Role Of Focal Adhesion Kinase Signaling Pathway In Nociceptive Synaptic Transmission In Spinal Cord Dorsal Horn

Objective:Focal adhesion kinase (FAK) is one of non-receptor protein tyrosin & kinases that are critical for the dynamic regulation of cell adhesion structures. Recent studies have demonstrated that FAK is also localized at excitatory glutamatergic synapses and involved in long-term modification of synaptic strength. However, whether FAK is engaged in nociceptive processing in spinal dorsal horn remains to be elucidated. Extracellular signal regulating kinase 1/2 (ERKl/2) and N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is closely related to the chronic inflammatory pain. The current study was designed to investigate the regulatory effect of FAK on spinal ERKl/2 activity and NMDA receptor-mediated synaptic nociceptive transmission.Method:We established the pain model by intraplantar injection of Complete Freund’s Adjuvant (CFA) in mice, and used western blot, behavioral tests, patch clamp recording and immunohistochemistry to explore the regulatory effects of peripheral tissue injury on FAK signaling pathways in spinal dorsal horn as well as its functional significance.Results:(1) Immunohistochemistry results showed that FAK was localized in spinal cord dorsal horn neurons; (2) Intraplantar injection of CFA in mice significantly increased FAK autophosphorylation at Tyr397, indicating a close correlation of FAK activation with inflammatory pain; (3) When NMDA receptor antagonist D-APV (5 ug) or the group I metabotropic glutamate receptor (mGluR) antagonist AIDA (25 jig) was intrathecally applied, FAK(Y397) phosphorylation in CFA-injected mice was significantly repressed, indicating FAK activation was attributed to the activities of NMDA receptor and mGluR; (4) To directly evaluate the role of FAK in spinal nociceptive processing, we monitored the pain thresholds of naive mice after intrathecal injection of pEGFP-N1 vector encoding FAK(WT) or its catalytically inactive FAK(Y397F) mutant. We found that FAK(WT) induced thermal hyperalgesia and mechanical hyperalgesia in normal mice, while spinal FAK(Y397F) expression had no effects; (5) Western blot results showed that one of the important functions of FAK was to activate ERK1/2 in spinal dorsal horn, as evidenced by their enhanced phosphorylation levels. Intrathecal application of MEK inhibitor U-0126 (0.2μg、0.5μg and 1.0μg) elevated the paw withdrawal threshold values of FAK(WT)-injected mice in a dose-dependent manner, suggesting that FAK might act through ERK1/2 signaling pathway to evoke central sensitization; (6) Through ERK1/2 singaling, FAK(WT) significantly increased the protein contents of NMDA receptor GluN1 and GluN2B subunits at the synaptosomal membrane fraction, while NMDA receptor GluN2A subunit and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPA receptor) GluA1 subunit underwent no detectable changes; (7) Inhibition of FAK activity by spinal expression of a kinase-dead FAK(Y397F) mutant repressed ERK1/2 hyperactivity in CFA-injected mice; (8) Electrophysiological recording demonstrated that intracellular loading of specific anti-FAK antibody, but not non-specific IgG，through the recording pipettes reduced the amplitudes of NMDA receptor-mediated excitatory postsynaptic currents on lamina Ⅱ neurons from inflamed mice; (9) Behavior results showed that spinal expression of FAK(Y397F) to inhibit FAK activity generated a long lasting alleviation of CFA-induced mechanical allodynia and thermal hyperalgesia.Conclusion:FAK might induce NMDA receptor hyperfunction and pain hypersensitivity through the FAK/ERK/GluN2B signaling pathways. Direct expression of inactive FAK (Y397F) mutant could effectively alleviate symptoms of chronic inflammatory pain.