNF-a (black bars) treatment was compared with unstimulated cultures (control, open bars). Total cell counts (N ¼ number) are indicated below each data set (x-axis). The data are from three independent experiments for each condition are shown. that TNF-a activity was likely to involve this pathway. The Ad-CMV-MKP-1 there was no statistical difference between approach we took was to test whether inhibition of MAP pre-stimulus and post-stimulus luciferase activity following kinases would lead to complete or partial inhibition of TNF-a TNF-a treatment (Figs 9b and c). These results demonstrate stimulation of CGRP promoter activity. MAP kinases were that overexpression of MKP-1 is able to completely block inhibited by complementary pharmacological and gene- TNF-a stimulation of the CGRP promoter. transfer strategies. The first strategy was to determine whether TNF-a Discussion stimulation of CGRP promoter activity could be repressed by pretreatment with selective inhibitors of JNK and In this report we provide the first evidence that TNF-a p38 MAP kinases. Treatment of trigeminal cultures with induces pro-inflammatory signal cascades that coordinately 100 ng/mL TNF-a resulted in a marked increase in CGRP increase the synthesis and release of CGRP in trigeminal promoter activity (> 3-fold) compared with vehicle-treated ganglion neurons. The TNF-a receptors TNFR1 and TNFR2 control values (Fig. 8). Pretreatment with the p38 inhibitor were detected primarily on CGRP-containing neuronal cells. SB 203580 (1 lM) or JNK inhibitor SP 600125 (1 lM) However, the TNF-a stimulatory effects on CGRP gene significantly repressed TNF-a stimulation, while the combi- expression likely are mediated by TNFR1, as the soluble nation of both inhibitors further reduced promoter activity to form of TNF-a used in our studies has been reported to baseline levels. No effect of the inhibitors alone was preferentially activate TNFR1 (Grell et al. 1995). The observed. These data provide evidence that the stimulatory mechanism of TNF-a action on CGRP synthesis and release effect of TNF-a on CGRP synthesis involves activation of appears to involve selective activation of the JNK and p38 the p38 and JNK MAP kinase pathways. MAP kinases. The role of MAP kinases was shown using The second strategy was to overexpress MAP kinase pharmacological inhibitors and by treatment with ceramide, phosphtase-1 (MKP-1). Cultures were sequentially infected which is a downstream signal from TNFR1 and has been with the Ad-CGRP-Luc reporter, then overnight with the reported to activate MAP kinase pathways in non-neuronal Ad-CMV-MKP-1 expression vector (Fig. 9). As a control, cells (Falluel-Morel et al. 2004). The involvement of MAP the cultures were infected with a virus lacking MKP-1 kinases builds on our previous findings that NGF and 5-HT1 (Ad-CMV-Empty). Luciferase activity was measured in the receptor agonists regulate CGRP gene expression by modu- same cultures before and after a 4 h TNF-a treatment. As lating MAP kinase activity (Durham and Russo 2003; described above for the NF-jB responsive reporter, the use Durham et al. 2004b). Given the importance of MAP kinase of viral vectors insured sufficient gene transfer for detection control of CGRP expression, we then demonstrated the of light production by a sensitive CCD camera imaging efficacy of gene transfer with an adenoviral vector encoding system. We observed a 4-fold decrease in CGRP promoter MKP-1. The MKP-1 vector was able to block TNF-a activity when MKP-1 was overexpressed (Fig. 9). Stimula- induction of CGRP promoter activity to a similar degree as tion of the control cultures with 50 ng/mL TNF-a for 4 h seen using pharmacological inhibitors of MAP kinases. yielded a 1.8-fold increase in CGRP promoter activity. The The connection between cytokines and CGRP may have reason for the smaller increase than seen with the plasmid implications for understanding the craniofacial diseases of reporter is not known, but the increase was statistically migraine and TMD that involve elevated CGRP. Trigeminal significant. In contrast, when the cultures were infected with nerves could be exposed to cytokines released from locations

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(a)

(b)

Fig. 8 MAP kinase inhibitors repress TNF-a-mediated stimulation of CGRP promoter. Trigeminal cultures transfected with the CGRP– luciferase reporter were either untreated (CON), treated 2 h with (c) 100 ng/mL TNF-a, or pretreated for 30 min prior to addition of TNF-a with the MAP kinase inhibitors, SB 203580 and/or SP 600125, and reporter activity measured. Mean luciferase activity (normalized to b-galactosidase activities) per 20 lg protein with SEM from ﬁve experiments is shown. *p<0.05 when compared with control levels; p<0.05 when compared with stimulated levels.

(Vaughan 1927). Trigeminal activation and neurogenic inflammation is now a generally accepted model for migraine pathogenesis (Buzzi et al. 1995; Reuter et al. 2001; Bolay et al. 2002; Goadsby et al. 2002; Parsons and Strijbos 2003). Cytokines are known to play an important role in inflammatory and pain conditions. Recently, the levels of the cytokines TNF-a and IL-1b were reported to be significantly elevated during migraine attacks in comparison with their levels outside attacks (Perini et al. 2005). However, evidence that Fig. 7 TNF-a induction of an NF-jB-responsive reporter gene. Lucif- cytokines are elevated in migraine remains controversial, erase activity from trigeminal ganglion cells was detected by a CCD perhaps in part to the difficulties in sampling and/or the camera and IVIS imaging system. (a) Overlay images of the pre-stimulus and post-stimulus bioluminescence signals on the culture dish inherent heterogeneity of the disease. Genetic studies suggest immediately before addition of the vehicle control and 4 h after vehicle an association of migraine with IL-1a (Rainero et al. 2002), treatment. The pseudocolor scale bar in photons/s/steridan/cm2 is TNF-a (Rainero et al. 2004), and decreased soluble TNFR1 shown. (b) Overlay images from a culture immediately before addition receptor that could compromise the ability to dampen TNF-a of 50 ng/mL TNF-a and 4 h after TNF-a treatment. The pseudocolor effects (Empl et al. 2003). In addition, nitric oxide, which scale bar in photons/s/steridan/cm2 is shown. (c) Luciferase activities has been implicated in migraine, induces IL-1b in the dura following vehicle or TNF-a treatments are shown as the ratio of post- mater (Reuter et al. 2001), and IL-1b contributes to stimulus to pre-stimulus signals from the same culture dishes. In these nociceptive fiber sensitization (Herbert and Holzer 1994; experiments, only a single vehicle control dish was analyzed in parallel Opree and Kress 2000). In addition to the potential relevance with the TNF-a treatments that were performed on two dishes to migraine, the connection between cytokines and CGRP is (mean ± range). *p < 0.05 when compared with vehicle levels. also applicable to TMD, as elevated levels of TNF-a are such as dural mast cells and ganglion microglia (Reuter et al. associated with the inflammation and pain of TMD (Fu et al. 2001; Mori et al. 2003). During migraine, a connection with 1995; Kacena et al. 2001). In this study, we have focused on the immune system was first proposed over 75 years ago TNF-a, which is generally viewed as ‘pro-inflammatory’ and

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Fig. 9 MKP-1 overexpression blocks induction of the CGRP promoter by TNF-a. Luciferase activity from trigeminal ganglion cells was detected by a CCD camera and IVIS imaging system. (a) Schematics of the adenoviral CGRP promoter–luciferase reporter vector and the adenoviral CMV promoter–MKP-1 expression vector are shown. (b) Overlay images of the pre-stimulus and post-stimulus bioluminescence signals on the culture dish immediately before addition of 50 ng/mL TNF-a and 4 h after treatment. Cultures were infected with Ad-CGRP-Luc and the control vector Ad-CMV-Empty (left images) or the Ad- CMV-MKP-1 vector (right images), as indicated. The pseudocolor scale bar in photons/s/steridan/cm2 is shown. The light signal from the cultures containing Ad- CMV-MKP-1 was 10-fold above the background light from non-infected cultures. (c) Luciferase activities pre-stimulus and post-stimulus TNF-a treatments are shown as the ratio of post-stimulus to pre- stimulus signals from the same culture dishes normalized to control pre-stimulus luciferase activity, which was set at 1. The means from four culture dishes in two independent experiments are shown with the SD. The p-values between the indicated samples are given. is released in response to inflammation, tissue damage, and identify additional consequences of TNF-a action on trige- microbial infection (Cavaillon 2001; Hanada and Yoshimura minal neurons beyond the pro-inflammatory induction of 2002; Palladino et al. 2003). Elevated levels of TNF-a are CGRP. linked to several painful conditions (Cunha et al. 1992). Studies in both neuronal and non-neuronal cells have shown However, cytokines cannot be easily classified by these that TNF-a can trigger activation of the JNK and p38 MAP labels (Vitkovic et al. 2000; Cavaillon 2001). Rather, kinases (Raingeaud et al. 1995; Chen and Goeddel 2002; cytokines appear to have context-dependent roles in the Pollock et al. 2002; Schafers et al. 2003). We have previously nervous system. For example, TNF-a can induce neuronal shown that MAP kinases regulate CGRP gene expression in death in the absence of neurotrophins or following injury trigeminal neurons via the distal cell-specific enhancer (Dur- (Barker et al. 2001; Shinoda et al. 2003), yet it can also have ham and Russo 2003). In this report we show that TNF-a a neuroprotective function (Cheng et al. 1994; Bruce et al. treatment leads to activation of JNK and p38 MAP kinases in 1996). Thus, it should prove interesting in the future to trigeminal neurons. We also show that TNF-a leads to

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 TNF-a stimulation of CGRP expression 75 activation of its well-established target, the NF-jB transcrip- deBustros A., Baylin S., Levine M. and Nelkin B. (1986) Cyclic tion factor (Chen and Goeddel 2002). The importance of the AMP and phorbol esters separately induce growth inhibition, MAP kinase activation was demonstrated by pretreating calcitonin secretion, and calcitonin gene transcription in cultured human medullary thyroid carcinoma. J. Biol. Chem. 261, 8036– the cultures with pharmacological inhibitors and by infecting 8041. the cells with a viral vector encoding MKP-1. The ability of Buzzi M., Bonamini M. and Moskowitz M. (1995) Neurogenic model of these complementary inhibitory schemes to completely blunt migraine. Cephalalgia 15, 277–280. TNF-a activation of the CGRP promoter indicates that the Cavaillon J. (2001) Pro- versus anti-inflammatory cytokines: myth or MAP kinase, not the NF-jB pathways, are required for CGRP reality. Cell Mol. Biol. 47, 695–702. Chen G. and Goeddel D. (2002) TNF-R1 signaling: a beautiful pathway. promoter activation. The repression of MAP kinase pathway Science 296, 1634–1635. activation as a means to regulate CGRP gene expression has Chen W., West A., Tao X., Corfas G., Szentirmay M., Sawadogo M., previously been demonstrated in a neuronal cell line (Durham Vinson C. and Greenberg M. (2003) Upstream stimulatory factors and Russo 1998, 2000). Activation of serotonin type 1 are mediators of Ca2+-responsive transcription in neurons. receptors caused a sustained increase in intracellular calcium J. Neurosci. 23, 2572–2581. Cheng B., Christakos S. and Mattson M. (1994) Tumor necrosis levels as well as the levels of MKP-1, which is known to factors protect neurons against metabolic-excitotoxic insults and negatively regulate JNK and p38 activity. These results promote maintenance of calcium homeostasis. Neuron 12, provide evidence that cell-type selective and regulated 139–153. suppression of CGRP synthesis by inhibiting MAP kinase Covelli V., Munno I., Pellegrino N., Marinaro M., Gesario A., Massari activity may be an effective treatment for inflammatory F., Savastano S. and Jirillo E. (1992) In vivo administration of propranolol decreases exaggerated amounts of serum TNF-a in conditions of the head and face involving trigeminal nerves. patients with migraine without aura. Possible mechanism of action. Acta Neurol. 14, 313–319. Cunha F., Poole S., Lorenzetti B. and Ferreira S. (1992) The pivotal role Acknowledgements of tumour necrosis factor a in the development of inflammatory We would like to thank Christine Niemann for technical assistance. hyperalgesia. Br. J. Pharmacol. 107, 660–664. Durham P. and Russo A. (1998) Serotonergic repression of mitogen- Supported by grants from the NIH DE015385 and National activated protein kinase control of the calcitonin gene-related Headache Foundation to PLD and NIH DE016511 and HL14388 peptide enhancer. Mol. Endocrinol. 12, 1002–1009. to AFR. Durham P. and Russo A. (1999) Regulation of calcitonin gene-related peptide secretion by a serotonergic antimigraine drug. J. Neurosci. 19, 3423–3429. References Durham P. and Russo A. (2000) Differential regulation of mitogen- Adam-Klages S., Schwandner R., Adam D., Kreder D., Bernardo K. and activated protein kinase-responsive genes by the duration of a Kronke M. (1998) Distinct adapter proteins mediate acid versus calcium signal. Mol. Endocrinol. 14, 1570–1582. neutral sphingomyelinase activation through the p55 receptor for Durham P. and Russo A. (2003) Stimulation of the calcitonin gene- tumor necrosis factor. J. Leukoc. Biol. 63, 678–682. related peptide enhancer by mitogen-activated protein kinases and Appelgren A., Appelgren B., Kopp S., Lundeberg T. and Theodorsson E. repression by an antimigraine drug in trigeminal ganglia neurons. (1993) Relation between intra-articular temperature of the arthritic J. Neurosci. 23, 807–815. temporomandibular joint and presence of calcitonin gene-related Durham P., Cady R. and Cady R. (2004a) Regulation of calcitonin gene- peptide in the joint fluid. A clinical study. Acta Odontol. Scand. 51, related peptide secretion from trigeminal nerve cells by botulinum 285–291. toxin type A: implications for migraine therapy. Headache 44, 35– Appelgren A., Appelgren B., Kopp S., Lundeberg T. and Theodorsson E. 42. (1995) Neuropeptides in the arthritic TMJ and symptoms and signs Durham P., Dong P., Belasco K., Kasperski J., Gierasch W., Edvinsson from the stomatognathic system with special considerations to L., Heistad D., Faraci F. and Russo A. (2004b) Neuronal expres- rheumatoid arthritis. J. Orofac. Pain 9, 215–225. sion and regulation of CGRP promoter activity following viral Barbin G., Roisin M. and Zalc B. (2001) Tumor necrosis factor a gene transfer into cultured trigeminal ganglia neurons. Brain Res. activates the phosphorylation of ERK, SAPK/JNK, and P38 997, 103–110. kinase in primary cultures of neurons. Neurochem. Res. 26, Edvinsson L., Mulder H., Goadsby P. and Uddman R. (1998) Calcitonin 107–112. gene-related peptide and nitric oxide in the trigeminal ganglion: Barker V., Middleton G., Davey F. and Davies A. (2001) TNFa con- cerebral vasodilation from trigeminal nerve stimulation involves tributes to the death of NGF-dependent neurons during develop- mainly calcitonin gene-related peptide. J. Auton. Nerv. Syst. 70, ment. Nat. Neurosci. 4, 1194–1198. 15–22. Bolay H., Reuter U., Dunn A., Huang Z., Boas D. and Moskowitz M. Empl M., Sostak P., Riedel M., Schwarz M., Muller N., Forderreuther S. (2002) Intrinsic brain activity trigger trigeminal meningeal affer- and Straube A. (2003) Decreased sTNF-RI in migraine patients? ents in a migraine model. Nat. Med. 8, 136–142. Cephalalgia 23, 55–58. Brain S., Williams T., Tippins J., Morris H. and MacIntyre I. (1985) Falluel-Morel A., Aubert N., Vaudry D., Basille M., Fontaine M., Calcitonin gene-related peptide is a potent vasodilator. Nature 313, Fournier A., Vaudry H. and Gonzalez B. (2004) Opposite regula- 54–56. tion of the mitochondrial apoptotic pathway by C2-ceramide and Bruce A., Boling W., Kindy M., Peschon J., Kraemer P., Carpenter M., PACAP through a MAP-kinase-dependent mechanism in cerebellar Holtsberg F. and Mattson M. (1996) Altered neuronal and granule cells. J. Neurochem. 91, 1231–1243. microglial responses to excitotoxic and ischemic brain injury in Fanciullacci M., Alessandri M., Figini M., Geppetti P. and Michelacci S. mice lacking TNF receptors. Nat. Med. 2, 788–794. (1995) Increase in plasma calcitonin gene-related peptide from the

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 76 E. J. Bowen et al.

extracerebral circulation during nitroglycerin-induced cluster Parsons A. and Strijbos P. (2003) The neuronal versus vascular hypo- headache attack. Pain 60, 119–123. thesis of migraine and cortical spreading depression. Curr. Opin. Fu K., Ma X., Zhang Z. and Chen W. (1995) Tumor necrosis factor in Pharmacol. 3, 73–77. synovial fluid of patients with temporomandibular disorders. Perini F., D’Andrea G., Galloni E., Pignatelli F., Billo G., Alba S., J. Oral Maxillofac. Surg. 53, 424–426. Bussone G. and Toso V. (2005) Plasma cytokine levels in Galibert M., Carreira S. and Goding C. (2001) The Usf-1 transcription migraineurs and controls. Headache 45, 926–931. factor is a novel target for the stress-responsive p38 kinase and Pollock J., McFarlane S., Connell M., Zehavi U., Vandenabeele P., mediates UV-induced tyrosinase expression. EMBO J. 20, 5022– MacEwan D. and Scott R. (2002) TNF-a receptors simultaneously 5031. activate Ca2+ mobilisation and stress kinases in cultured sensory Goadsby P. and Edvinsson L. (1993) The trigeminovascular system neurones. Neuropharmacology 42, 93–106. and migraine: studies characterizing cerebrovascular and neuro- Rainero I., Pinessi L., Salani G., Valfre W., Rivoiro C., Savi L., Gentile peptide changes seen in humans and cats. Ann. Neurol. 33, 48– S., Giudice R. and Grimaldi L. (2002) A polymorphism in the 56. interleukin-1a gene influences the clinical features of migraine. Goadsby P. and Edvinsson L. (1994) Human in vivo evidence for Headache 42, 337–340. trigeminovascular activation in cluster headache. Neuropeptide Rainero I., Grimaldi L., Salani G., Valfre W., Rivoiro C., Savi L. and changes and effects of acute attacks therapies. Brain 117, Pinessi L. (2004) Association between the tumor necrosis factor- 427–434. a-308 G/A gene polymorphism and migraine. Neurology 62, Goadsby P., Lipton R. and Ferrari M. (2002) Migraine – current 141–143. understanding and treatment. N. Engl. J. Med. 346, 257–270. Raingeaud J., Gupta S., Rogers J., Dickens M., Han J., Ulevitch R. and Grell M., Douni E., Wajant H. et al. (1995) The transmembrane form of Davis R. (1995) Pro-inflammatory cytokines and environmental tumor necrosis factor is the prime activating ligand of the 80-kDa stress cause p38 mitogen-activated protein kinase activation by tumor necrosis factor receptor. Cell 83, 793–802. dual phosphorylation on tyrosine and threonine. J. Biol. Chem. Hanada T. and Yoshimura A. (2002) Regulation of cytokine signaling 270, 74–20–7426. and inflammation. Cytokine Growth Factor Rev. 13, 413–421. Reuter U., Bolay H., Jansen-Olesen I., Chiarugi A., Sanchez del Rio M., Herbert M. and Holzer P. (1994) Interleukin-1b enhances capsaicin- Letourneau R., Theoharides T., Waeber C. and Moskowitz M. induced neurogenic vasodilatation in the rat skin. Br. J. Pharmacol. (2001) Delayed inflammation in rat meninges: implications for 111, 681–686. migraine pathophysiology. Brain 124, 2490–2502. Hopkins S. and Rothwell N. (1995) Cytokines and the nervous system. I: Rothwell N. and Hopkins S. (1995) Cytokines and the nervous system Expression and recognition. Trends Neurosci. 18, 83–88. II: Actions and mechanisms of action. Trends Neurosci. 18, Kacena M., Merrel G., Konda S., Wilson K., Xi Y. and Horowitz M. 130–136. (2001) Inflammation and bony changes at the temporomandibular Sanlioglu S., Williams C., Samavati L., Butler N., Wang G., McCray P. joint. Cells Tissues Organs 169, 257–264. J., Ritchie T., Hunninghake G., Zandi E. and Engelhardt J. (2001) Kayali A., Austin D. and Webster N. (2000) Stimulation of MAPK Lipopolysaccharide induces Rac1-dependent reactive oxygen cascades by insulin and osmotic shock: lack of an involvement of species formation and coordinates tumor necrosis factor-a secre- p38 mitogen-activated protein kinase in glucose transport in 3T3- tion through IKK regulation of NF-jB. J. Biol. Chem. 276, L1 adipocytes. Diabetes 49, 1783–1793. 30 188–30 198. Kemper R., Meijler W., Korf J. and Ter Horst G. (2001) Migraine and Schafers M., Svensson C., Sommer C. and Sorkin L. (2003) Tumor function of the immune system: a meta-analysis of clinical lit- necrosis factor-a induces mechanical allodynia after spinal nerve erature published between 1966 and 1999. Cephalalgia 21, 549– ligation by activation of p38 MAPK in primary sensory neurons. 557. J. Neurosci. 23, 2517–2521. Kopp S. (2001) Neuroendocrine, immune, and local responses related to Schramek H. (2002) MAP kinases: from intracellular signals to physi- temporomandibular disorders. J. Orofac. Pain 15, 9–28. ology and disease. News Physiol. Sci. 17, 62–67. Lanigan T. and Russo A. (1997) Binding of upstream stimulatory factor Sessle B. (2001) New insights into peripheral chemical mediators of and a cell-specific activator to the calcitonin/calcitonin gene-related temporomandibular pain and inflammation. J. Orofac Pain 15,5. peptide enhancer. J. Biol. Chem. 272, 18316–18324. Shinoda S., Skradski S., Araki T., Schindler C., Meller R., Lan J., Taki Li Y., Ji A., Weihe E. and Schafer M. (2004) Cell-specific expression and W., Simon R. and Henshall D. (2003) Formation of a tumour lipopolysaccharide-induced regulation of tumor necrosis factor a necrosis factor receptor 1 molecular scaffolding complex and (TNFa) and TNF receptors in rat dorsal root ganglion. J. Neurosci. activation of apoptosis signal-regulating kinase 1 during seizure- 24, 9623–9631. induced neuronal death. Eur. J. Neurosci. 17, 2065–2076. Mori I., Goshima F., Koshizuka T. et al. (2003) Iba1-expressing Tabuchi A., Sakaya H., Kisukeda T., Fushiki H. and Tsuda M. (2002) microglia respond to herpes simplex virus infection in the mouse Involvement of an upstream stimulatory factor as well as cAMP- trigeminal ganglion. Brain Res. Mol. Brain Res. 120, 52–56. responsive element-binding protein in the activation of brain-de- Nicolodi M. and Del Bianco E. (1990) Sensory neuropeptides (substance rived neurotrophic factor gene promoter I. J. Biol. Chem. 277, P, calcitonin gene-related peptide) and vasoactive intestinal poly- 35 920–35 931. peptide in human saliva: Their pattern in migraine and cluster Taylor P., Williams R. and Feldmann M. (2004) Tumour necrosis factor headache. Cephalalgia 10, 39–50. a as a therapeutic target for immune-mediated inflammatory dis- Opree A. and Kress M. (2000) Involvement of the proinflammatory eases. Curr. Opin. Biotechnol. 15, 557–563. cytokines tumor necrosis factor-a, IL-1b, and IL-6 but not IL-8 in Thiagalingam A., deBustros A., Borges M., Jasti R., Compton D., the development of heat hyperalgesia: effects on heat-evoked Diamond L., Mabry M., Ball D., Baylin S. and Nelkin B. (1996) calcitonin gene-related peptide release from rat skin. J. Neurosci. RREB-1, a novel zinc finger protein, is involved in the differen- 20, 6289–6293. tiation response to ras in human medullary thyroid carcinomas. Palladino M., Bahjat F., Theodorakis E. and Moldawer L. (2003) Anti- Mol. Cell Biol. 16, 5335–5345. TNF-a therapies: the next generation. Nat. Rev. Drug Discov. 2, Tverberg L. and Russo A. (1993) Regulation of the calcitonin/calcitonin 736–746. gene-related peptide gene by cell-specific synergy between

2005 The Authors Journal Compilation 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 65–77 TNF-a stimulation of CGRP expression 77

helix-loop-helix and octamer-binding transcription factors. J. Biol. USF and the Foxa2 forkhead protein. J. Biol. Chem. 279, 49948– Chem. 268, 15 965–15 973. 49955. Vaughan W. (1927) Allergic migraine. J. Am. Med. Assoc. 88, 1383– Vitkovic L., Bockaert J. and Jacque C. (2000) ‘Inflammatory’ cytok- 1386. ines: neuromodulators in normal brain? J. Neurochem. 74, Viney T., Schmidt T., Gierasch W., Sattar A., Yaggie R., Kuburas A., 457–471. Quinn J., Coulson J. and Russo A. (2004) Regulation of the Williamson D. and Hargreaves R. (2001) Neurogenic inflammation in cell-specific calcitonin/calcitonin gene-related peptide enhancer by the context of migraine. Microsc. Res. Tech. 53, 167–178.

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