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

Abstract

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

Fig. 1

Expression of TNFR1 and TNFR2. Day-2 trigeminal ganglia cultures were stained for expression of CGRP (a,b) and co-stained for CGRP and TNFR1 (c-e) or TNFR2 (f-h). Phase microscopy was used to identify neuronal (large arrows) and non-neuronal cells (small arrows) (a, c, f). CGRP was expressed exclusively in neuronal cells (b). The same cultures were stained for CGRP (d) and TNFR1 (e) or CGRP (d) and TNFR2 (h). Scale bar, 50 μm. TNFR1 and TNFR2 were easily detected on the cell body of CGRP-positive trigeminal neurons. TNFR1 and TNFR2 staining of some non-neuronal cells was observed.

Fig. 2

TNFα increases CGRP secretion from primary trigeminal neurons. CGRP secretion from untreated cultures (CON) or cultures treated for 1 h with vehicle (VEH), 60 mM KCl, or increasing amounts of TNFα was determined. The mean basal rate of CGRP release was 132 ± 13 pg/h/well. The secretion rate for each condition was normalized to the basal rate for each well. The means and the SE from five independent experiments are shown. ^*p< 0.05 when compared with control levels; †p< 0.01 when compared with control levels.

Fig. 3

Stimulation of CGRP secretion by ceramide. The relative amount of CGRP secreted from control cultures (CON) or cultures treated with vehicle (VEH) or increasing amounts of ceramide for 1 h. The mean basal rate of CGRP release was 107 ± 9 pg/h/well. The secretion rate for each condition was normalized to the basal rate for each well. The means and the SE from four independent experiments are shown. ^*p< 0.05 when compared with control levels.

Fig. 4

TNFα stimulates CGRP promoter and c-Jun activity. The -1250-bp CGRP promoter fragment contains proximal cAMP- and ras-responsive regions (gray box) and a distal enhancer that contains both cell-specific (open box) and non-cell-specific (striped box) elements. (a) Trigeminal cultures transfected with the CGRP-luciferase reporter were either untreated (CON) or treated 2 h with 50 or 100 ng/mL TNFα and reporter activity measured. (b) c-Jun reporter activity in cultures treated with 50 ng/mL TNFα for 2 h. Mean luciferase activity (normalized to β-galactosidase activities) per 20 μg protein with SEM from four experiments is shown. ^*p < 0.05 when compared with control levels.

Fig. 5

TNFα stimulation of JNK and p38 MAP kinases and NF-κB. Unstimulated trigeminal ganglia cultures (control, CON) or cultures treated with TNFα (TNF) were stained with antibodies directed against the active (phosphorylated) forms of ERK (a), JNK (b), and p38 (c), as well as NF-κB (d) (left panels). Increased expression of active JNK and p38, but not ERK, was observed in the nucleus of TNFα-treated cultures. Nuclear expression of NF-κB was also increased in response to TNFα. The same cultures were co-stained with DAPI to identify nuclei (right panels). Cell bodies of neurons are indicated by arrows.

Fig. 6

Percentage of trigeminal neurons expressing the active form of ERK, JNK, p38 or NF-κB in response to TNFα treatment. The number of neuronal cells in which nuclear staining was detected following sorbitol (positive control, gray bars) or TNFα (black bars) treatment was compared with unstimulated cultures (control, open bars). Total cell counts (N = number) are indicated below each data set (x-axis). The data are from three independent experiments for each condition are shown.

Fig. 7

TNFα induction of an NF-κB-responsive reporter gene. Luciferase activity from trigeminal ganglion cells was detected by a CCD camera and IVIS imaging system. (a) Overlay images of the pre-stimulus and post-stimulus bioluminescence signals on the culture dish immediately before addition of the vehicle control and 4 h after vehicle treatment. The pseudocolor scale bar in photons/s/steridan/cm² is shown. (b) Overlay images from a culture immediately before addition of 50 ng/mL TNFα and 4 h after TNFα treatment. The pseudocolor scale bar in photons/s/steridan/cm² is shown. (c) Luciferase activities following vehicle or TNFα treatments are shown as the ratio of post-stimulus to pre-stimulus signals from the same culture dishes. In these experiments, only a single vehicle control dish was analyzed in parallel with the TNFα treatments that were performed on two dishes (mean ± range). ^*p < 0.05 when compared with vehicle levels.

Fig. 8

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

Fig. 9

MKP-1 overexpression blocks induction of the CGRP promoter by TNFα. 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 TNFα 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 TNFα 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.