Regulation Of STEP61 Signaling Pathway By RCAN1.4 In The Spinal Dorsal Horn

Objective: The dysfunction of striatum-enriched protein tyrosine phosphatase(61kD Striatal-enriched protein tyrosine phosphatase,STEP61)contribute to the formation of the hypersensitivity in chronic pain.However,the molecular mechanism of its dysfunction has not been elucidated yet.The purpose of this study was to explore the possible functional regulation of STEP61 by RCAN1.4,an endogenous inhibitor of CaN in the spinal dorsal horn,and to explore the therapeutic significance of intervention with the "RCAN1.4-CaN-STEP61" pathway in inflammatory chronic pain.Method: To investigate the effect of overexpression or knockdown of RCAN1.4in spinal dorsal horn neurons on the STEP61 signaling pathway,the adeno-associated virus or siRNA was injected in the dorsal horn of the spinal cord or intrathecally applied.A chronic inflammatory pain model was prepared by a posterior foot subcutaneous injection of the complete Freund’s adjuvant(CFA).Changes in CaN activity were detected by using a colorimetric assay.Virus expression was visualized by using immunofluorescence staining.Protein-protein interactions was investigated using the co-immunoprecipitation method.Protein expression and phosphorylation levels were detected by the Western Blot assay.The pain behavior was examined by detecting the paw withdrawal thresholds(PWTs).Results:(1)The phosphorylation levels of serine 221(Ser221)of STEP61 in the membrane fractions from the dorsal horn of the spinal cord were significantly increased after subcutaneous injection of CFA for 24 h,and this effect of CFA could be reversed by the intrathecal supplementation of recombinant CaN,suggesting that the weakened CaN activity may be an important reason for the hyperphosphorylation of STEP61-Ser221 under inflammatory conditions.(2)Intrathecal injection of the CaN inhibitor FK506 could mimic the effect of CFA,that is,FK506 significantly increased the phosphorylation level of STEP61-Ser221 and induced mechanical pain hypersensitivity.(3)The activity of CaN in the dorsal horn of the spinal cord was decreased significantly after subcutaneous injection of CFA 24 h into the posterior foot of mice,and this effect of CFA could be inhibited by Knockdown of RCAN1.4 using siRNA,suggesting an important role of RCAN1.4 in the regulating CaN activity.Overexpression of RCAN1.4 in the dorsal horn of normal mice also induced mechanical pain hypersensitivity and decreased the activity of CaN.Intrathecal administration of CaN relieved the pain behavior in RCAN1.4 overexpressing mice.(4)RCAN1.4 was immunoprecipitated from the lysate of synaptosome components with the specific antibody against STEP61,suggesting that RCAN1.4 can form a complex with STEP61.(5)Overexpression of RCAN1.4 in the spinal dorsal horn significantly increased the phosphorylation of STEP61-Ser221 and reduced the interactions of STEP61 with its substrates ERK 1/2 and Fyn as well as p38,while intrathecal supplementation of CaN could reverse the hyperphosphorylation of STEP61-Ser221 caused by RCAN1.4overexpression.Overexpression of RCAN1.4 also increased phosphorylation levels of the threonine 202 and tyrosine 204(Thr202/Tyr204),Fyn tyrosine 418(Tyr418)and tyrosine at 180 and 182(Tyr180/182)of p38.(6)Subcutaneous CFA injection significantly reduced the interaction of STEP61 with ERK 1/2 and Fyn,as well as p38,while enhancing the phosphorylation of ERK1/2,Fyn,and p38.However,prior knockdown of RCAN1.4 inhibited the above effects of CFA.(7)Overexpression of the spinal dorsal horn also significantly increased the phosphorylation of tyrosine at position 1472(Y1472)of the NMDA receptor GluN2 B subunit and the synaptic expression of the GluN2 B receptor.Intrathecal supplementation with CaN reversed the hyperphosphorylation of GluN2 B caused by RCAN1.4.(8)Subcutaneous injection of CFA significantly increased the phosphorylation of GluN2 B as well as the synaptic expression of GluN2 B.Knockdown of RCAN1.4 could inhibit the GluN2 B hyperphosphorylation evoked by CFA.(9)Intrathecal administration of an exogenous cell-penetrating peptide TATSTEP that mimicked the action of STEP61 effectively reversed the hyperphosphorylation of GluN2B-Y1472.(10)Intrathecal administration of TAT-STEP inhibited the pain hypersensitivity induced by RCAN1.4 overexpression or CFA injection.Conclusion: RCAN1.4 in the spinal dorsal horn can regulate STEP61 function by changing the activity of CaN.Under peripheral inflammatory conditions,RCAN1.4causes hyperphosphorylation of STEP61-Ser221 through excessive inhibiting CaN activity,as a result,the activity of STEP61 is dysfunctional.Interventions of the "RCAN1.4-CaN-STEP61" pathway probably have therapeutic implications for the treatment of inflammatory chronic pain.