Src Family Kinases Facilitate the Crosstalk between CGRP and Cytokines in Sensitizing Trigeminal Ganglion via Transmitting CGRP Receptor/PKA Pathway

Abstract

The communication between calcitonin gene-related peptide (CGRP) and cytokines plays a prominent role in maintaining trigeminal ganglion (TG) and trigeminovascular sensitization. However, the underlying regulatory mechanism is elusive. In this study, we explored the hypothesis that Src family kinases (SFKs) activity facilitates the crosstalk between CGRP and cytokines in sensitizing TG. Mouse TG tissue culture was performed to study CGRP release by enzyme-linked immunosorbent assay, cytokine release by multiplex assay, cytokine gene expression by quantitative polymerase chain reaction, and phosphorylated SFKs level by western blot. The results demonstrated that a SFKs activator, pYEEI (YGRKKRRQRRREPQY(PO3H2)EEIPIYL) alone, did not alter CGRP release or the inflammatory cytokine interleukin-1β (IL-1β) gene expression in the mouse TG. In contrast, a SFKs inhibitor, saracatinib, restored CGRP release, the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release, and IL-1β, CCL2 gene expression when the mouse TG was pre-sensitized with hydrogen peroxide and CGRP respectively. Consistently with this, the phosphorylated SFKs level was increased by both hydrogen peroxide and CGRP in the mouse TG, which was reduced by a CGRP receptor inhibitor BIBN4096 and a protein kinase A (PKA) inhibitor PKI (14-22) Amide. The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization.

Keywords: C-C motif ligand 2; C-X-C motif ligand 1; Src family kinases; calcitonin gene-related peptide; interleukin-1β; migraine; protein kinase A; trigeminal ganglion.

Figure 1

pYEEI alone did not alter CGRP release and IL-1β gene expression in the mouse TG. (A,B) Effects of 1 mM pYEEI or 1 mM YEEI (n = 8 per group) on CGRP release (pg/mL) and IL-1β mRNA level at 20 min post treatment. IL-1β mRNA level was present in the fold change relative to the geometric mean of β-actin and PPIA mRNA levels. Two-tailed unpaired t-test was used for the comparison in CGRP release and IL-1β mRNA level between the YEEI group and the pYEEI group.

Figure 2

Saracatinib reduced CGRP release induced by H₂O₂ in the mouse TG. Effects of Kreb’s (n = 8); 1 mM H₂O₂ (n = 8); 1.5 μM (n = 7), 4 μM (n = 7), or 10 μM (n = 7) saracatinib in the presence of 1 mM H₂O₂ at 20 min post treatment on CGRP release (pg/mL). Abbreviations: saracatinib (SRCT). Two-tailed unpaired t-test was used for the comparison in CGRP release between the H₂O₂ group and either the Kreb’s group or the saracatinib in the presence of H₂O₂ group. Significant differences were labeled as * p < 0.05.

Figure 3

Saracatinib reduced IL-1β, CCL2, and CXCL1 release induced by CGRP in the mouse TG. (A–D) Effects of Kreb’s, 3 µM CGRP, or 1.5 μM saracatinib in the presence of 3 μM CGRP (n = 8 per group) at 20 min post treatment on IL-1β, CCL2, CXCL1, and IL-10 release (pg/mL). Abbreviations: saracatinib (SRCT). Two-tailed unpaired t-test was used for the comparison in IL-1β, CCL2, CXCL1, and IL-10 release between the CGRP group and either the Kreb’s group or the saracatinib in the presence of CGRP group. Significant differences were labeled as * p < 0.05 or ** p < 0.01.

Figure 4

Saracatinib reduced IL-1β, CCL2 and CCL1 gene expression induced by CGRP in the mouse TG. (A–C) Effects of Kreb’s, 3 µM CGRP, 1.5 μM, or 4 μM saracatinib in the presence of 3 μM CGRP (n = 8 per group) at 60 min post treatment on mRNA level of IL-1β, CCL2 and CCL1 in respective order. IL-1β, CCL2, and CCL1 mRNA levels were present in the fold change relative to the geometric mean of β-actin and PPIA mRNA levels. Abbreviations: saracatinib (SRCT). Two-tailed unpaired Mann–Whitney test was used for the comparison in IL-1β and CCL2 mRNA levels between the CGRP group and either the Kreb’s group or the saracatinib in the presence of CGRP group. Significant differences were labeled as * p < 0.05 or *** p < 0.001.

Figure 5

The protein level of phosphorylated SFKs at Y416 was increased by H₂O₂ in the mouse TG. (A) The representative Western blot bands of phosphorylated SFKs at Y416, SFKs, and β-actin subjected to the treatment with Kreb’s, 1 mM H₂O₂, or 10 µM BIBN4096 in the presence of 1 mM H₂O₂. (B–D) Effects of Kreb’s, 1 mM H₂O₂, or 10 µM BIBN4096 in the presence of 1 mM H₂O₂ (n = 7 per group) at 20 min post treatment on the protein levels of phosphorylated SFKs at Y416 and SFKs relative to that of β-actin and on the protein level of phosphorylated SFKs at Y416 relative to that of SFKs, all of which were presented in the absolute ratio. Abbreviations: BIBN4086 (BIBN), phosphorylated SFKs at Y416 (pSFKs). Two-tailed unpaired t-test was used for the comparison in the protein level of phosphorylated SFKs at Y416 between the H₂O₂ group and either the Kreb’s group or the BIBN4096 in the presence of 1 mM H₂O₂ group. Significant differences were labeled as * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001. Original western blot images for the representative images in Figure 5 was shown in Supplementary Figure S2.

Figure 6

The protein level of phosphorylated SFKs at Y416 was increased by CGRP, which was reduced by both PKI (14-22) Amide and BIBN4096 in the mouse TG. (A) The representative Western blot bands of phosphorylated SFKs at Y416, SFKs, and β-actin subjected to the treatment with Kreb’s, 3 µM CGRP, 30 µM PKI (14-22) Amide or 10 µM BIBN4096 in the presence of 3 µM CGRP for 20 min. (B–D) Effects of Kreb’s (n = 9), 3 μM CGRP (n = 9), 30 μM PKI (14-22) Amide (n = 8) or 3 µM BIBN4096 (n = 8) in the presence of 3 μM CGRP on the protein levels of phosphorylated SFKs at Y416 and SFKs relative to that of β-actin and on the protein level of phosphorylated SFKs at Y416 relative to that of SFKs, all of which were presented in the absolute ratio. Abbreviations: PKI (14-22) Amide (PKI), BIBN4086 (BIBN), phosphorylated SFKs at Y416 (pSFKs). Two-tailed unpaired t-test was used for the comparison in the protein level of phosphorylated SFKs at Y416 between the CGRP group and either the Kreb’s group, the PKI (14-22) Amide, or the BIBN4096 in the presence of CGRP groups. Significant differences were labeled as * p < 0.05, ** p < 0.01, or *** p < 0.001. Original western blot images for the representative images in Figure 6 were shown in Supplementary Figures S3 and S4.

Figure 7

The levels of phosphorylated SFKs at Y416 and released cytokines induced by CGRP were correlated in the mouse TG. Correlation analysis between the levels of phosphorylated SFKs at Y416 and IL-1β (A), CCL2 (B), CXCL1 (C) release in the mouse TG treated by Kreb’s and 3 µM CGRP (n = 8 per group) for 20 minutes.

Figure 8

SFKs co-localized with both CGRP and RAMP1 in the mouse TG. (A) The representative images of double staining with the anti-CGRP antibody and anti-SFKs antibody in the TG. (B) The representative images of double staining with the anti-RAMP1 antibody and anti-SFKs antibody in the TG. CGRP and RAMP1 were stained with the anti-CGRP antibody and anti-RAMP1 antibody, respectively, and are shown in red; SFKs were stained with the anti-SFKs antibody and are shown in green; nucleus was stained with DAPI and is shown in blue. Co-localization of SFKs and CGRP or RAMP1 is shown in yellow and is indicated by the white arrows.

Figure 9

Model of SFKs activity facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor signaling to potentiate TG sensitization. SFKs are activated in response to ROS to induce CGRP release in small to medium neurons; released CGRP binds to CGRP receptor (dotted line with arrow) to activate SFKs in large neurons and satellite glial cells, which causes IL-1β, CCL2, CXCL1 release and IL-1β, CCL2 gene expression, thus leading to TG sensitization.