IL-1β stimulates COX-2 dependent PGE₂ synthesis and CGRP release in rat trigeminal ganglia cells

Objective

Pro-inflammatory cytokines like Interleukin-1 beta (IL-1β) have been implicated in the pathophysiology of migraine and inflammatory pain. The trigeminal ganglion and calcitonin gene-related peptide (CGRP) are crucial components in the pathophysiology of primary headaches. 5-HT1B/D receptor agonists, which reduce CGRP release, and cyclooxygenase (COX) inhibitors can abort trigeminally mediated pain. However, the cellular source of COX and the interplay between COX and CGRP within the trigeminal ganglion have not been clearly identified.

Methods and Results

1. We used primary cultured rat trigeminal ganglia cells to assess whether IL-1β can induce the expression of COX-2 and which cells express COX-2. Stimulation with IL-1β caused a dose and time dependent induction of COX-2 but not COX-1 mRNA. Immunohistochemistry revealed expression of COX-2 protein in neuronal and glial cells. 2. Functional significance was demonstrated by prostaglandin E2 (PGE₂) release 4 hours after stimulation with IL-1β, which could be aborted by a selective COX-2 (parecoxib) and a non-selective COX-inhibitor (indomethacin). 3. Induction of CGRP release, indicating functional neuronal activation, was seen 1 hour after PGE₂ and 24 hours after IL-1β stimulation. Immunohistochemistry showed trigeminal neurons as the source of CGRP. IL-1β induced CGRP release was blocked by parecoxib and indomethacin, but the 5-HT1B/D receptor agonist sumatriptan had no effect.

Conclusion

We identified a COX-2 dependent pathway of cytokine induced CGRP release in trigeminal ganglia neurons that is not affected by 5-HT1B/D receptor activation. Activation of neuronal and glial cells in the trigeminal ganglion by IL-β leads to an elevated expression of COX-2 in these cells. Newly synthesized PGE₂ (by COX-2) in turn activates trigeminal neurons to release CGRP. These findings support a glia-neuron interaction in the trigeminal ganglion and demonstrate a sequential link between COX-2 and CGRP. The results could help to explain the mechanism of action of COX-2 inhibitors in migraine.     

Epoxyeicosatrienoic acids are endogenous regulators of vasoactive neuropeptide release from trigeminal ganglion neurons

Epoxyeicosatrienoic acids (EETs) are bioactive eicosanoids produced from arachidonic acid by cytochrome P450 epoxygenases. We previously described the expression of cytochrome P450-2J epoxygenase in rat trigeminal ganglion neurons and that EETs signaling is involved in cerebrovascular dilation resulting from perivascular nerve stimulation. In this study, we evaluate the presence of the EETs signaling pathway in trigeminal ganglion neurons and their role in modulating the release of calcitonin gene-related peptide (CGRP) by trigeminal ganglion neurons. Liquid chromatography tandem mass spectrometry identified the presence of each of the four EETs regio-isomers within primary trigeminal ganglion neurons. Stimulation for 1 h with the transient receptor potential vanilloid-1 channel agonist capsaicin (100 nmol/L) or depolarizing K(+) (60 mmol/L) increased CGRP release as measured by ELISA. Stimulation-evoked CGRP release was attenuated by 30 min pre-treatment with the EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol/L). K(+) stimulation elevated CGRP release 2.9 ± 0.3-fold above control levels, whereas in the presence of 14,15-EEZE K(+)-evoked CGRP release was significantly reduced to 1.1 ± 0.2-fold above control release (p < 0.01 anova, n = 6). 14,15-EEZE likewise attenuated capsaicin-evoked CGRP release from trigeminal ganglion neurons (p < 0.05 anova, n = 6). Similarly, pre-treatment with the cytochrome P450 epoxygenase inhibitor attenuated stimulation-evoked CGRP release. These data demonstrate that EETs are endogenous constituents of rat trigeminal ganglion neurons and suggest that they may act as intracellular regulators of neuropeptide release, which may have important clinical implications for treatment of migraine, stroke and vasospasm after subarachnoid hemorrhage.     

Regulation of the cell-specific calcitonin/calcitonin gene-related peptide enhancer by USF and the Foxa2 forkhead protein

An 18-bp enhancer controls cell-specific expression of the calcitonin/calcitonin gene-related peptide gene. The enhancer is bound by a heterodimer of the bHLH-Zip protein USF-1 and -2 and a cell-specific factor from thyroid C cell lines. In this report we have identified the cell-specific factor as the forkhead protein Foxa2 (previously HNF-3beta). Binding of Foxa2 to the 18-bp enhancer was demonstrated using electrophoretic mobility shift assays. The cell-specific DNA-protein complex was selectively competed by a series of Foxa2 DNA binding sites, and the addition of Foxa2 antiserum supershifted the complex. Likewise, a complex similar to that seen with extracts from thyroid C cell lines was generated using an extract from heterologous cells expressing recombinant Foxa2. Interestingly, overexpression of Foxa2 activated the 18-bp enhancer in heterologous cells but only in the presence of the adjacent helix-loop-helix motif. Likewise, coexpression of USF proteins with Foxa2 yielded greater activation than by Foxa2 alone. Unexpectedly, Foxa2 overexpression repressed activity in the CA77 thyroid C cell line, suggesting that Foxa2 may interact with additional cofactors. The stimulatory role of Foxa2 at the calcitonin/calcitonin gene-related peptide gene enhancer was confirmed by short interfering RNA-mediated knockdown of Foxa2. As seen with Foxa2 overexpression, the effect of Foxa2 knockdown also required the adjacent helix-loop-helix motif. These results provide the first evidence for combinatorial control of gene expression by bHLH-Zip and forkhead proteins.     

CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogen-activated protein kinase pathways

Clinical and basic science data support an integral role of calcitonin gene-related peptide (CGRP) in the pathophysiology of temporomandibular joint disorders. Recently, we have shown that CGRP can stimulate the synthesis and release of nitric oxide (NO) from trigeminal ganglion glial cells. The goal of this study was to determine the role of mitogen-activated protein kinase (MAPK) signaling pathways in CGRP regulation of iNOS expression and NO release from cultured trigeminal ganglion glial cells from Sprague–Dawley rats. CGRP treatment for 2 h significantly increased activity of the MAPK reporter genes, Elk, ATF-2, and CHOP. In addition, CGRP increased nuclear staining for the active forms of the MAPKs: extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38. This stimulatory event was not observed in cultures pre-treated with the CGRP receptor antagonist peptide CGRP_8–37. Similarly, pre-treatment with selective MAPK inhibitors repressed increases in reporter gene activity as well as CGRP-induced increases in iNOS expression and NO release mediated by MAPKs. In addition, over-expression of MAPK kinase 1 (MEK1), MEK3, MEK6, and MEK kinase significantly increased iNOS expression and NO production in glial cells. Results from our study provide evidence that CGRP binding to its receptor can stimulate iNOS gene expression via activation of MAPK pathways in trigeminal ganglion glial cells.