| Opioids,such as morphine,have powerful analgesic effects on acute or chronic pain and induce antinociception tolerance and dependence after repeated use,which limits the clinical utility of morphine.The ? opioid receptor(MOR)is the main receptor mediated opioids antinociception.The mechanism of tolerance induced by opioids includes G protein-dependent signaling and ?-arrestin-mediated signaling.While super-activation or sensitization of adenylate cyclase(AC)in G protein-dependent signaling by chronic agonist treatment are considered as classic theories of opioid tolerance,?-arrestin-mediated signaling also plays a crucial role in morphine tolerance.Agonist-induced phosphorylation of MOR is thought to be a trigger for ?-arrestin-mediated signaling.The antinociception induced by opioids is strongly potentiated and the antinociceptive tolerance is decreased obviously in MOR phosphorylation-deficient mice.?-arrestins,including ?-arrestin1 and ?-arrestin2,are highly expressed in the central nervous system(CNS)and regulate MOR signaling pathway.It has been demonstrated that ?-arrestins expression,especially ?-arrestin2 involved in the receptor internalization.In cells,?-arrestin2 is recruited after MOR phosphorylation and then causes a sequestration of MOR under morphine exposure.Ubiquitinated ?-arrestins are crucial for downstream endocytic and signaling processes.Due to the vital role of ?-arrestin2 on MOR regulation,mice lacking ?-arrestin2 did not develop antinociceptive tolerance in a hot plate test following chronic morphine treatment.Thereby several proteins have been found to mediate morphine tolerance by regulating ?-arrestin signaling.Recently,MOR-associated proteins that negatively regulation of ?-arrestin signaling pathway are thought to be therapeutic targets and molecular probes to resolve the limitations of morphine.According to these studies,19 proteins associated with MOR were identified by the bacterial two-hybrid system using C-terminal domain of MOR as bait in our previous study.ABIN-1 and Hsp90? were included in our study.ABIN-1 is well known as a ubiquitin-binding protein that regulates processes in autoimmune inflammatory diseases by inhibiting NF-?B activation.It has been demonstrated that ABIN-1 was up-regulated in morphine-tolerant mouse and could interact with MOR functionally in our previous work,but its effects on morphine tolerance and dependence have rarely been reported.Hsp90?,one isoform of Hsp90,regulates many physiological functions in the cell.Recent studies indicated a strongly relationship between Hsp90 and pain.Hsp90 inhibitor 17-AAG could affect the antinociception induced by morphine.Therefore,we tried to demonstrate the mechanism of ABN-1 and Hsp90?on morphine tolerance and dependence.Objective: To explore the role of ABN-1 and Hsp90? on morphine tolerance and dependence,and to find out the underlying molecular mechanisms for providing a theoretical basis for the development of new drug targets.Methods: 1.The function and mechanism of ABIN-1 on morphine tolerance and dependence 1.1.The effect of ABIN-1 on morphine tolerance and dependence: AAV-ABIN-1 or AAV-ABIN-1-sh RNA crossing the blood-brain barrier was generated to interfere with ABIN-1 in mouse brain.The analgesic effects of morphine were assessed by the hotplate test(55?);The physical dependence of morphine was assessed in morphine-tolerant mice by the naloxone-induced withdrawal test;The psychological dependence of morphine was assessed by the conditioned place preference test.1.2.The mechanism of ABIN-1 on morphine tolerance 1.2.1.The effect of ABIN-1 on ?-arrestin signaling of MOR: we preformed time-dependently phosphorylation of MOR and ERK by DAMGO or morphine treatment in stable expression MOR-CHO cell or MOR-ABIN-1-CHO cell;using HCS analysis of ?-arrestin2 translocation to plasma membrane on MOR-?-arrestin2-EGFP-CHO cell;the plasma membrane MOR was detected by plasma membrane protein isolation kit in morphine-tolerant mouse brain or MOR-CHO cell with DAMGO treatment after overexpressing ABIN-1;1.2.2.The effect of ABIN-1 on ?-arrestin2: the interaction between ABIN-1 and ?-arrestin2 was detected by immunofluorescence and coimmunoprecipitation assays in HEK293 cell transiently transfected of ABIN-1,MOR and ?-arrestin2;the ubiquitination of ?-arrestin2 was detected by immunoprecipitation in HEK293 cell transiently expression of ABIN-1 and ?-arrestin2;the expression of ?-arrestin2 was detected in N2 A cell or in morphine-tolerant mouse brain by immunoblotting or immunohistochemistry;1.2.3.The effect of ABIN-1 in ?-arrestin2-knockout mice: the analgesic effect of morphine was detected by hotplate test after ABIN-1 overexpression or knockdown in ?-arrestin2-knockout mice.1.3.The structure domain of ABIN-1 interaction with MOR 1.3.1.The construction of ABIN-1 mutant:the construction of ABIN-1-AHD1-DEL,ABIN-1-AHD2-DEL,ABIN-1-AHD3-DEL,ABIN-1-AHD4-DEL were confirmed by DNA sequencing and immunoblotting;1.3.2.The structure domain of ABIN-1 interaction with MOR: the interaction between mutants of ABIN-1 and MOR was detected by coimmunoprecipitation assay in HEK293 cell transfected mutants of ABIN-1and MOR;Molecular Docking predicted the structure of key region of ABIN-1 and MOR;the phosphorylation of MOR was detected by transfecting mutants of ABIN-1 into MOR-CHO cell;1.3.3.The function of key regions of ABIN-1 on morphine tolerance: we synthetized the peptide of ABIN-1 mutant and performed hotplate assay.2.The function and mechanism of Hsp90 on morphine tolerance and dependence 2.1.The effect of Hsp90 on morphine tolerance and dependence: the analgesic effects of morphine were assessed by hotplate test(55?)after pretreatment with 17-AAG(Hsp90 inhibitor,10 nmol/5 ?l);the physical dependence was assessed by naloxone-induced withdrawal test in morphine-tolerant mice;the psychological dependence of morphine was assessed by CPP test.2.2.The function of Hsp90? on MOR signaling 2.2.1.The interaction between Hsp90? and MOR: the interaction between Hsp90 and MOR was detected by immunofluorescence and coimmunoprecipitation assay in SH-SY5 Y cell or in HEK293 cell transfected Hsp90?/Hsp90? and MOR;2.2.2.The effect of Hsp90? on MOR signaling: the accumulation of c AMP was detected in HEK293 cell transiently transfection of Hsp90? and MOR under treatment of agonists;HCS detected the activity of PKA in PKAcat-EGFP-MOR-CHO cell transfected Hsp90?;the phosphorylation of MOR/PKA/ERK/CREB/JNK was detected by immunoblotting in stable expression MOR-CHO cell transfected Hsp90? after DAMGO pretreatment;MOR in plasma membrane was detected by immunofluorescence and immunoblotting in MOR-CHO cell transiently overexpressed Hsp90? under DAMGO pretreatment.Results: 1.The function and mechanism of ABIN-1 on morphine tolerance and dependence 1.1.The effect of ABIN-1 on morphine tolerance and dependence The hot plate results showed that the analgesic maximal possible effect(%MPE)decreased gradually over time after 7 consecutive days treatment of morphine(10 mg/kg,s.c.).Compared with the control group,the(%)MPE was significantly increased in ABIN-1overexpression group at day 1-day 5(Day 1:P =0.0194,Day 3:P = 0.0051,Day 5:P = 0.0325,F(1,7)= 71.75,P < 0.0001);mice overexpressing ABIN-1 showed a remarkable decrease in naloxone-precipitated(10 mg/kg,i.p.)jumping behavior(P = 0.0081)and had less prone to the box that always injected morphine in CPP test(P = 0.0092);knockdown of ABIN-1 in brain reduced the(%)MPE of morphine analgesia(Day1:P = 0.0032,Day 3:P = 0.0004,Day 5:P = 0.0156,F(1,8)= 15.39,P < 0.0044),but increased the jumping counts induced by naloxone(P = 0.0022)and had more prone to the box that always injected morphine in CPP test significantly(P = 0.0323).ABIN-1 overexpression or knockdown group had no effect on nociceptive latencies.ABIN-1 was up-regulated or down-regulated in the Hipp and NAc of mice brain respectively via stereotactic injection AAV-ABIN-1 and AAV-ABIN-1-sh ABIN-1.Compared with the control group,up-regulation of ABIN-1 in Hipp had no effect on nociceptive latencies and significantly increased the(%)MPE of morphine analgesia after continuous administration of morphine(Day 1:P = 0.0253,Day 3:P = 0.0050,F(1,7)= 75.95,P = 0.0142),but decreased withdrawal syndrome(P = 0.0437);down-regulation of ABIN-1 in the Hipp decreased the(%)MPE of morphine analgesia(Day 1:P = 0.0057,Day 3: P = 0.0010,F(1,6)= 12.27,P = 0.0128)and increased withdrawal syndrome significantly(P = 0.0056).Compared with the control group,up-regulation of ABIN-1 in the NAc increased the nociceptive latencies of mice(Day 3:P = 0.0350,Day 7:P = 0.0145,F(1,7)= 10.98,P = 0.0129)and the(%)MPE of morphine analgesia significantly(Day 3:P = 0.0170,Day 5:P = 0.0195,Day 7:P = 0.0443),but decreased withdrawal syndrome significantly(P = 0.0415);the nociceptive latencies of mice(Day 1:P = 0.0002,Day 3:P = 0.0076,Day 5:P = 0.0008,Day 7:P = 0.0053,F(1,6)= 201.7,P < 0.0001)and the(%)MPE of morphine analgesia were significantly decreased by ABIN-1 knockdown in the NAc(Day 3:P = 0.0262,Day 5:P = 0.0099,Day 7:P = 0.0041),but the withdrawal syndrome was significantly increased compared with the control group(P = 0.0454).ABIN-1 knockdown in ACC had no effect on the nociceptive latencies of mice,the(%)MPE of morphine analgesia and the naloxone-induced jumping counts(P > 0.05).1.2.The mechanism of ABIN-1 on morphine tolerance 1.2.1.The effect of ABIN-1 on ?-arrestin signaling of MOR: the phosphorylation of MOR and ERK were significantly decreased by ABIN-1 overexpression after acute or chronic DAMGO treatment;overexpressing ABIN-1 decreased the phosphorylation of MOR and ERK after acute morphine treatment significantly;ABIN-1 promoted a rapid translocation of ?-arrestin2 to the plasma membrane after DAMGO(10?M)or morphine(10?M)treatment 5min,although without agonist activation;ABIN-1 overexpression increased the MOR on plasma membrane significantly in morphine-tolerant mice or MOR-CHO cell pretreatment DAMGO(10 m M,P < 0.05);1.2.2.The effect of ABIN-1 on ?-arrestin2: ABIN-1 could interact with ?-arrestin2,which was augment by DAMGO pretreatment;?-arrestin2 ubiquitination was significantly increased by ABIN-1 overexpression with or without DAMGO treatment(10 m M,P < 0.05);ABIN-1 overexpression reduced ?-arrestin2 expression in morphine-tolerant mice or N2 A cell remarkedly(P < 0.05);1.2.3.The effect of ABIN-1 in ?-arrestin2-knockout mice: ABIN-1 overexpression or knockdown in ?-arrestin2-knockout mice did not affect the nociceptive latencies of mice,the(%)MPE of morphine analgesia and the naloxone-induced jumping counts.1.3.The structure domain of ABIN-1 interaction with MOR 1.3.1.The construction of ABIN-1 mutant: DNA sequencing and immunoblotting results showed that ABIN-1-AHD1-DEL,ABIN-1-AHD2-DEL,ABIN-1-AHD3-DEL,ABIN-1-AHD4-DEL mutants were successfully constructed;1.3.2.The structure domain of ABIN-1 interaction with MOR: ABIN-1-AHD2-DEL damaged the interaction between ABIN-1and MOR,ABIN-1-AHD1-DEL or ABIN-1-AHD3-DEL or ABIN-1-AHD4-DEL still interacted with MOR respectively;molecular Docking method results showed that the Leu462-Asp472 segment of AHD2 bound to the MOR protein pocket,while the Phe473-Arg482 segment attached to the protein surface outside the pocket;AHD2 could interact with MOR;MOR phosphorylation was significantly decreased by AHD2 overexpression after DAMGO treatment(10 m M,P < 0.05);1.3.3.The effect of key regions of ABIN-1 on morphine tolerance: TAT-AHD2-FITC or TAT-AHD3-FITC penetrating peptide was synthesized;the hot plate results suggested the(%)MPE was remarkedly decreased by AHD2 overexpression after 7 consecutive days treatment of morphine(Day 1: P = 0.0058,Day 5: P = 0.0039,Day 7: P = 0.1747,F(1,9)= 46.58,P < 0.0001).2.The function and mechanism of Hsp90 on morphine tolerance and dependence 2.1.The effect of Hsp90? on morphine tolerance and dependence: the(%)MPE decreased for approximately 25% under the pretreatment of 17-AAG(Hsp90 inhibition,10 nmol/5 ?l)after morphine(10 mg/kg,s.c.)addition(P < 0.05);17-AAG pretreatment decreased the phosphorylation of ERK and CREB,but increased the phosphorylation of JNK in mice treated with morphine for 30 min(P < 0.05);under chronic morphine administration(100 mg/kg,s.c.),the rate of the inhibition of(%)MPE in the 17-AAG pretreatment group was significantly lower than solvent pretreatment group;17-AAG decreased naloxone-induced withdrawal signs significantly(P < 0.05);the time spent in morphine-matched compartments of 17-AAG pretreatment group was significantly decreased compared with solvent group during CPP testing(P < 0.05);2.2.The function of Hsp90? on MOR singling 2.2.1.The interaction between Hsp90? and MOR: Hsp90 interacted with MOR via Hsp90?,not Hsp90?,which was augmented by morphine pretreatment and was blocked by 17-AAG pretreatment;2.2.2.The effect of Hsp90? on MOR signaling: the accumulation of c AMP was significantly decreased by Hsp90? overexpression under morphine or DAMGO treatment in HEK293 cell(10 m M,P < 0.05);HCS results showed the activity of PKA was significantly increased by Hsp90? overexpression under agonist treatment in PKAcat-EGFP-MOR-CHO cell(10 m M,P < 0.05);MOR phosphorylation was significantly increased by overexpressing Hsp90? after DAMGO treatment in MOR-CHO cell(P < 0.05);17-AAG pretreatment could decrease the phosphorylation of MOR(P < 0.05);immunofluorescence and immunoblotting results showed that Hsp90? overexpression diminished the MOR in plasma membrane and promoted MOR internalization induced by DAMGO(10 m M,P < 0.05);Hsp90? overexpression increased the phosphorylation of ERK and CREB,but decreased the phosphorylation of JNK and PKA under treatment of DAMGO(10 m M,P < 0.05).Conclusion: 1.In summary,ABIN-1 in brain alleviated morphine tolerance and dependence.The ABIN-1-?-arrestin2-MOR complexes promoted ?-arrestin2 degradation by ubiquitination and promoted ?-arrestin2 recruitment,which might decrease the phosphorylation and internalization of MOR.Attenuation of morphine tolerance by ABIN-1 was abolished in ?-arrestin-2 knockout mice.All that illustrated ABIN-1 targeted ?-arrestin2 to regulate MOR signaling pathway.Furthermore,the ABIN-1-mediated regulation of MOR function required its AHD2 region.Thus,ABIN-1-targeted strategy may have therapeutic potentiality as an auxiliary process that alleviated morphine tolerance.2.Inhibition of Hsp90 by 17-AAG decreased morphine analgesia and alleviated morphine tolerance and dependence.Hsp90 regulated MOR signaling positively via the interaction between Hsp90? and MOR.Hsp90? is a new regulators of morphine analgesia. |
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