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

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.


Introduction
Migraine is a recurrent primary headache disorder that afflicts approximately 15% of the population worldwide [1]. A key mechanism by which nearly all migraine triggers induce migraine attacks is the activation and sensitization of the trigeminovascular pathway [2][3][4]. As an important peripheral component of the trigeminovascular pathway, trigeminal ganglion (TG) contains the cell bodies of meningeal nociceptors, the activation of which initiate trigeminovascular activation [5][6][7]. Active signaling mediated mainly by neuropeptides and inflammatory mediators occurs within the TG, among which calcitonin gene-related peptide (CGRP), the key drug target of migraine prevention and therapy, is a key player [8]. In the TG, released CGRP binds to CGRP receptor to facilitate neuronal excitability [9][10][11] and neuroinflammation, including elevated release and expression of inflammatory cytokines [11][12][13][14]. Importantly, cytokines can signal back to neurons, which promotes CGRP synthesis and release [15,16], thereby inducing a positive feedback loop of sensitization. Thus, the communication between CGRP and cytokines plays a prominent role in maintaining TG activation and sensitization as well as trigeminovascular sensitization [17][18][19], although the underlying regulatory mechanism is elusive.
Src family kinases (SFKs) activity has been previously found to mediate CGRP release in dorsal root ganglion neurons [20] and TG [21]. SFKs activity also mediates inflammatory cytokine release and expression in primary glial cells [22][23][24][25] and mediates interleukin-1 β (IL-1β) gene expression in the mouse TG [21]. Importantly, SFKs are known to play a key role in migraine pathogenesis. In an inflammatory soup-induced chronic migraine model, central inhibition of SFKs attenuates mechanical allodynia and synaptic plasticity [26]. In a genetic mouse migraine with aura model familial hemiplegic migraine type 2 (FHM2), deactivation of SFKs reduces the Ca 2+ sensitivity and contraction of the cerebral arteries, which contributes to vascular tone and brain perfusion abnormalities [27]. Similarly, systemic deactivation of SFKs reduces cortical spreading depression (CSD), a migraine with aura model, and CSD-induced cerebral cortical inflammatory cytokines interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) gene expression [28]. Taken together, it is likely that SFKs activity facilitates the communication between CGRP and cytokines to activate and sensitize TG, which requires clarification.
In the present study, we examined whether SFKs activity facilitates the crosstalk between CGRP release and cytokines release and gene expression to activate and sensitize the mouse TG. How SFKs activity mediates the communication between CGRP and cytokines in TG is also explored by investigating the involvement of CGRP receptor/protein kinase A (PKA) pathway.

Animals
A total of 143 adult male C57BL/6J mice (21.4 ± 0.17 g) were used and purchased from Shanghai SLAC Laboratory Animal Corporation Ltd. (Shanghai, China). All studies in this paper were carried out in male rodents so that the effect of hormonal fluctuation in females is minimized. Mice were housed in the Experimental Animal Centre of Soochow University for at least one week to be acclimated to the housing room before use. Animal procedures were approved by the Ethical Review Panels of Xi'an Jiaotong-Liverpool University (XJTLU) under the agreement with Soochow University and performed in accordance with relevant China national and provincial guidelines. For each experiment, randomization of experimental groups was performed to reduce bias. All animals used were randomly allocated to different experimental groups.

Mouse TG Tissue Culture
Isolated TG culture is a commonly used model to study TG molecular and neurophysiological properties. Signaling molecules produced in TG cell bodies are delivered to the peripheral and central terminals via axonal transport to give rise to sensory transduction and neurotransmission [6,[29][30][31]. Therefore, isolated TG culture is commonly used as a model of its peripheral or central endings to study meningeal nociceptors and trigeminal nociceptive transmission [17,19,32]. The method of TG tissue culture was established as reported previously [21]. Mice were sacrificed by rapid cervical dislocation. Both the left and right TG of each mouse were collected, and the merged TG were used for one individual experiment. The TG were recovered in 300 µL pre-oxygenated Kreb's solution (composition in mM: 126 NaCl, 2.5 KCl, 2.4 CaCl 2 ·2H 2 O, 1.3 MgCl 2 ·6H 2 O, 18 NaHCO 3 , 1.2 NaH 2 PO 4 , 10 glucose; pH 7.4) for 30 min at 37 • C and then washed with pre-oxygenated Kreb's solution every 5 min for 30 min. Subsequently, the TG were incubated with each drug for 20 min or 1 h at 37 • C.
In order to explore how SFKs activity mediates the communication between CGRP and cytokines in TG, the signaling pathway that SFKs transmit during these processes is investigated, for which three series of experiments were designed. Series 4: to ensure that SFKs activity is increased by H 2 O 2 in the mouse TG in Series 2, the TG treated with Kreb's and 1 mM H 2 O 2 were collected to measure SFKs activity represented by the level of phosphorylated SFKs at Y416 using western blot in order to minimize animal use. How SFKs activity is enhanced by H 2 O 2 was then investigated by examining whether inhibition of CGRP receptor reduces H 2 O 2 -enhanced SFKs activity in cultured mouse TG. To inhibit CGRP receptor, a CGRP receptor inhibitor, BIBN4096 (4561, Tocris, Bristol, UK), was used. The cultured TG was treated with 10 µM BIBN4096 in the presence of 1 mM H 2 O 2 for 20 min. One additional group was designed: 1 mM H 2 O 2 + 10 µM BIBN4096 (n = 7). Series 5: to ensure that SFKs activity is increased by CGRP in the mouse TG in Series 3, the TG treated with Kreb's and 3 µM CGRP for 20 min were collected to measure the level of phosphorylated SFKs at Y416 using Western blot. Next, how SFKs activity is enhanced by CGRP was investigated by examining whether SFKs activity transmits CGRP receptor/PKA pathway as PKA is known to transmit signaling downstream CGRP [52][53][54] and activate SFKs in several models [36,55,56]. Specifically, whether inhibition of CGRP receptor and deactivation of PKA reduce CGRP-enhanced SFKs activity was examined in cultured mouse TG. To deactivate PKA, a PKA inhibitor, PKI (14-22) Amide (476485, Sigma-Aldrich, St. Louis, MO, USA), was used. The cultured TG was treated with 3 µM BIBN4096 [57] or 30 µM PKI (14-22) Amide [58] in the presence of 3 µM CGRP for 20 min. Two additional groups were designed: 3 µM CGRP + 3 µM BIBN4096, 3 µM CGRP + 30 µM PKI (14-22) Amide (n = 8 for each). Series 6: whether SFKs co-localize with CGRP or receptor activity modifying protein 1 (RAMP1), the unique and essential functional CGRP receptor subunit [59,60], was also examined in mouse TG using immunohistochemistry.

ELISA
After TG tissue culture, the level of CGRP released into the culture medium was measured using a mouse CGRP ELISA kit (CSB-EQ027706MO, CUSABIO, Houston, TX, USA). Briefly, 100 µL medium and each of 8 serially diluted standard solutions were added into an assay plate pre-coated with CGRP antibody, which was then incubated at 37 • C for 2 h. Next, after removing the remaining liquid in the wells, 100 µL 1 × biotin-conjugated antibody specific for CGRP was added to each well followed by incubating at 37 • C for 1 h. The wells were then aspirated and washed, after which each well was added with 100 µL 1 × avidin conjugated horseradish peroxidase (HRP) and incubated at 37 • C for 1 h. Following further wash to remove any unbound substances, each well was added with 90 µL TMB substrate and incubated at 37 • C for 30 min in the dark. The reaction was stopped by adding 50 µL stop solution to each well and the OD of the wells was read at 450 nm, 540 nm, and 570 nm using a colorimetric microplate reader (BioTek, Winooski, VT, USA). The mean reading at 540 nm and 570 nm were subtracted from that at 450 nm, which corrected for optical imperfections. A standard curve relating the OD values to the concentration of CGRP (pg/mL) in the standard solutions was plotted and an equation of the curve was obtained. The OD values of the media were used to calculate their CGRP concentration (pg/mL) using the equation.

Multiplex Immunoassay
A multi-analyte flow assay kit (740621, Biolegend, San Diego, CA, USA) was used to detect the release of 12 pro-inflammatory cytokines, CCL2, CCL5, CXCL1, CXCL10, GM-CSF, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-12, TNFα, and one anti-inflammatory cytokine-IL-10-into the TG culture medium. First, 25 µL medium and each of 8 serially diluted standard solutions were added into an assay plate followed by adding 25 µL assay buffer and 25 µL mixed beads, which was then shaken at 500 rpm at room temperature for 2 h in the dark. After aspirating and washing the plate, 25 µL detection antibodies was added into the plate followed by shaking at 500 rpm at room temperature for 1 h. Next, 25 µL streptavidin-phycoerythrin (SA-PE) was added, and the plate was shaken at 500 rpm at room temperature for 30 min. After aspirating and washing the plate again, the beads in the plate were resuspended, and the plate was read on CytoFLEX S Flow Cytometer (C01161, Beckman Coulter, Brea, CA, USA). Each of the mixed beads was conjugated with a type of allophycocyanin (APC) fluorescence and an antibody specific to one of the 13 cytokines so that each cytokine in the medium was captured by its specific bead. The APC fluorescence conjugated to each bead had a differing level, which could be recognized by the flow cytometer at 660 nm to distinguish among different beads and identify the corresponding cytokine of each bead. The SA-PE bound to the detection antibody provided fluorescent signal in proportion to the amount of a certain cytokine bound to each bead, which was read as PE signal fluorescence intensity by the flow cytometer at 585 nm. Using LEGENDplexTM Data Analysis Software 8.0 (Biolegend, San Diego, CA, USA), a standard curve relating the PE signal fluorescence intensities to the concentration of each of the 13 cytokines (pg/mL) in the standard solutions was plotted and an equation of the curve was obtained. The PE signal fluorescence intensities of the media were used to calculate the concentration (pg/mL) of each of the 13 cytokines using the equation.

qPCR
After 60 min of TG tissue culture, total RNA of mouse TG was extracted using TRIZOL reagent (T9424 Sigma-Aldrich, St. Louis, MO, USA) and was reverse transcribed to cDNA by a GoScript Reverse Transcription System (A5001 Promega, Madison, WI, USA). The mRNA levels of specific genes were detected by qPCR using GoTaq qPCR Master Mix (A6002, Promega, Madison, WI, USA). The qPCR reaction was performed in QuantStudio 5 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) under the following thermal cycling conditions: 95 • C for 2 min, 95 • C for 15 s, 60 • C for 1 min, and 60-95 • C for 1 min. The mRNA level of each target gene was presented as relative fold change by normalizing the individual mRNA level of the gene to the geometric mean of the mRNA levels of two housekeeping genes, β-actin, and peptidylprolyl isomerase A (PPIA). Primers specific to the target genes were shown as follows: IL-1β forward 5 ACTACAGGCTCCGAGATGAACAAC3 , reverse 5 CCCAAGGCCACAGGTATTTT3 ; CCL2 forward 5 CACTCACCTGCTGCTACTCA3 , reverse 5 GCTTGGTGACAAAAACTACAGC3 ; ACTB forward 5 CTGTCCACCTTCCAGCAGAT3 , reverse 5 CGCAGCTCAGTAACAGTCCG3 ; PPIA forward, 5 TTGCTGCAGACATGGTCAAC3 , reverse 5 TGTCTGCAAACAGCTCGAAG3 .

Western Blot
Total protein of mouse TG was extracted using sodium dodecyl sulfate (SDS, 74255, Sigma-Aldrich, St. Louis, MO, USA), as described previously [21]. The concentration of the extracted protein was measured using Bicinchoninic Acid Protein Assay Kit (P0010, Beyotime, Shanghai, China). The protein levels of phosphorylated SFKs at Y416 and SFKs were analyzed by Western blot. Except for that, the protein level of β-actin was also analyzed, which was used as an internal control to calculate the relative expression levels of phosphorylated SFKs at Y416 and SFKs. Protein samples were denatured with SDS polyacrylamide (SDS-PAGE) sample loading buffer (P0015, Beyotime, Shanghai, China) at 100 • C for 5 min. The protein samples were separated on a 10% SDS-PAGE gel followed by transfer onto nitrocellulose membranes (66485, Pall, Pensacola, FL, USA). The membranes were incubated in 5% milk at room temperature for 1 h, followed by incubation with anti-phospho-Y416 SFKs antibody (1:200, 6943, CST, Beverly, MA, USA) and anti-β-actin antibody (1:2000, 4970, CST, Beverly, MA, USA) at 4 • C overnight. Subsequently, the membranes were incubated with IRDye 680RD donkey anti-rabbit secondary antibody (1:5000, 925-68073, LI-COR, Lincoln, NE, USA) for 1 h in the dark. Odyssey Near-Infrared Fluorescent Imaging System (LI-COR, Lincoln, NE, USA) was used to detect the protein levels of phosphorylated SFKs at Y416 and β-actin on the membranes by scanning fluorescent signals at 700 nm. Next, the anti-phospho-Y416 SFK antibody on the membranes was stripped off using 0.2 M NaOH (134070010, Acros Organics, Geel, Belgium) for 15 min. After incubating in 5% milk, the membranes were incubated with anti-SFK antibody (1:1000, 2109, CST) at 4 • C overnight followed by incubating with the anti-rabbit secondary antibody and imaging to detect the protein level of SFKs. The mean gray value of protein band intensity was quantified using Image Studio Lite 5.0 (LI-COR, Lincoln, NE, USA). The level of phosphorylated SFKs at Y416 was presented as absolute ratio in the band intensities between phosphorylated SFKs at Y416 and β-actin, phosphorylated SFKs at Y416 and SFKs, and SFKs and β-actin.

Statistical Analysis
For quantitative studies, all raw data generated in experiments were statistically analyzed using GraphPad Prism 7.0 (San Diego, CA, USA) for testing if each dataset followed normal distribution and if significant difference existed between the data of two comparable experimental groups. In order to choose a proper test for analyzing significant statistical difference between two groups, a Shapiro-Wilk test was performed for all the datasets to determine if they followed normal distribution. If the normality test was passed, the data were presented as mean ± standard error of the mean, and the significance of intergroup statistical difference was analyzed by two-tailed unpaired t-test; if not, the data were presented as median (interquartile range), and the significance of intergroup statistical difference was analyzed by two-tailed Mann-Whitney test. Four types of significant intergroup statistical difference were used: * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001. Detailed data presentation and statistical analysis for each quantitative study was described in the respective figure legend.

pYEEI Alone Did Not Increase CGRP Release and IL-1β Gene Expression in the Mouse TG
We examined whether activation of SFKs increases CGRP release and IL-1β gene expression in the TG. When treated the TG with 1 mM pYEEI, the SFKs activator, the CGRP level was 32.8 ± 1 pg/mL, which was not significantly different from the CGRP level at 35.1 ± 1.9 pg/mL in the YEEI group (n = 8 per group, Figure 1A). Similarly, 1 mM pYEEI did not affect the IL-1β mRNA level either, which was 1 ± 0.1 (vs. 1 ± 0.1 in the YEEI group, Figure 1B) (n = 8 per group). 35.1 ± 1.9 pg/mL in the YEEI group (n = 8 per group, Figure 1A). Similarly, 1 mM pYEEI did not affect the IL-1β mRNA level either, which was 1 ± 0.1 (vs. 1 ± 0.1 in the YEEI group, Figure 1B) (n = 8 per group).

Saracatinib Reduced CGRP Release Induced by H2O2 in the Mouse TG
This section determined whether deactivation of SFKs reduces CGRP release induced by ROS in the TG. H2O2 at 1 mM increased the level of CGRP in the TG culture medium to 28.4 ± 4.4 pg/mL in comparison with that at 13.7 ± 2.2 pg/mL in the Kreb's group (n = 8 per group, p = 0.0126, Figure 2). In the presence of 1 mM H2O2, 1.5 µ M saracatinib (n = 7), the SFKs inhibitor, slightly reduced the CGRP level from the TG to 19.8 ± 4 pg/mL, which was not significantly different from that in the H2O2 group ( Figure 2). When saracatinib was applied at 4 µ M (n = 7), a significant reduction in the CGRP level to 15.9 ± 1 pg/mL was seen compared to that in the H2O2 group (p = 0.024, Figure 2). Saracatinib at 10 µ M (n = 7) also significantly reduced the level of CGRP to 13.4 ± 0.9 compared to that in the H2O2 group (p = 0.0105, Figure 2). These data supported a concentration-response effect of saracatinib on CGRP release from the TG primed by H2O2.

Saracatinib Reduced CGRP Release Induced by H 2 O 2 in the Mouse TG
This section determined whether deactivation of SFKs reduces CGRP release induced by ROS in the TG. H 2 O 2 at 1 mM increased the level of CGRP in the TG culture medium to 28.4 ± 4.4 pg/mL in comparison with that at 13.7 ± 2.2 pg/mL in the Kreb's group (n = 8 per group, p = 0.0126, Figure 2). In the presence of 1 mM H 2 O 2 , 1.5 µM saracatinib (n = 7), the SFKs inhibitor, slightly reduced the CGRP level from the TG to 19.8 ± 4 pg/mL, which was not significantly different from that in the H 2 O 2 group (Figure 2). When saracatinib was applied at 4 µM (n = 7), a significant reduction in the CGRP level to 15.9 ± 1 pg/mL was seen compared to that in the H 2 O 2 group (p = 0.024, Figure 2). Saracatinib at 10 µM (n = 7) also significantly reduced the level of CGRP to 13.4 ± 0.9 compared to that in the H 2 O 2 group (p = 0.0105, Figure 2). These data supported a concentration-response effect of saracatinib on CGRP release from the TG primed by H 2 O 2 .

Saracatinib Reduced CGRP Release Induced by H2O2 in the Mouse TG
This section determined whether deactivation of SFKs reduces CGRP release induced by ROS in the TG. H2O2 at 1 mM increased the level of CGRP in the TG culture medium to 28.4 ± 4.4 pg/mL in comparison with that at 13.7 ± 2.2 pg/mL in the Kreb's group (n = 8 per group, p = 0.0126, Figure 2). In the presence of 1 mM H2O2, 1.5 µ M saracatinib (n = 7), the SFKs inhibitor, slightly reduced the CGRP level from the TG to 19.8 ± 4 pg/mL, which was not significantly different from that in the H2O2 group ( Figure 2). When saracatinib was applied at 4 µ M (n = 7), a significant reduction in the CGRP level to 15.9 ± 1 pg/mL was seen compared to that in the H2O2 group (p = 0.024, Figure 2). Saracatinib at 10 µ M (n = 7) also significantly reduced the level of CGRP to 13.4 ± 0.9 compared to that in the H2O2 group (p = 0.0105, Figure 2). These data supported a concentration-response effect of saracatinib on CGRP release from the TG primed by H2O2.  We addressed whether deactivation of SFKs reduces inflammatory cytokine release induced by CGRP in the TG. Among the 12 pro-inflammatory cytokines (CCL2, CCL5, CXCL1, CXCL10, GM-CSF, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-12, TNFα) and one antiinflammatory cytokine (IL-10), 3 µM CGRP promoted the levels of IL-1β to 4.6 ± 0.4 pg/mL (vs. 2.9 ± 0.5 pg/mL in the Kreb's group, p = 0.0288), CCL2 to 8.9 ± 1.7 pg/mL (vs. 3.9 ± 0.7 pg/mL in the Kreb's group, p = 0.0232), and CXCL1 to 2.6 ± 0.7 pg/mL (vs. 0.9 ± 0.2 pg/mL in the Kreb's group, p = 0.0303) in the TG culture medium (n = 8 per group, Figure 3A-C). Differently, 3 µM CGRP decreased the level of IL-10 to 0.2 ± 0.1 pg/mL (vs. 3.7 ± 0.9 pg/mL in the Kreb's group, p = 0.0017, n = 8 per group, Figure 3D) in the medium. It is noted that 3 µM CGRP did not alter the levels of the other 9 cytokines (Supplementary Figure S1). As expected, in the presence of 3 µM CGRP, 1.5 µM saracatinib decreased the levels of IL-1β to 1.9 ± 0.5 pg/mL (p = 0.001), CCL2 to 2.5 ± 0.5 pg/mL (p = 0.0069), and CXCL1 to 1 ± 0.3 pg/mL (p = 0.0476) when compared to the respective data in the CGRP group (n = 8 per group, Figure 3A-C). However, 1.5 µM saracatinib did not significantly affect the reduced level of IL-10 elicited by 3 µM CGRP, which was 1.2 ± 0.5 (n = 8 per group, Figure 3D). 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.

Saracatinib Reduced IL-1β and CCL2 Gene Expression Induced by CGRP in the Mouse TG
Our data demonstrated that saracatinib reduced IL-1β, CCL2, and CXCL1 release promoted by CGRP in the TG ( Figure 3A-C). All these proteins are associated with pain hypersensitivity and migraine [61][62][63][64][65]. We therefore further investigated whether SFKs activity promotes cytokines production machinery by examining the effect of the SFKs inhibitor saracatinib on their CGRP-induced gene expression. CGRP at 3 µM increased the mRNA levels of IL-1β to 2.1 (2.1) (vs. 1.1 (0.9) in the Kreb's group, p = 0.0003) and CCL2 to 1.5 (4.5) (vs. 0.9 (1.2) in the Kreb's group, p = 0.0207) in the TG (n = 8 per group, Figure 4A,B). In contrast, 3 µM CGRP did not affect the CXCL1 mRNA level, which was 1.1 ± 0.2 compared to that at 1 ± 0.1 in the Kreb's group (n = 8 per group, Figure 4C). When 1.5 µM saracatinib was applied in the presence of 3 µM CGRP, the IL-1β mRNA level was 4 (1.2), which was insignificantly different from that in the CGRP group ( Figure 4A). Saracatinib at 4 µM, however, resulted in a pronounced reduction in the mRNA levels of IL-1β to 1 (1.1) (p = 0.0148) and CCL2 to 0.8 (0.3) (p = 0.0002) in comparison with that in the CGRP group (n = 8 per group, Figure 4A,B). 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.

Saracatinib Reduced IL-1β and CCL2 Gene Expression Induced by CGRP in the Mouse TG
Our data demonstrated that saracatinib reduced IL-1β, CCL2, and CXCL1 release promoted by CGRP in the TG ( Figure 3A-C). All these proteins are associated with pain hypersensitivity and migraine [61][62][63][64][65]. We therefore further investigated whether SFKs activity promotes cytokines production machinery by examining the effect of the SFKs inhibitor saracatinib on their CGRP-induced gene expression. CGRP at 3 µ M increased the mRNA levels of IL-1β to 2.1 (2.1) (vs. 1.1 (0.9) in the Kreb's group, p = 0.0003) and CCL2 to 1.5 (4.5) (vs. 0.9 (1.2) in the Kreb's group, p = 0.0207) in the TG (n = 8 per group, Figure  4A,B). In contrast, 3 µ M CGRP did not affect the CXCL1 mRNA level, which was 1.1 ± 0.2 compared to that at 1 ± 0.1 in the Kreb's group (n = 8 per group, Figure 4C). When 1.5 µ M saracatinib was applied in the presence of 3 µ M CGRP, the IL-1β mRNA level was 4 (1.2), which was insignificantly different from that in the CGRP group ( Figure 4A). Saracatinib at 4 µ M, however, resulted in a pronounced reduction in the mRNA levels of IL-1β to 1 (1.1) (p = 0.0148) and CCL2 to 0.8 (0.3) (p = 0.0002) in comparison with that in the CGRP group (n = 8 per group, Figure 4A,B).  . 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.

The Protein Level of Phosphorylated SFKs at Y416 Was Increased by H 2 O 2 , Which Was Reduced by BIBN4096 in the Mouse TG
We examined whether SFKs activity is increased by H 2 O 2 and whether such elevation can be reversed by inhibition of CGRP receptor using BIBN4096. When the protein level of phosphorylated SFKs at Y416 was normalized to that of β-actin, exposure to 1 mM H 2 O 2 increased the protein level of phosphorylated SFKs at Y416 to 0.48 ± 0.06 compared to that at 0.13 ± 0.02 in the Kreb's group (n = 7 per group, p = 0.0010, Figure 5B). BIBN4096 at 10 µM reduced the H 2 O 2 -enhanced protein level of phosphorylated SFKs at Y416 to 0.24 ± 0.04 compared to that in the H 2 O 2 group (n = 7 per group, p = 0.0082, Figure 5B). In contrast, the protein level of SFKs was unchanged among the three groups ( Figure 5C), suggesting that the protein level of SFKs was insensitive to H 2 O 2 or BIBN4096. When the protein level of phosphorylated SFKs at Y416 was normalized to that of SFKs, consistently, the protein level of phosphorylated SFKs at Y416 was increased to 0.32 ± 0.03 by H 2 O 2 in comparison with that at 0.07 ± 0.01 in the Kreb's group (n = 7 per group, p < 0.0001, Figure 5D). BIBN4096 reduced H 2 O 2 -enhanced protein level of phosphorylated SFKs at Y416 to 0.19 ± 0.03 in comparison with that in the H 2 O 2 group (n = 7 per group, p = 0.0117, Figure 5D).

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
We next investigated whether SFKs activity can be elevated by CGRP and whether such elevation is sensitive to PKA or CGRP receptor inhibition using PKI (14)(15)(16)(17)(18)(19)(20)(21)(22) Amide and BIBN4096, respectively. When the protein level of phosphorylated SFKs at Y416 was normalized to that of β-actin, 3 µM CGRP markedly increased the protein level of phosphorylated SFKs at Y416 to 0.13 ± 0.01 compared to that at 0.06 ± 0.01 in the Kreb's group (n = 9 per group, p = 0.0001, Figure 6B). In the presence of 3 µM CGRP, both 30 µM PKI (14-22) Amide (n = 8) and 3 µM BIBN4096 (n = 8) reduced the protein level of phosphorylated SFKs at Y416 to 0.07 ± 0.01 compared to that in the CGRP group (p = 0.0023 and 0.002 respectively, Figure 6B). In contrast, the protein level of SFKs was unchanged among the four groups ( Figure 6C). Consistently, when the protein level of phosphorylated SFKs at Y416 was normalized to that of SFKs, the protein level of phosphorylated SFKs at Y416 was increased to 0.11 ± 0.01 by CGRP in comparison with that at 0.06 ± 0.01 in the Kreb's group (n = 9 per group, p = 0.0158, Figure 6D). In the presence of CGRP, both PKI (14-22) Amide (n = 8) and BIBN4096 (n = 8) reduced the protein level of phosphorylated SFKs at Y416 to 0.06 ± 0.01 and 0.05 ± 0.01 in comparison with that in the CGRP alone group (p = 0.0283 and 0.0063, respectively, Figure 6D).

The Protein Levels of Phosphorylated SFKs at Y416 and Released Cytokines Induced by CGRP Were Positively Correlated in the Mouse TG
We then carried out further analysis to explore whether the SFKs activity and cytokine release enhanced by CGRP are correlated. A positive relationship between the increased levels of phosphorylated SFKs at Y416 and IL-1β release (r = 0.7261, p = 0.0014, Figure 7A), CCL2 release (r = 0.7462, p = 0.0009, Figure 7B), and CXCL1 release (r = 0.7768, p = 0.0004, Figure 7C) was seen in the TG treated by both Kreb's and 3 µM CGRP (n = 8 per group). 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. 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 H 2 O 2 group and either the Kreb's group or the BIBN4096 in the presence of 1 mM H 2 O 2 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  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 Figure S3 and S4. . 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. kine release enhanced by CGRP are correlated. A positive relationship between the increased levels of phosphorylated SFKs at Y416 and IL-1β release (r = 0.7261, p = 0.0014, Figure 7A), CCL2 release (r = 0.7462, p = 0.0009, Figure 7B), and CXCL1 release (r = 0.7768, p = 0.0004, Figure 7C) was seen in the TG treated by both Kreb's and 3 μM CGRP (n = 8 per group).

SFKs Co-Localized with CGRP and RAMP1 in the Mouse TG
Consistent with the previous reports [10,[66][67][68], we were able to demonstrate that CGRP immunoreactivity was present in small to medium-sized neurons and C fibers (Figure 8A) while RAMP1 immunoreactivity was present in large neurons and Aδ fibers (Figure 8B) in the TG. Further, SFKs immunoreactivity was present in the neurons of nearly all sizes and fibers in the TG (Figure 8A,B). Double staining with the anti-SFKs antibody and anti-CGRP antibody or anti-RAMP1 antibody demonstrated that SFKs co-localized with both CGRP and RAMP1 proteins in their respective cell types and fibers of the TG ( Figure 8A,B).

SFKs Co-Localized with CGRP and RAMP1 in the Mouse TG
Consistent with the previous reports [10,[66][67][68], we were able to demonstrate that CGRP immunoreactivity was present in small to medium-sized neurons and C fibers ( Figure 8A) while RAMP1 immunoreactivity was present in large neurons and Aδ fibers ( Figure 8B) in the TG. Further, SFKs immunoreactivity was present in the neurons of nearly all sizes and fibers in the TG ( Figure 8A

Discussion
Our data demonstrate that SFKs activity facilitates the crosstalk between CGRP and cytokines in sensitizing trigeminal ganglion by transmitting CGRP receptor/PKA signaling. These findings uncover an unprecedented role of SFKs in migraine pain transmission.
We have previously demonstrated that SFKs mediate CGRP release and IL-1β gene expression and SFKs inhibition by saracatinib reduces the stress-sensing cation channel transient receptor potential ankyrin 1 (TRPA1)-activated CGRP release and IL-1β gene expression in the mouse TG [21]. We therefore postulated that direct SFKs activation may induce CGRP release and neuroinflammation from the TG thus triggering TG activation. Unexpectedly in this communication, activation of SFKs using pYEEI does not alter CGRP release or IL-1β gene expression in the mouse TG ( Figure 1). pYEEI, the SFKs activator, activates SFKs by binding to their SH2 domains, which disrupts the closing conformation of inactive SFKs ensuing their open active conformation [34,37,38]. Given that the concentration (1 mM) of pYEEI applied in this study is high enough to trigger neuronal activation [35,36], the current data suggest that SFKs activation by pYEEI alone is least likely to be a stimulus for TG activation, which is unlike other stimuli such as KCl or TRPA1 activator that can activate multiple pathways triggering mass activation and sensitization of TG [21].
We then explored whether modulating SFKs activity may be only effective when the TG is pre-primed. Indeed, in the mouse TG that is pre-sensitized by H2O2, the SFKs inhibitor, saracatinib, markedly reduces CGRP release from TG in a concentration-dependent manner ( Figure 2). Consistent with this, SFKs activity is increased by H2O2 in the mouse TG ( Figure 5). These data highlight a key role of SFKs activity in facilitating TG sensitization by mediating endogenous CGRP release. Moreover, these data extend the previous

Discussion
Our data demonstrate that SFKs activity facilitates the crosstalk between CGRP and cytokines in sensitizing trigeminal ganglion by transmitting CGRP receptor/PKA signaling. These findings uncover an unprecedented role of SFKs in migraine pain transmission.
We have previously demonstrated that SFKs mediate CGRP release and IL-1β gene expression and SFKs inhibition by saracatinib reduces the stress-sensing cation channel transient receptor potential ankyrin 1 (TRPA1)-activated CGRP release and IL-1β gene expression in the mouse TG [21]. We therefore postulated that direct SFKs activation may induce CGRP release and neuroinflammation from the TG thus triggering TG activation. Unexpectedly in this communication, activation of SFKs using pYEEI does not alter CGRP release or IL-1β gene expression in the mouse TG ( Figure 1). pYEEI, the SFKs activator, activates SFKs by binding to their SH2 domains, which disrupts the closing conformation of inactive SFKs ensuing their open active conformation [34,37,38]. Given that the concentration (1 mM) of pYEEI applied in this study is high enough to trigger neuronal activation [35,36], the current data suggest that SFKs activation by pYEEI alone is least likely to be a stimulus for TG activation, which is unlike other stimuli such as KCl or TRPA1 activator that can activate multiple pathways triggering mass activation and sensitization of TG [21].
We then explored whether modulating SFKs activity may be only effective when the TG is pre-primed. Indeed, in the mouse TG that is pre-sensitized by H 2 O 2 , the SFKs inhibitor, saracatinib, markedly reduces CGRP release from TG in a concentration-dependent manner ( Figure 2). Consistent with this, SFKs activity is increased by H 2 O 2 in the mouse TG ( Figure 5). These data highlight a key role of SFKs activity in facilitating TG sensitization by mediating endogenous CGRP release. Moreover, these data extend the previous findings that deactivation of SFKs reverses CGRP release promoted by nerve growth factor and capsaicin in dorsal root ganglion neurons [20] and by a TRPA1 activator, umbellulone, in the TG [21].
Similar to the release of CGRP, the SFKs inhibitor saracatinib also reduces the release of IL-1β, CCL2, and CXCL1 from the mouse TG pre-sensitized by exogenous CGRP (Figure 3), which consistently increases SFKs activity ( Figure 6). The levels of all these three cytokines released show positive correlations with the level of respective phosphorylated SFKs induced by CGRP (Figure 7), highlighting the importance of SFK activity in promoting TG neuroinflammation. Moreover, these data extend the previous finding that SFKs activity contributes to the release of inflammatory cytokines in astrocytes [25] and microglia [22][23][24]. Among these inflammatory cytokines, IL-1β potentiates the excitability of nociceptive neurons in the TG and directly causes the hypersensitivity to nociception ensuing the nociceptive behaviors, hyperalgesia, and allodynia [69]. While IL-1β has a well-identified role in migraine pathogenesis [65], CXCL1 [61,62,70,71] and CCL2 [63,64,72] are associated with pain hypersensitivity and more recently with migraine. It is therefore concluded that SFKs activity can synergistically elevate CGRP and cytokine release to reinforce TG sensitization and facilitate pain transmission. It is noted that, unlike the three inflammatory cytokines, the anti-inflammatory cytokine, IL-10, release is insensitive to SFK inhibition. This might suggest that SFKs activity plays a more prominent role in controlling inflammatory cytokine but not anti-inflammatory cytokine release.
One question to ask from our data is how the SFKs-mediated TG sensitization is sustained. In our study, besides cytokine release by CGRP being reduced by saracatinib at 20 min post treatment (Figure 3), the induction of IL-1β and CCL2 gene expression by exogenous CGRP was also reduced by SFKs deactivation at 1 h post treatment in the mouse TG ( Figure 4). These data are consistent with our previous finding that SFKs activity contributes to IL-1β gene expression induced by the TRPA1 activator umbellulone in the mouse TG [21]. It is highly likely that the SFKs-induced transcriptional machinery activation of these cytokines is crucial to sustain the TG sensitization, especially in that IL-1β mRNA expression and protein expression are well correlated [73]. The possible mechanism by which SFKs mediate cytokines gene expression could be via transcription factors or histone modification. SFKs are known to activate the transcription factor NFκB in models of neurodegenerative diseases [25,74], which is important for inflammatory responses [75]. Interestingly, SFKs-mediated CCL2 gene expression and histone H3 acetylation at the CCL2 promoter are associated in macrophages [76]. Furthermore, CGRP is a potent neuroinflammatory mediator that induces the expression and release of cytokines in the TG [11][12][13][14], including IL-1β, CCL2, CXCL1, all of which in turn stimulate CGRP release [16,77], thereby inducing a positive feedback loop of TG sensitization. Similar to CGRP, IL-1β can activate SFKs in several cell lines [78][79][80][81], which suggests that released cytokines are highly likely to strengthen SFKs activity again to induce CGRP release and aggravate TG sensitization. This is consistent with the significant positive correlation between the elevated SFKs activity and cytokine release induced by CGRP, which supports the model that SFKs activity facilitates the crosstalk between CGRP and cytokines ( Figure 7). Taken together, we propose a feedback mechanism by which SFKs activity facilitates the crosstalk and intraganglionic signaling between CGRP and cytokines in stress-primed TG to potentiate TG sensitization and ultimately trigeminovascular sensitization.
The molecular mechanism underlying the SFKs-mediated crosstalk between CGRP and cytokines in sensitizing TG has yet to be fully defined. Interestingly, SFKs activation induced by either H 2 O 2 ( Figure 5) or exogenous CGRP ( Figure 6) can be reduced by the CGRP receptor inhibitor, BIBN4096, in the mouse TG, supporting CGRP receptor-dependent SFKs activation in TG. As SFKs co-localize with CGRP in small-to medium-sized neurons and C fibers, whilst RAMP1 in large neurons, Aδ fibers, and satellite glial cells of the mouse TG (Figure 8), we can conclude that the SFKs-mediated crosstalk between CGRP and cytokines is dependent on CGRP/CGRP receptor signaling. Notably, PKA is known to actively transmit CGRP/CGRP receptor signaling to initiate downstream signaling cascades, thus leading to TG activation [52][53][54]. PKA robustly increases SFKs activity in cell lines [55], spinal dorsal horn [56], and hypothalamic arcuate nucleus neurons [36], and PKA/SFKs pathway facilitates neuronal firing [56] and pain sensitivity [36]. This can be compared with our previous study which demonstrates that SFKs activity is elevated by PKA upon TRPA1 activation to promote CGRP release in the mouse TG [21]. Furthermore, in the present study, the enhanced phosphorylated SFKs at Y416 induced by CGRP is also reduced by the PKA inhibitor PKI (14)(15)(16)(17)(18)(19)(20)(21)(22) Amide, which is similar to that by the CGRP receptor inhibitor BIBN4096 ( Figure 6). Taken together, these data pinpoint that SFKs activity is increased downstream of CGRP/CGRP receptor signaling via PKA activity in the TG, thereby contributing to CGRP-cytokines crosstalk and TG sensitization (Figure 9). Given that PKA promotes the phosphorylation of SFKs at the S17 site followed by autophosphorylation at their Y416 site [55], it is likely that SFKs are activated directly by PKA downstream of CGRP/CGRP receptor signaling in the TG, which awaits future validation. Future work should also examine whether CGRP co-localizes with PKA in the TG.
Cells 2022, 11, x FOR PEER REVIEW 18 of 22 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.

Conclusions
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. These findings shed light on the SFKs-mediated peripheral mechanism of migraine pathogenesis and support the promising efficacy of drugs targeting SFKs for migraine therapy. 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.
In this study, only male mice are used to explore the role of SFKs in TG sensitization so that the effect of hormonal fluctuation in females can be minimized. Similarly, the previous studies on investigating the role of SFKs in the CSD-induced migraine with aura model [82] and the inflammatory soup-induced chronic migraine model [26] only use male rodents. Interestingly, SFKs deactivation does not show sex difference in brain perfusion abnormalities in the genetic migraine with aura model FHM2 [27]. Nevertheless, future work should explore whether SFKs mediate different migraine pathogenesis in females in order to understand if there are gender-specific effects of targeting SFKs.

Conclusions
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. These findings shed light on the SFKs-mediated peripheral mechanism of migraine pathogenesis and support the promising efficacy of drugs targeting SFKs for migraine therapy.