Molecular Mechanisms For STEP61in Spinal Dorsal Horn To Inhibit Chronic Inflammatory Pain

Objective:GABAergic disinhibition caused by peripheral tissue damage in spinal dorsal horn activates cAMP-dependent protein kinase (PKA), which phosphorylates61kD striatal-enriched protein tyrosine phosphatase (STEP61) and breaks its interaction with Src family protein tyrosine kinase member Fyn and extracellular regulated kinases1and2(ERK1/2), leading to the initiation of a variety of intracellular signaling pathways. This study aimed to investigate whether reinforcing GABAergic tone was able to resume STEP61activity to relieve chronic inflammatory pain.Method:The present study induced the chronic inflammatory pain by intraplantar injection of Complete Freund’s Adjuvant (CFA) in mice, and utilized nociceptive behavioral tests, western blot, immunoprecipitation, co-immunoprecipitation and immunohistochemistry to explore the the role of STEP61in GABAergic disinhibition-exaggerated chronic inflammatory pain.Results:(1) Spinal dorsal horn neurons expressed green fluorescent protein (GFP)-tagged exogenous STEP61in a time-dependent manner after intrathecal injection of recombinant adenovirus;(2) Spinal expression of catalytically-inactive STEP61(C472S) in intact mice not only elicited a robust allodynia, but also entirely occluded the pronociceptive effects of bicuculline (BIC;0.1μg);(3) The important function of bicuculline was to disrupt STEP61binding to its substrates Fyn and ERK1/2, which was blocked by N-methyl-D-aspartate receptor (NMDAR) antagonist D-APV (100μM) and high Mg2+(10mM), but not by Na+channel blocker lidocaine (Lido;500μM), suggesting that GABAergic disinhibition perturbed STEP61complex through spontaneous activation of synaptic NMDA receptor;(4) CFA, via GABAergic disinhibition, activated cAMP-dependent protein kinase (PKA) in spinal dorsal horn to dissociate STEP61complex;(5) Intrathecal injection of GABAA receptor agonist muscimol (Mus;0.1μg) in mice with inflammatory pain could resume the interaction of STEP61with Fyn and ERK1/2, and inhibit phosphorylation of Fyn and ERK1/2;(6) In order to restore the function of STEP61, we delivered exogenous wild-type STEP61[STEP(WT)] to the spinal cord, finding that STEP61(WT) completely prevented abnormal Fyn and ERK1/2phosphorylation induced by CFA;(7) Meanwhile, expression of STEP61(WT) completely prevented NR2B phosphorylation at Tyr1472(NR2B-Y1472) induced by CFA, and decreased NR2B receptor content at synaptosomal membrane fraction;(8) Importantly, overexpression STEP(WT) effectively inhibited the induction and maintenance of chronic inflammatory pain.Conclusion:Peripheral tissue damage, via GABAergic disinhibition, disturbed the inhibitory control by STEP61over its substrates Fyn and ERK1/2, leading to NMDA receptor hyperfunction and allodynia. Direct expression of exogenous wild-type STEP61could effectively ameliorate the symptoms of chronic inflammatory pain.