Corticosteroid-dependent increased expression of CGRP and its receptor subunits within the rodent trigeminal ganglion does not prompt cephalic allodynia

Abstract

Background

Calcitonin gene-related peptide (CGRP) has a causative role in migraine pathogenesis but its effects on trigeminal afferents are still unclear. Corticosteroids represent a very useful tool in headache therapy with an unknown mechanism of action. Despite the widespread effects of corticosteroids on gene transcription, whether they regulate CGRP expression within the trigeminovascular system remains to be investigated.

Methods

The effects of dexamethasone on expression of CGRP and its receptor subunits receptor activity-modifying protein (RAMP1) and calcitonin receptor-like receptor (CLR) have been evaluated in rat thyroid parafollicular CA77 and human neuroblastoma SHSY-5Y cell cultures, as well as in isolated human peripheral blood mononuclear cells. The effects of dexamethasone on the rat and human CGRP promoter were also evaluated. In vivo, rats and mice were treated with betamethasone (320 µg/kg for 10 days) to investigate whether the drug altered the expression of CGRP, RAMP1 and CLR in the trigeminal ganglion (TG). We also evaluated the effect of betamethasone on CGRP mRNA stability and release from the mouse TG, as well as on mouse spontaneous or nitroglycerin-induced cephalic allodynia.

Results

We report that dexamethasone triggered transcriptional activation of the rat and human CGRP gene, also increasing transcript levels of RAMP1 but not of CLR in cultured cells. These effects were paralleled in the TG of rats and mice challenged with betamethasone, with mice also showing increased expression levels of CLR. Of note, although a 13-fold increase of the CGRP releasable pool occurred in the TG of betamethasone-treated mice, the animals were not sensitized to cephalic allodynia.

Conclusions

In keeping with the emerging immunosuppressing effects of CGRP, corticosteroids increase its expression in rat and human cell lines, as well as in rodent TG. Evidence that a substantial increase of releasable CGRP in the TG does not reduce orofacial pain thresholds suggests that basal release of endogenous CGRP differs from its exogenous administration in terms of trigeminal afferent sensitization.

Graphical abstract

This is a visual representation of the abstract.

Keywords

allodynia CGRP corticosteroids RAMP1 trigeminal ganglion

Introduction

Calcitonin gene-related peptide (CGRP) exerts pleiotypic functions in the human body (1). Being first identified as an alternative product of the calcitonin gene with potent vasodilating properties, CGRP has then been shown to play a key role in migraine pathogenesis. The recent development of anti-CGRP monoclonals and CGRP receptor antagonists with excellent antimigraine efficacy unequivocally confirms the central role of the CGRP in the pathogenesis of migraine (2,3-5,6), how CGRP promotes migraine waits to be unequivocally understood. A major focus has been directed at elucidating the proinflammatory effects of CGRP and its role in development of sterile inflammation within the meninges, apparently a key event in migraine pain generation. Indeed, upon antidromic release by trigeminal terminals, CGRP prompts vasodilation and plasma extravasation, two events with key roles in meningeal neurogenic inflammation. The latter is also promoted by CGRP-dependent degranulation of meningeal mastocytes (7-8).

Interestingly, CGRP is emerging as a pleiotypic anti-inflammatory peptide. Specifically, the CGRP reduces tumor necrosis factor α and interleukin-6 release by cultured macrophages (9), as well as pre-B cell colony formation (10-12), in keeping with its ability to counteract T-lymphocyte proliferation (13-14,15-16,17-20,21). A key role of CGRP in suppressing allergic inflammation by inhibiting group-2 innate lymphoid cell activity has also been reported (22-24).

Corticosteroids are potent anti-inflammatory drugs and of therapeutic relevance to different types of headaches such as acute migraine attack (25-27). In light of the potent anti-inflammatory effects of corticosteroids, their efficacy in headache treatment suggests the potential involvement of immune cell activation in cephalic pain. However, the mode of action of corticosteroids in headache pain relief still waits to be understood. Likewise, whether/how corticosteroids affect CGRP transcriptional homeostasis needs to be clarified. Indeed, one would expect two opposite relationships between corticosteroids and CGRP. On the one hand, given the role of CGRP in meningeal neurogenic inflammation, corticosteroids are expected to negatively regulate its expression. On the other, in light of the emerging immunosuppressive properties of CGRP, corticosteroids might increase its expression. The present study attempted to answer these questions by investigating the impact of corticosteroids on expression of CGRP and its receptor subunits both in cell lines and the rodent trigeminal ganglion (TG). The effect of corticosteroids on the mouse cephalic pain thresholds has also been investigated.

Methods

Animals and drug administration

Adult male or female Wistar rats, weighing 125–150 g, and male or female C57Bl/6J mice, weighing 25–30 g (Charles River, Milan, Italy) were housed in groups with free access to food (Harlan Global Diet 2018; Harlan Laboratories, Udine, Italy) and water and maintained under a 12:12 hour light/dark cycle at 21 °C. All animal manipulations were performed according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive 86/609/EEC). Animals were randomized (generating groups by the RAND function of Excel; Microsoft Corp., Redmond, WA, USA) and daily treated (at 10:00 am) orally with betamethasone (Merck, Milan, Italy) 320 µg/kg for 10 days. This dose equals that of 24 mg dexamethasone adopted for migraine treatment (28). We preferred to avoid considering the interspecies mouse-to-human dose conversion factor of 12 (29) because of the chronic treatment schedule adopted in the in vivo experiments. Vehicle animals received the same volume of water. At the end of the experiments, animals were euthanized, and TGs and dorsal root ganglia (DRG) were collected and processed for real-time PCR analysis or CGRP release assay.

Cell culture

Rat medullary thyroid carcinoma CA77 and human neuroblastoma SHSY-5Y cell lines were obtained from American Type Culture Collection (#catalog number CRL-3234 and CRL-2266; American Type Culture Collection, Manassas, VA, USA). CA77 cells were grown in Dulbecco's modified Eagle's medium (DMEM) low glucose:Ham's F12 (1:1) supplemented with 2 mm glutamine, 1 mm pyruvate, 10% fetal bovine serum and antibiotics. SHSY-5Y cells were grown in DMEM high glucose supplemented with 2 mm glutamine, 1 mm pyruvate, 10% fetal bovine serum and antibiotics. Cultures were brought to 50–70% confluence and exposed to different dexamethasone concentrations. Dexamethasone was purchased by Merck (Milan, Italy). All results are expressed as the percentage of the control (untreated cells); each sample was normalized by the protein content.

Peripheral blood mononuclear cell culture

Peripheral blood mononucleated cells (PBMC) were isolated from the blood samples obtained from healthy donors and discarded from the Blood Drawn Laboratory of the Careggi University Hospital. Briefly, 10 ml of whole blood was diluted with phosphate-buffered saline to a final volume of 35 ml. The sample was carefully layered over 15 ml of Ficoll-Hypaque in a 50 ml conical tube and centrifuged at 377 g for 30 seconds at room temperature. PBMCs were recovered and washed three times before culturing. Cells were maintained in RPMI 1640 supplemented with 1% penicillin/streptomycin, 1% sodium pyruvate, 10% FBS (all from EuroClone, Pero, Italy) and 2% glutamine (Merck) and exposed to dexamethasone. At 12, 24 and 48 hours after dexamethasone exposure, cells were collected and transcript levels were performed.

CGRP release from TG and enzyme-linked immunosorbent assay (ELISA) quantitation

CGRP release from TG was evaluated as previously described (30) with minor modifications. TG were collected from mice, weighted, treated or not with betamethasone. Each TG was placed in an Eppendorf tube containing 50 µl of artificial cerebrospinal fluid (aCSF) containing: NaCl 130 mm, KCl 3.5 mm, NaH₂PO₄ 1.25 mm, NaHCO₃ 24 mm, glucose 10 mm, CaCl₂ 2 mm, MgSO₄ 1 mm and incubated at 37 °C. Two hours later, CGRP release was induced by adding an aCSF solution containing 50 mm KCl. Thirty min later CGRP concentrations were determined in the medium by means of an ELISA kit (Bertin Pharma, Montigny le Bretonneux, France). The same kit was adopted to measure CGRP in the plasma of female rats and mice at the end of the 10-day treatment protocol.

Cell transfection and luciferase expression analysis

100 ng of CGRP promoter-luciferase plasmids (rCGRP-Luc and hCGRP-Luc) (31), a kind gift from Professor Andrew Russo (Department of Neurology, University of Iowa, Iowa City, IA, USA) were used for CA77 and SHSY-5Y cells transfection. Luciferase expression was evaluated 48 hours later by Dual-Glo luciferase assay system (Promega, Milan, Italy) and a luminometer. Firefly luciferase activity was normalized to Renilla luciferase and total protein levels.

RT-PCR and evaluation of mRNA stability

Total RNA was isolated using Trizol Reagent (Life Technologies, Carlsbad, CA, USA). One microgram of RNA was retrotranscribed using iScript (Bio-Rad, Hercules, CA, USA). Real-time PCR was performed using Rotor-Gene 3000 (Qiagen, Hilden, Germany) as reported previously (32). The primers used were: mCGRP FW: 5′-GCTAAGTGCAGAATAAGTTGCCTATTGTG-3′, RV: 5′-CAACATTACCATGTGTCCCCAGAAGAGC-3′; mRAMP1 FW: 5′-TGCTGCTGGCTACCATCTCTTCA-3′, RV: 5′-AGCCAATCGTGTGCGCCACGTGCT-3′; mCLR FW: 5′-TGCCCTGACTACTTCCCGGACTTT-3′, RV: 5′-GACAAGGAGTGACCCACAAGAGCC-3′; rCGRP FW: 5′-GGCATGCGCCCTCCAGGCAG-3′, RV: 5′-AGAGCCCTCAGCCTCCTGTCCCT-3′; rRAMP1 FW: 5′-CTCACTGCACCAAACTCGTGGCAAAC-3′, RV: 5′-CACCAGGGCAGTCATGAGCAGTG-3 ′; rCLR FW: 5′-CCTGTGAGCTGCAAGGTGTCCCA-3′, RV: 5′-GTAATACAAGCTTCTGGCGATGGCATG-3′; hCGRP FW: 5′-GAAAGCAATTTGTAGGAAAGGCTCCATG-3′, RV: 5′-GACGGGGCCTAGATTTGCTACCAG-3′; hRAMP1 FW: 5′-AGCTTCTGGAGCCTTGGGACAGAG-3′, RV: 5′-AAGCAGTCTAGGCTGGGACCCTTTC-3′; hCLR FW: 5′-GGCCCAATTTGTGCTGCTTTACTGGT-3′, RV: 5′-TCCTCTGCAATCTTTCCTTCAGGTCG-3′. For CGRP mRNA stability assessment, PCR amplification was carried out as previously described previously (33), by means of the following primers: rPre-CT/CGRP (Ex-ln) FW: 5′-TGAGGCAAAGGATGGTATCTGG-3′, RV: 5′- GGGCTACATCAATGACCACCA-3′; rPre-CT/CGRP (Ex-Ex) FW: 5′-CTCTCTGGCTGTTCTCTGGG-3′, RV: 5′-TTGTCCTTCACCACACCTCC-3′; Primers were purchased from Integrated DNA Technologies (Iowa, USA).

Von Frey testing

Measurement of periorbital allodynia was conducted by an experimenter blind to treatment. Measuremen4ts were taken on the day before treatment and on days 5, 10 and 15 during betamethasone exposure in male mice. On the testing day, the animals were brought into the behaviour room one hour before the test session. Periorbital allodynia was measured by means of the Von Frey test as previously reported (34).

Grimace test

Grimace score test was conducted on female mice as reported (35,36). Grimace was measured before nitroglycerin (NTG) induction (basal) and after NTG induction (3 mg/kg, i.p.). NTG was administered one time at the end of 10 days treatment with vehicle or betamethasone (320 µg/kg, oral). Briefly, animals were placed individually in a plexiglas chamber for 60 minutes and orbital tightening, nose bulge, cheek bulge, ear position were evaluated from 30 to 60 minutes after NTG treatment by two blinded experimenters. The mean grimace scores were calculated as the average score across all the analysed parameters.

Statistical analysis

Data are presented as the mean ± SEM. All differences among groups were performed using two-way analysis of variance (ANOVA) followed by Tukey's W-test. p < 0.05 was considered statistically significant. Statistical analyses were carried out using Prism, version 8 (GraphPad Software Inc., San Diego, CA, USA).

Results

Effects of dexamethasone on expression of CGRP and its receptor subunits by cultured cells

Reportedly, splicing of calcitonin pre-mRNA in thyroid parafollicular cells (C cells) leads to significant amounts of mature CGRP mRNA (37), indicating a constitutively-active basal transcriptional machinery at the CGRP gene promoter. Given that this is instrumental to investigate whether/how corticosteroids impact CGRP expression when its gene is transcriptionally active, we first evaluated the effects of dexamethasone on the expression of CGRP and its receptor subunits receptor activity-modifying protein (RAMP1) and calcitonin receptor-like receptor (CLR) by the medullary thyroid carcinoma CA77 cell line originating from rat parafollicular cells. To this end, we adopted the dexamethasone concentrations of 10 and 100 nM (38,39). We found that 12 hours of exposure did not alter transcript levels of CGRP and its receptor (data not shown). In contrast, 24 hours of exposure to dexamethasone dose-dependently increased transcript levels of both CGRP and RAMP1, while having no significant effects on those of CLR. These effects were maintained at 48 hours, with CLR transcript showing a tendency to increase (Figure 1(a)–(c)). Accordingly, CGRP contents in the culture medium doubled 48 hours after dexamethasone exposure, showing identical increases at the concentrations of 10 and 100 nm (Figure 1(d)). The ability of dexamethasone to increase CGRP release at 10 n40). As shown in Figure 1(e), we found that dexamethasone increased luciferase expression at 100 nm (Figure 1(e)). Further substantiating the ability of dexamethasone to activate CGRP transcription, the corticosteroid also increased luciferase transcripts in human neuroblastoma SHSY-5Y cultured cells transfected with a luciferase reporter plasmid driven by the human CGRP promoter (40) (Figure 1(f)). Next, to understand whether these effects of corticosteroids were specific of cancer cell lines or were also shared by primary cell cultures, we evaluated the effects of dexamethasone on cultured human peripheral blood mononuclear cells. Data showed that 10 and 100 nm dexamethasone increased CGRP transcript levels at 12 and 24 hours of incubation, whereas, at 48 hours, only 100 nm dexamethasone prompted CGRP mRNA accumulation (Figure 1(g)).

Figure 1.

Effects of dexamethasone on transcript levels for calcitonin gene-related peptide (CGRP), receptor activity-modifying protein (RAMP1) and calcitonin receptor-like receptor (CLR) in cell cultures. (a–c) Expression levels of transcripts for CGRP, RAMP1 and CLR in CA77 cells exposed to dexamethasone (10 and 100 nm) for 24 and 48 hours are shown. (d) Release of CGRP in the medium of CA77 cells exposed to dexamethasone (10 and 100 nm) for 48 hours. The effects of 48 hours of exposure to dexamethasone on luciferase expression in CA77 (e) and SHSY-5Y (f) cells transfected with a reporter plasmid driven by the CGRP promoter are shown. (g) Effects of dexamethasone (10 and 100 nm) on CGRP transcript levels in human peripheral blood mononuclear cell culture exposed for 12, 24 and 24 hours. Each column is the mean ± SEM of at least three experiments conducted in triplicate. *p < 0.05, **p < 0.01 two-way analysis of variance plus Tukey's test.

Effects of betamethasone on expression of CGRP and its receptor subunits in trigeminal and DRG of rats

Next, we investigated whether corticosteroids also induce CGRP expression within the TG in rats. We therefore evaluated transcripts of the CGRP and its receptor subunits in the TG of rats daily exposed to a commercially available formulation of oral betamethasone, a stereoisomer of dexamethasone with identical potency. The animals have been treated orally at the dose of 320 µg/kg for 10 consecutive days. In keeping with data obtained in vitro, we found that transcript levels of CGRP and RAMP1 increased in the TG of male and female rats, whereas CLR mRNAs showed only a tendency to increase in both sexes (Figure 2(a)–(c)). It is known that CGRP also regulates neurotransmission at peripheral and central terminals of DRG neurons. Intriguingly, however, exposure to betamethasone did not significantly affect transcripts for CGRP, RAMP1 or CLR in the DRG of male and female rats (Figure 2(d)–(f)) (41). Somehow consistent with the selective induction of CGRP in the TG, plasma levels of CGRP did not differ between control and 10 days betamethasone-treated rats (53.3 ± 15.4 and 49.5 ± 7.1 pg/ml, respectively). Given that corticosteroids are capable of regulating gene expression both at the transcriptional and post-transcriptional (i.e. by regulating mRNA stability) level (42), we investigated whether the stability of CGRP mRNA was affected by betamethasone exposure. Specifically, by means of intron- and exon-specific primers we evaluated the ratio between mature and immature (heteronuclear) CGRP RNA contents in TG of control and betamethasone-treated rats (Figure 2(g)). Typically, the ratio rises when mRNA stability increases. Findings showed that the ratio increased upon betamethasone exposure (Figure 2(h)), thereby indicating that, corticosteroids also stabilize CGRP mRNA. Next, we took advantage of the Eukaryotic Promoter Database (https://epd.expasy.org/epd) to understand whether promoters of CGRP, RAMP1 and CLR have glucocorticoid recognition elements (GREs). In the rat and human CGRP promoters we did not find canonical (i.e. palindromic) GRE sequences recognizing the glucocorticoid receptor GRα (also known as NR3C1). Conversely, we found one GRE and two GREs in the rat and human RAMP1 promoter, respectively. No GREs were found in both the rat and human CLR promoters.

Figure 2.

Effects of betamethasone on CGRP and its receptor subunits in TG and DRG of mice

We also evaluated whether an identical exposure to oral betamethasone (320 µg/kg) for 10 days also affects CGRP expression homeostasis in the TG of mice. Figure 3(a)–(c) shows that, in keeping with data obtained in rats, exposure to the corticosteroid increased trigeminal CGRP and RAMP1 mRNA levels in male and female mice. At variance with rats, however, trigeminal CLR transcripts increased upon betamethasone exposure in male and female mice, revealing a higher inducibility of the mouse CGRP signaling system by corticosteroids. In keeping with this, CGRP and RAMP1 transcripts also increased in the DRG of male mice exposed to betamethasone, whereas, in females, only CGRP expression was induced by the corticosteroid (Figure 3(d) and (e)). No changes were found for CLR mRNAs in both mouse sexes (Figure 3(f)). The partial discrepancy with respect to what obtained in rats may well be due to species difference in transcriptional regulation. However, consistent with our findings in rats, even in mice CGRP plasma contents did not differ between control and 10 days betamethasone-treated animals (89.2 ± 12.3 and 93.7 ± 9.4 pg/ml, respectively). Analysis of the promoter sequences by means of the Eukaryotic Promoter Database (https://epd.expasy.org/epd), confirmed that, akin to the rat, no GREs are present in the mouse CGRP promoter, whereas one GRE is present in that of RAMP1. In the mouse, one GRE was also found CLR promoter, in keeping with the increased expression of CLR in TG of mice challenged with betamethasone (Figure 3(c)).

Figure 3.

Effects of betamethasone treatment on transcript levels for calcitonin gene-related peptide (CGRP), receptor activity-modifying protein (RAMP1) and calcitonin receptor-like receptor (CLR) in the trigeminal and dorsal root ganglia and on periorbital allodynia in mice. Effects of betamethasone on expression levels of transcripts for CGRP, RAMP1 and CLR in the trigeminal ganglion (a–c) and dorsal root ganglia (d–f) of male and female mice exposed to daily oral treatment (320 µg/kg) for 10 days are shown. (g) Evaluation of CGRP release from female mouse trigeminal ganglia incubated in vitro in artificial CSF under control conditions or after exposure to 60 mM K⁺ in the absence or presence of fremanezumab 10 µm. (h) Evaluation of CGRP release from trigeminal ganglia of female of mice exposed to daily oral treatment with betamethasone (320 µg/kg) or vehicle for 10 days. (i) Time course of mechanical thresholds (von Frey test) in the periorbital region of male mice exposed to daily oral treatment with betamethasone (320 µg/kg) or vehicle for 10 days. (j) Schematic representation of the timeline of betamethasone and nitroglycerin (NTG) treatment. (k) Grimace score of female mice exposed to daily oral treatment with betamethasone (320 µg/kg) or vehicle for 10 days in the absence or presence of a pretreatment (30 minutes) with nitroglycerin (3 mg/kg i.p.). Each column/point is the mean ± SEM of two experiments with at least five mice per group. In (g) and (h), each column is the mean ± SEM of two experiments conducted in triplicate. *p < 0.05, **p < 0.01 two-way analysis of variance plus Tukey's test.

To understand whether induction of transcription correlated to CGRP production in the TG of betamethasone-treated mice, we adopted the method of K⁺-induced CGRP release from TG neurons 42). As shown in Figure 3(g), TG from control male mice incubated in vitro promptly released CGRP upon exposure to a depolarizing, 60 mm K⁺-containing incubation buffer. Of note, the signal revealed by the ELISA assay was abrogated by the presence of the anti-CGRP antibody fremanezumab in the incubation buffer (Figure 3(g)), indicating the affinity of the humanized antibody for mouse CGRP, as well as specificity of the signal. We then compared the CGRP releasable pool in TG from control and betamethasone-treated mice. In keeping with data on CGRP transcript increase, we found that K⁺-induced release of the CGRP from TG of betamethasone treated male mice was, on average, 13-fold higher than that from TG of control animals (Figure 3(h)).

Effects of betamethasone on cephalic allodynia in mice

Next, in light of the role of CGRP in pronociceptive trigeminovascular sensitization (2,43-44), we considered whether mice challenged with betamethasone showed reduced basal, perifacial pain thresholds. The latter were therefore evaluated by means of the von Frey filament assay before and at different time points during and after betamethasone treatment. We found no evidence of reduced cephalic pain thresholds/allodynia in male mice challenged with betamethasone both during and at the end of treatment (Figure 3(i)), a time point corresponding to TG CGRP contents 13-fold higher than control levels (Figure 3(h)). To confirm this finding, withdrawal thresholds were also evaluated at a later time point by prolonging betamethasone exposure from 10 to 15 days, in light of the possibility that a more prolonged exposure to increased CGRP contents are required to sensitize the trigeminovascular system and prompt cephalic allodynia. Even at these delayed time points, however, no evidence of reduced perifacial withdrawal thresholds in corticosteroid-challenged mice was recorded Figure 3(i) (45). The latter was therefore injected intraperitoneally in control or betamethasone-treated female mice and the severity of cephalic pain was evaluated by means of the grimace score (35). We found that the increase of grimace score from 30 to 60 minutes after NTG injection did not differ between control and betamethasone challenged mice Figure 3(j) and (k).

Discussion

We report here that corticosteroids prompt expression of CGRP and its receptor subunits in different cell lines as well as in the rodent TG. We build upon prior work on thyroid parafollicular cells (46,47), showing that corticosteroids, activate CGRP transcription also in human neuroblastoma SHSY-5Y cells and human peripheral blood mononuclear cells, as well as in the rat and mouse TG and in the mouse DRG. These findings suggest that CGRP signaling plays a functional role in the pleiotypic effects prompted by glucocorticoids. Also, having extended data from parafollicular cells to human cell types, on the one hand is relevant to the understanding that steroids induce the CGRP system (peptide and receptor) in cells of different origin, and, on the other hand, corroborates the hypothesis that our findings are pertinent to humans. Intriguingly, however, we have been unable to find promoter sequences recognizing the glucocorticoid receptor (the so-called GREs), thereby suggesting that corticosteroids activate CGRP gene expression not by regulating glucocorticoid receptor binding to DNA, but assisting the formation of transcriptionally active supramolecular complexes. Indeed, corticosteroids have pleiotypic modes of action on gene expression regulation. In addition to classic binding to GREs, they regulate basal transcriptional machinery by affecting (both positively and negatively) recruitment of transcription factors and epigenetic regulators at the supramolecular complexes forming at gene promoters (i.e. a GRE independent mode of action). In this regard, our finding showing that betamethasone increases CGRP mRNA stability in the rat TG further increases the complexity of the mechanisms brought about by corticosteroids to increase CGRP signaling. CGRP mRNA stabilization is in keeping with the well-known ability of steroids to post-transcriptionally regulate gene expression (42). We report that corticosteroids also induce expression of the CGRP receptor component RAMP1 in vitro as well as in the mouse and rat TG. This is in keeping with the presence in the human, rat and mouse RAMP1 promoters of specific GREs (one in rodents and two in humans). Conversely, CLR, the other key CGRP receptor subunit, only shows modest, non-significant induction in vitro, being highly induced in the mouse TG (about 10- and 5-fold in males and females, respectively) and only modestly in the TG of female rats (about 2-fold). Accordingly, the mouse is the sole, among the species evaluated, showing a GRE in the CGRP promoter sequence (https://epd.expasy.org/epd). As for the finding that plasma levels of CGRP did not differ between control and betamethasone-treated rats or mice, we reason that this might be ascribed to a tissue/cell specific induction of the neuropeptide unable to trigger a rise in the serum CGRP content. Of note, different proteases including the neutral endopeptidase might well contribute to blunt tissue, and therefore serum, CGRP accumulation in animals exposed to corticosteroids.

The ability of betamethasone to increase CGRP expression within the rodent TG appears somehow paradoxical. Indeed, given the therapeutic properties of corticosteroids in medication-overuse headache (26-27), as well as the causative role of CGRP in their pathogenesis, one would expect that corticosteroids reduce rather than increase CGRP expression in the TG. Evidence that CGRP release from TG of betamethasone-exposed mice is approximately 13-fold more abundant than that from TG of control mice emphasizes the apparent paradox of a corticosteroid-dependent CGRP increase in the TG. This inconsistency is further strengthened by our findings showing that betamethasone-challenged mice bearing a remarkable increase of releasable CGRP in the TG develop neither spontaneous not nitroglycerin-induced cephalic allodynia. Evidence that perifacial injection of CGRP triggers cephalic allodynia (44-49). Hence, based on the study by Zhang et al. (49) the increased expression of RAMP1 by betamethasone should have contributed to prompt spontaneous as well as nitroglycerin-induced mouse cephalic allodynia. We emphasize, however, that the sensitizing effect of increased RAMP1 expression to neurogenic inflammation is not spontaneous but, again, occurs upon peripheral injection of exogenous CGRP (49). Endogenous CGRP release and exogenous GGRP injections may differently affect trigeminovascular responses. In contrast with our findings, stress-dependent corticosterone production reduces facial withdrawal thresholds in mice (50). Although the reason for this apparent inconsistency may reside in the intricacy of the neurohumoral response to stress, it is worth noting that corticosteroids do not prompt headache when acutely or chronically administered to patients. Given the widespread effects of steroids on gene transcription, expression of proteins capable of counteracting CGRP-dependent trigeminovascular afferent sensitization might explain lack of facial allodynia in betamethasone-treated mice. It can be also hypothesized that the increased expression of CGRP, RAMP1 and CLR in the TG of rodents exposed to betamethasone does not occur in humans. In this regard, Neeb et al. (51), it is unclear why steroids reduce plasma CGRP only in CH patients. Apparently, more work needs to be done to clarify the impact of corticosteroids on CGRP expression in humans. In particular, it will be worth investigating the impact of steroids on CGRP levels of the trigeminovascular system of both healthy and migraine/CH subjects.

Limitations of the study

We acknowledge that our study presents some limitations. First, it remains unknown whether steroids also induce CGRP in human TG. Indeed, the only primary human cells that we have evaluated are peripheral blood mononuclear cells that are not pertinent to the nervous system. An additional limitations is that we did not investigate the cell type(s) in which induction of CGRP and its receptor subunits occurs within TG. Still, evidence that CGRP release from TG is obtained by incubation in high K⁺ suggests an involvement of excitable cells. Furthermore, each functional experiment aimed at evaluating the effects of betamethasone on cephalic pain thresholds has been conducted in a single gender (male mice for spontaneous cephalic allodynia, and female mice for sensitization to NTG-induced facial pain). Furthermore, the experiments with males and females were not conducted simultaneously. As for limitations of the in vitro experiments, we emphasize that only two concentrations of dexamethasone have been tested on the different cell cultures, and only two time points of 24 and 48 hours have been evaluated.

Conclusions

Notwithstanding the apparent inconsistency of the present findings with the current interpretation of migraine pathogenesis, data are in keeping with the emerging anti-inflammatory/immunosuppressive roles of CGRP (9-20,22,23). Indeed, it makes sense that the anti-inflammatory effects of corticosteroids take advantage of the immune-suppressing effects of CGRP by increasing its expression as well as that of its receptor. The present study suggests that the interplay among endogenous CGRP signaling, corticosteroids and migraine is more complex than previously envisaged. It also provides hints that the role of CGRP-dependent meningeal inflammation in migraine pathogenesis might be reevaluated (8). Overall, our findings are at odds with the ability of chronically administered corticosteroids to counteract CH and medication-overuse headache. Given that we did not adopt an acute-administration protocol, that obviously could not affect CGRP expression, the present findings do not allow us to draw conclusions on the antimigraine effects of acutely administered corticosteroids.

Article highlights

Corticosteroids increase CGRP expression in rat and human cell lines as well as in rodent TG

The increase of CGRP in the TG does not prompt or sensitize to cephalic pain

Data suggest that the interplay among CGRP signaling, corticosteroids and migraine is more complex than previously envisaged

Footnotes

Acknowledgments

We are grateful to Professor Andrew F. Russo for providing us with the luciferase plasmids driven by the human and rat CGRP promoter.

Author contributions

All authors made substantial contributions to conception and study design. AP, DB, ST, AL, AM, LL and RN performed the experiments and collected data. AC and DB wrote the manuscript.

Data availability

Data and materials are available upon request.

Funding

The study was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), Italian Foundation for Multiple Sclerosis (FISM, 2022/R-single/023), The Health Project from the Region of Tuscany and the PNRR project (CN3-Gene therapy and RNA technology Drugs; THE - Health Ecosystem).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

ORCID iD

Alessandra Pistolesi

Simone Tuniz

Andrea Lapucci

Alice Molli

Francesco De Cesaris

Romina Nassini

Daniela Buonvicino

References

Russell

King

Smillie

S-J

, et al. Calcitonin gene-related peptide: physiology and pathophysiology. Physiol Rev 2014; 94: 1099–1142.

Edvinsson

Haanes

Warfvinge

, et al. CGRP As the target of new migraine therapies - successful translation from bench to clinic. Nat Rev Neurol 2018; 14: 338–350.

Charles

Pozo-Rosich

. Targeting calcitonin gene-related peptide: a new era in migraine therapy. Lancet 2019; 394: 1765–1774.

Olesen

Burstein

Ashina

, et al. Origin of pain in migraine: evidence for peripheral sensitisation. Lancet Neurol 2009; 8: 679–690.

Goadsby

Holland

Martins-Oliveira

, et al. Pathophysiology of migraine: a disorder of sensory processing. Physiol Rev 2017; 97: 553–622.

Brennan

Pietrobon

. A systems neuroscience approach to migraine. Neuron 2018; 97: 1004–1021.

Ashina

Moller Hansen

, et al. Migraine and the trigeminovascular system-40 years and counting. Lancet Neurol 2019; 18: 795–804.

Charles

Nwaobi

Goadsby

. Inflammation in migraine…or not…: a critical evaluation of the evidence. Headache 2021; 61: 1575–1578.

Liu

Chen

Wang

. Calcitonin gene-related peptide inhibits lipopolysaccharide-induced interleukin-12 release from mouse peritoneal macrophages, mediated by the cAMP pathway. Immunology 2000; 101: 61–67.

10.

Fernandez

Knopf

McGillis

. Calcitonin-gene related peptide (CGRP) inhibits interleukin-7-induced pre-B cell colony formation. J Leukoc Biol 2000; 67: 669–676.

11.

Mikami

Matsushita

Kato

, et al. Calcitonin gene-related peptide is an important regulator of cutaneous immunity: effect on dendritic cell and T cell functions. J Immunol 2011; 186: 6886–6893.

12.

Voedisch

Rochlitzer

Veres

, et al. Neuropeptides control the dynamic behavior of airway mucosal dendritic cells. PLoS One 2012; 7: e45951.

13.

Boudard

Bastide

. Inhibition of mouse T-cell proliferation by CGRP and VIP: effects of these neuropeptides on IL-2 production and cAMP synthesis. J Neurosci Res 1991; 29: 29–41.

14.

Gomes

Castro-Faria-Neto

Bozza

, et al. Calcitonin gene-related peptide inhibits local acute inflammation and protects mice against lethal endotoxemia. Shock 2005; 24: 590–594.

15.

Kroeger

Erhardt

Abt

, et al. The neuropeptide calcitonin gene-related peptide (CGRP) prevents inflammatory liver injury in mice. J Hepatol 2009; 51: 342–353.

16.

Xiong

Wang

Bai

, et al. Calcitonin gene-related peptide: a potential protective agent in cerebral ischemia-reperfusion injury. Front Neurosci 2023; 17: 1184766.

17.

Liu

Cai

, et al. Calcitonin gene-related peptide prevents blood-brain barrier injury and brain edema induced by focal cerebral ischemia reperfusion. Regul Pept 2011; 171: 19–25.

18.

Y-Z

Nayer

Singh

, et al. CGRP sensory neurons promote tissue healing via neutrophils and macrophages. Nature 2024; 628: 604–611.

19.

Pinho-Ribeiro

Deng

Neel

, et al. Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion. Nature 2023; 615: 472–481.

20.

Sardi

Zambusi

Finardi

, et al. Involvement of calcitonin gene-related peptide and receptor component protein in experimental autoimmune encephalomyelitis. J Neuroimmunol 2014; 271: 18–29.

21.

Pistolesi

Molli

De Cesaris

, et al. The anti-CGRP mAb Fremanezumab reverts the anti-inflammatory effects of CGRP in vitro but does not alter disease evolution in a mouse model of progressive multiple sclerosis. Eur J Pharmacol 2025; 995: 177415.

22.

Nagashima

Mahlakoiv

Shih

H-Y

, et al. Neuropeptide CGRP limits group 2 innate lymphoid cell responses and constrains type 2 inflammation. Immunity 2019; 51: 682–695.e6.

23.

Wallrapp

Burkett

Riesenfeld

, et al. Calcitonin gene-related peptide negatively regulates alarmin-driven type 2 innate lymphoid cell responses. Immunity 2019; 51: 709–723.e6.

24.

Ike

Kawano

Takahashi

, et al. Calcitonin gene-related peptide receptor antagonist suppresses allergic asthma responses via downregulation of group 2 innate lymphoid cells in mice. Int Immunopharmacol 2023; 122: 110608.

25.

Woldeamanuel

Rapoport

Cowan

. The place of corticosteroids in migraine attack management: a 65-year systematic review with pooled analysis and critical appraisal. Cephalalgia 2015; 35: 996–1024.

26.

Lee

Park

H-K

S-Y

, et al. Effect of prednisolone for the treatment of medication-overuse headache: a 3-month result from a multicenter REgistry for Load and management of mEdicAtion overuSE headache (RELEASE) study. Headache 2024; 64: 149–155.

27.

Peng

Burish

. Management of cluster headache: treatments and their mechanisms. Cephalalgia 2023; 43: 3331024231196808.

28.

Innes

, et al. Dexamethasone prevents relapse after emergency department treatment of acute migraine: a randomized clinical trial. CJEM 1999; 1: 26–33.

29.

Nair

Jacob

. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016; 7: 27–31.

30.

Belinskaia

Zurawski

Kumar Kaza

, et al. NGF Enhances CGRP release evoked by capsaicin from rat trigeminal neurons: differential inhibition by SNAP-25-cleaving proteases. Int J Mol Sci 2022; 23: 892.

31.

Viney

Schmidt

Gierasch

, et al. Regulation of the cell-specific calcitonin/calcitonin gene-related peptide enhancer by USF and the Foxa2 forkhead protein. J Biol Chem 2004; 279: 49948–49955.

32.

Buonvicino

Ranieri

Pittelli

, et al. SIRT1-dependent Restoration of NAD+ homeostasis after increased extracellular NAD+ exposure. J Biol Chem 2021; 297: 100855.

33.

Lapucci

Lulli

Amedei

, et al. Zeta-Crystallin is a bcl-2 mRNA binding protein involved in bcl-2 overexpression in T-cell acute lymphocytic leukemia. FASEB J 2010; 24: 1852–1865.

34.

Urru

Buonvicino

Pistolesi

, et al. Histone deacetylase inhibitors counteract CGRP signaling and pronociceptive sensitization in a rat model of medication overuse headache. J Pain 2022; 23: 1874–1884.

35.

Langford

Bailey

Chanda

, et al. Coding of facial expressions of pain in the laboratory mouse. Nat Methods 2010; 7: 447–449.

36.

Benbow

Ekbatan

Wang

GHY

, et al. Systemic administration of monosodium glutamate induces sexually dimorphic headache- and nausea-like behaviours in rats. Pain 2022; 163: 1838–1853.

37.

Russo

Clark

Durham

. Thyroid parafollicular cells. An accessible model for the study of serotonergic neurons. Mol Neurobiol 1996; 13: 257–276.

38.

Beta

RAA

Arsenopoulou

Kanoura

, et al. Core clock regulators in dexamethasone-treated HEK 293T cells at 4h intervals. BMC Res Notes 2022; 15: 23.

39.

Lou

Murtola

Tuohimaa

. Regulation of aromatase and 5alpha-reductase by 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3), dexamethasone and progesterone in prostate cancer cells. J Steroid Biochem Mol Biol 2005; 94: 151–157.

40.

Lanigan

Russo

. Binding of upstream stimulatory factor and a cell-specific activator to the calcitonin/calcitonin gene-related peptide enhancer. J Biol Chem 1997; 272: 18316–18324.

41.

Buonvicino

Urru

Muzzi

, et al. Trigeminal ganglion transcriptome analysis in 2 rat models of medication-overuse headache reveals coherent and widespread induction of pronociceptive gene expression patterns. Pain 2018; 159: 1980–1988.

42.

Ing

. Steroid hormones regulate gene expression posttranscriptionally by altering the stabilities of messenger RNAs. Biol Reprod 2005; 72: 1290–1296.

43.

Diener

H-C

Dodick

Evers

, et al. Pathophysiology, prevention, and treatment of medication overuse headache. Lancet Neurol 2019; 18: 891–902.

44.

De Logu

Landini

Janal

, et al. Migraine-provoking substances evoke periorbital allodynia in mice. J Headache Pain 2019; 20: 18.

45.

Wei

Kim

McKemy

. Transient receptor potential melastatin 8 is required for nitroglycerin- and calcitonin gene-related peptide-induced migraine-like pain behaviors in mice. Pain 2022; 163: 2380–2389.

46.

Russo

Nelson

Roos

, et al. Differential regulation of the coexpressed calcitonin/alpha-CGRP and beta-CGRP neuroendocrine genes. J Biol Chem 1988; 263: 5–8.

47.

Tverberg

Russo

. Cell-specific glucocorticoid repression of calcitonin/calcitonin gene-related peptide transcription. Localization to an 18-base pair basal enhancer element. J Biol Chem 1992; 267: 17567–17573.

48.

Levy

Burstein

Strassman

. Calcitonin gene-related peptide does not excite or sensitize meningeal nociceptors: implications for the pathophysiology of migraine. Ann Neurol 2005; 58: 698–705.

49.

Zhang

Winborn

de Prado

, et al. Sensitization of calcitonin gene-related peptide receptors by receptor activity-modifying protein-1 in the trigeminal ganglion. J Neurosci 2007; 27: 2693–2703.

50.

Souza

Muthuraman

, et al. Glucocorticoid signaling mediates stress-induced migraine-like behaviors in a preclinical mouse model. Cephalalgia 2024; 44: 3331024241277941.

51.

Neeb

Anders

Euskirchen

, et al. Corticosteroids alter CGRP and melatonin release in cluster headache episodes. Cephalalgia 2015; 35: 317–326.