title: Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund's adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats.
abstract: The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum- and glucocorticoid-inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1)/Rab4-dependent GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund's adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4-5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)-GRASP-1, GRASP-1-Rab4, and Rab4-GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA-enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP-1, Rab4, and GluR1 by immunofluorescence. GSK-650394 (an SGK1 activation antagonist, 1, 10, and 30 μM, 10 μL/rat, intrathecally) dose-dependently prevented CFA-induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein-protein interactions at 1 day after CFA administration. Intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPAR antagonist, 1, 3, and 10 μM, 10 μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA-induced hyperalgesia and the associated GluR1 trafficking and Rab4-GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP-1/Rab4 cascade, plays a pivotal role in CFA-induced inflammatory pain by regulating GluR1-containing AMPAR recycling in the dorsal horn.
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei, Taiwan Department of Urology, China Medical University Hospital, China Medical University, Taichung, Taiwan Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, Chung-Shan Medical University, Taichung, Taiwan Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Physiology, College of Medicine, China Medical University, Taichung, Taiwan Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan.
authors.forename: Hsien-Yu
authors.initials: HY
authors.lastname: Peng
authors.forename: Gin-Den
authors.initials: GD
authors.lastname: Chen
authors.forename: Ming-Chun
authors.initials: MC
authors.lastname: Hsieh
authors.forename: Cheng-Yuan
authors.initials: CY
authors.lastname: Lai
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Huang
authors.forename: Tzer-Bin
authors.initials: TB
authors.lastname: Lin
mesh_terms: D000276:Adjuvants, Immunologic; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D005620:Freund's Adjuvant; D006930:Hyperalgesia / Q000139:chemically induced / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D020671:Posterior Horn Cells / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000378:metabolism*; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism*; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D000276:Adjuvants, Immunologic; D002352:Carrier Proteins; D005620:Freund's Adjuvant; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D017346:Protein Serine-Threonine Kinases; D018091:Receptors, AMPA; D020695:rab4 GTP-Binding Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C574553:GRASP protein, rat