RAMP1-dependent hormonal regulation of CGRP and its receptor in the trigeminal ganglion

Abstract

Objectives: Menstrual migraine (MM) is a debilitating neurological disorder triggered by fluctuations in ovarian hormones, particularly estrogen. While the calcitonin gene-related peptide (CGRP) system is central to migraine pathophysiology, the molecular mechanisms linking hormonal changes to CGRP signaling remain unclear. This study investigates how sex hormones regulate CGRP-related gene expression in the trigeminal ganglion (TG), with a focus on the receptor activity-modifying protein 1 (RAMP1), using both wild-type (WT) and Ramp1 knockout (KO) mice.

Methods: We analyzed gene expression in the TG across the estrous cycle and following systemic estrogen or progesterone administration. Expression of Ramp1, Calca (encoding CGRPα), Calcrl (encoding CRLR), estrogen receptors, and related genes was assessed by RT-qPCR. WT and Ramp1 KO mice of both sexes were used to explore hormone-dependent gene regulation and RAMP1’s functional role.

Results: Ramp1 expression varied across the estrous cycle, peaking in proestrus and declining in diestrus, inversely correlated with Calca. Calcrl levels remained unchanged. Ramp1 expression correlated significantly with Esr2 (encoding ERβ), suggesting estrogen receptor–mediated regulation. Estrogen treatment upregulated Ramp1 in both sexes; Calca was downregulated in females but upregulated in males. Progesterone had more modest effects, primarily altering Ramp3 expression. In Ramp1 KO mice, the cyclical variation of Calca, Ramp2, and Ramp3 seen in WT mice was absent, and basal Calca expression was elevated in males, indicating that RAMP1 is essential for hormonal regulation of the CGRP system. Additionally, our findings support a role for estrogen-driven epigenetic mechanisms, such as DNA methylation, in the long-term regulation of Ramp1.

Conclusion: This study highlights RAMP1 as a key mediator linking hormonal fluctuations to CGRP signaling in the TG. Hormone-dependent gene expression changes were sex-specific and disrupted in Ramp1 KO mice, supporting its role in migraine susceptibility. These findings provide mechanistic insight into hormonal migraine and suggest that both acute hormone signaling and long-term epigenetic regulation shape individual sensitivity to CGRP-based therapies.

Supplementary Information: The online version contains supplementary material available at 10.1186/s10194-025-02071-7.

Keywords: Calca; Migraine; Ramp1; Sex hormones; Trigeminal ganglion.

Fig. 1

Gene expression of Calca and its receptor (A) Schematic representation of fluctuation in circulating estradiol and progesterone across the estrous cycle– Fig. 1A was created by Biorender and modified from Pestana et al. [18]. Endogenous expression level of (B) Calca, (C) Ramp1, and (D) Calcrl in TG during the estrous cycle (proestrus, estrus, and diestrus) in female WT mice. Data were obtained by RT-qPCR and are shown as mean fold change ± SEM compared to proestrus. * p-value < 0.05, ** p-value < 0.01, ***p-value < 0.001; one-way ANOVA with Tukey’s post hoc test. N.s = not significant. E Correlation of expression levels of Ramp1 and Esr2 across nine different organs. r = 0.40; p-value = 0.001; simple linear regression analysis

Fig. 2

Gene expression of Calca and its receptor in WT and Ramp1 KO mice. Endogenous expression level of (A) Ramp1, (B) Calca, (C) Calcrl, (D) Ramp2, (E) Ramp3, (F) Adm, and (G) Esr2 in TG from female WT and Ramp1 KO mice during the estrous cycle (proestrus, estrus, and diestrus). Data were obtained by RT-qPCR and are shown as mean fold change ± SEM compared to proestrus. * p-value < 0.05, ** p-value < 0.01, ***p-value < 0.001; one-way ANOVA with Tukey’s post hoc test. N.s = not significant

Fig. 3

Exogenous administration of 17-β-estradiol in female and male WT and Ramp1 KO mice. Body weight changes after administration of 17-β-estradiol in (A) female and (B) male WT and Ramp1 KO mice. * p-value < 0.05, ** p-value < 0.01, ***p-value < 0.001; one-way ANOVA with Tukey’s post hoc test. Gene expression of (C) Ramp1, (D) Calca, (E) Calcrl, (F) Ramp2, (G) Ramp3, (H) Esr1, and (I) Esr2 in TG from vehicle or 17-β-estradiol treated female and male WT and RAMP1 KO mice. Data were obtained by RT-qPCR and are shown as mean fold change ± SEM compared to vehicle. * p-value < 0.05, ** p-value < 0.01; Mann-Whitney U test. N.s = not significant

Fig. 4

Exogenous administration of progesterone in female and male WT and RAMP1 KO mice. Gene expression of (A) Ramp1, (B) Calca, (C) Calcrl, (D) Ramp2, (E) Ramp3, and (F) Adm in TG from vehicle or progesterone treated female and male WT and RAMP1 KO mice. Data were obtained by RT-qPCR and are shown as mean fold change ± SEM compared to vehicle. * p-value < 0.05, ** p-value < 0.01; Mann-Whitney U test

Fig. 5

RAMP1 and CRL protein level in TG of WT and Ramp1 KO male and female mice. Differences in RAMP1 immunoreactivity were observed between groups differing in sex and genotype. A RAMP1 protein was detected in neuron somas (arrowhead) in male WT TG. However, no clear distinction between larger and smaller neurons, as previously reported in rat TG [22], was observed. Patchy and inconsistent RAMP1 staining was also seen in Aδ-fibres (arrow). B In male RAMP1 KO TG, no specific RAMP1 staining was observed, and the pattern did not differ from the negative control, apart from some nonspecific binding to neuronal somas (arrowhead). C In female WT TG, RAMP1 immunoreactivity was also observed in neuron somas (arrowhead), again without a clear size-based distribution. Fibre staining (arrow) appeared more pronounced than in male WT mice. D In female Ramp1 KO TG, RAMP1 staining did not differ from the negative control. E–H CRLR immunoreactivity appeared consistent across groups, with no obvious differences related to sex or genotype. CRLR protein was predominantly observed in larger neuron somas (arrowheads) and in A-type fibres (arrows), likely Aδ-type, consistent with patterns previously reported in rat TG [22]. CRLR immunostaining of TG in (E) male WT, (F) male Ramp1 KO, (G) female WT, and (H) female Ramp1 KO

Fig. 6

Estrogen and DNA methylation regulate RAMP1 and CALCA expression in human neuronal cells and rat TG. Gene expression of (A) RAMP1 and (B) CALCA in human neuronal cells treated with vehicle, 17-β-estradiol (10 nM), DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC, 50 µM), or the combination of estradiol and 5-aza-dC Gene expression of (C) Ramp1 and (D) Calca in TG organ cultures from male rats treated with vehicle, E2, 5-aza-dC, or the combination. Data were obtained by RT-qPCR and are shown as mean fold change ± SEM compared to vehicle. *p-value < 0.05, **p-value < 0.01; Mann-Whitney U test. N.s = not significant