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Mechanistic/mammalian target of rapamycin (mTOR) activation by μ-opioid receptor (OPRM1) participates in antinociceptive tolerance, hyperalgesia, physical dependence. Our previous study also showed that mTOR activation by OPRM1 could attenuate β amyloid oligomers-induced neurotoxicity. OPRM1 is demonstrated to interact with FK506 binding protein 12 (FKBP12). It is our great interest to investigate whether OPRM1-mediated mTOR signaling is related to receptor-FKBP12 association.
The activities of mTOR and its downstream effector p70 S6K were measured by immunoblotting their phosphorylation status. The interaction of receptor with mTOR was detected by co-immunoprecipitation and immunofluorescence.
OPRM1 activation by morphine induced time-dependent mTOR activation. PI3K specific inhibitor LY294002 only blocked the late phase of mTOR activation. However, morphine-induced mTOR activation was totally blocked at all time points in cells expressing FKBP12 association-deficient mutant receptor. FKBP12 knockdown also blocked morphine-induced mTOR activation. Further analysis demonstrated that morphine treatment enhanced the association of receptor with phosphorylated mTOR, whereas, decreased association was observed after FKBP12 knockdown, mTOR inhibition or in cells expressing FKBP12 association-deficient mutant.
OPRM1-FKBP12 association played a key role in OPRM1-mediated mTOR activation, which could underlie the mechanisms of multiple physiological and pathological processes. Thus our findings provide new avenue to modulating these processes.
μ-opioid receptor (OPRM1) plays a decisive role in antinociception . Opioids are still the most effective medication for pain control. However, the clinical use of opioids, especially for long-term use, is hampered by the development of antinociceptive tolerance, hyperalgesia, physical dependence and addiction. Although the mechanisms underlying these phenomena are not fully resolved and still under intensive investigation, the importance of mechanistic target of rapamycin (mTOR) in these processes gradually emerged [2–4].
mTOR, a serine/threonine protein kinase, primarily controls transcription, protein synthesis, and cell growth, cell proliferation, cell motility [5, 6]. In central nervous system (CNS), mTOR signaling has emerged as a critical integrator of neuronal activity and synaptic inputs and plays a pivotal role in neuronal plasticity and the process of learning and memory [7, 8]. mTOR forms two structurally and functionally distinct complexes: mTOR complex 1 (mTORC1) and mTORC2. mTORC1 contains the defining components Raptor and proline-rich protein kinase B (Akt) substrate 40 kDa. mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinases (p70 S6K) . Phosphorylation of p70 S6K at Thr389 serves as a characterized downstream effector of mTOR [10, 11]. Another well-characterized downstream effector is the eukaryotic translation initiation factor-4E binding protein 1 (4E-BP1) . Both p70 S6K and 4E-BP1 interact with mRNAs and regulate mRNA translation initiation and progression, which are the control steps of protein synthesis .
By controlling the protein synthesis in the CNS, mTOR has been found to regulate multiple biological processes which are mediated by opioid receptor. It is shown to play a key role in addiction-related behaviors such as reward seeking and excessive drug intake . The activation of mTOR and p70 S6K in hippocampus by morphine is shown to be positively correlated with the acquisition of morphine conditioned place preference (CPP), a learning paradigm for assessing drug reward . Rapamycin, a selective inhibitor of mTOR, prevents the acquisition of CPP [4, 15]. Activation of OPRM1 in rat spinal dorsal horn neurons results in an increase in mTOR and p70 S6K, 4E-BP1 activities and the inhibition of mTOR activity by rapamycin or siRNA blocks both induction and maintenance of morphine tolerance and hyperalgesia . Our recent study has demonstrated that the attenuation of β amyloid oligomers-induced neurotoxicity by OPRM1 activation is mediated through mTOR signaling .
mTOR belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase family. The best-characterized activation input to mTORC1 is through PI3K/Akt signaling pathway. On the other hand, rapamycin can specifically and effectively block the activity of mTORC1 by binding to FK506-binding protein 1A, 12 kD (FKBP12) to form a complex with mTOR . FKBP12 belongs to the family of the immunophilins which are protein chaperones to guide proper folding and assembly that provide functional stability to multiprotein macromolecules . Moreover, FKBP12 is expressed 10–50 fold more highly in CNS and peripheral nervous system than in immune system [19, 20].
In our previous study, we have demonstrated that OPRM1 specifically interacts with FKBP12 and the receptor-FKBP12 complex increases after morphine treatment . Moreover, we have showed that PI3K signaling pathway is involved in mTOR signaling induced by OPRM1 activation . In current study, we further demonstrated that the association of FKBP12 with OPRM1 played a key role in mTOR activation.
Morphine and naloxone were supplied by the National Institute on Drug Abuse. Rapamycin, H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and mouse monoclonal anti- β-actin antibody were purchased from Sigma Chemical Co (St. Louis, MO, USA). Mouse HA.11 monoclonal antibody (clone 16B2) was purchased from Covance (Berkeley, CA, USA). All the other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All the cell culture reagents were purchased from GIBCO (Grand Island, NY, USA).
HEK293 cells stably transfected with hemagglutinin (HA) epitope tagged rat OPRM1 (HA-OPRM1) or its mutant at Pro353 to Ala (HA-OPRM1P353A) were maintained in Eagle’s minimum essential medium (MEM) with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin plus 200 μg/ml G418 at 37°C in a humidified atmosphere of 95% air and 5% CO2.
After treatment, cells were extracted with cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 1 μg/ml leupeptin and 1 × protease inhibitor cocktail (Sigma)). After centrifuging at 12,000 ×g for 5 min, sample loading buffer was added to the supernatants and boiled for 5 min. Approximately 30–40 μg of protein was subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% milk and then incubated with primary antibodies against mTOR phosphorylated at Ser2448 (1:1000), mTOR (1:1000), p70 S6K phosphorylated at Thr389 (1:1000), p70 S6K (1:1000) and β-actin (1:5000) overnight at 4°C. Membranes were then incubated with goat anti-rabbit/mouse HRP-conjugated secondary antibody for 1 h at room temperature, visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical, Rockford, IL, USA) and analyzed by Image J (National Institutes of Health) software.
HEK293 cells stably expressing OPRM1 were transfected with short interfering RNA (siRNA) corresponding to the target sequence GCTTGAAGATGGAAAGAAA of FKBP12 gene (Qiagen, Valencia, CA, USA) or a scrambled siRNA as control at the final concentration of 50 nM using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions , 24 h later, cells were analyzed as indicated. The effect of siRNA on protein expression was determined by western blot.
After treatment, cells in 100 mm dish were extracted with 300 μl lysis buffer (0.1% Triton X-100, 50 mM Tris·HCl pH 8.0, 100 mM NaCl, 10% glycerol, 10 mM EDTA, 10 mM NaF, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate and 1 × protease inhibitor). After centrifuging at 12,000 ×g for 5 min, 1 μl mouse anti-HA antibodies and 30 μl protein-G-agarose (Pierce, Rockford, IL, USA) were added to the supernatants and rotated overnight. Antibodies specific for phospho-mTOR Ser2448, mTOR and HA were used for immunoblotting.
HEK293 cells stably expressing OPRM1, OPRM1P353A and OPRM1 with FKBP12 knockdown were treated with 1 μM morphine for 5 min, fixed with pre-warmed 3%–4% formaldehyde for 20 min at room temperature. Then the cells were washed with PBS three times and blocked with 10% normal goat serum in PBS. Then the cells were permeabilized by 0.3% Triton X-100 and stained with rabbit anti-phospho-mTOR Ser2448 antibody (Clone D9C2, 1:200) and mouse anti-HA antibody (1:1000), washed with PBS for five times and stained with Alexa 488-conjugated goat anti-mouse and Alexa 594-conjugated goat anti-rabbit antibodies (1:500, Molecular Probes, Eugene, OR, USA). Cells were viewed and captured with Laser Confocal Microscope (Carl Zeiss LSM 510, USA). Colocalization was analyzed using MetaMorph Microscopy Automation and Image Analysis Software (Sunnyvale, CA, USA) to determine the percentage of receptor overlapping phosphorylated mTOR.
Data were presented as mean ± standard error of the mean (SEM). The results were analyzed by one-way analysis of variances (ANOVA) followed by Dunnett’s test to compare with control or Bonferroni’s test to compare all pairs or two-way ANOVA followed by Bonferroni’s test by using GraphPad Prism 5.0 statistical analysis software. P < 0.05 was considered statistically significant.
In a previous study we have demonstrated that morphine induces mTOR activation . Here we continue to characterize this morphine action by investigating the time-dependent effect of morphine on mTOR activation. When OPRM1-expressing HEK293 cells were treated with 1 μM morphine, marked increase of mTOR phosphorylation was demonstrated (Fig. 1A upper panel and 1B). Robust increase of mTOR phosphorylation started within 5 min of morphine exposure and lasted for at least 2 h. 5min, 15 min, 30 min, 1 h, and 2 h of morphine treatment resulted in 1.43 ± 0.11, 1.50 ± 0.05, 1.57 ± 0.09, 1.61 ± 0.09, 1.50 ± 0.09 folds increase of mTOR phosphorylation, respectively. To confirm the effect of PI3K signaling pathway, PI3K specific inhibitor LY294002 was employed. Unexpectedly, in the presence of the inhibitor, 5 min of morphine treatment still resulted in 1.42 ± 0.06 fold increase of mTOR phosphorylation. But LY294002 did block the phosphorylation of mTOR when the cells were treated with morphine for more than 15 min (Fig. 1A middle panel and and1B).1B). Further analyses demonstrated that Akt was activated after 15 and 30 min of morphine treatment (Supplementary Fig. 1A and 1B). These data demonstrated that the morphine-induced mTOR activation was not fully mediated by PI3K signaling pathway. The morphine-induced mTOR activation could be divided into two phases and only the late phase was related to PI3K signaling pathway.
Our previous data identified that FKBP12 is an OPRM1 association protein in mammalian cells and Pro353 residue in the carboxyl tail of OPRM1 is involved in the interaction . Site mutation of Pro353 to Ala (OPRM1P353A) abolished the association of FKBP12 with OPRM1 . FKBP12 binding to rapamycin regulates the activities of mTOR and its downstream effectors, which is highly conserved from yeast to humans [5, 6]. Thus we investigated whether the interaction of FKBP12 with OPRM1 could affect the morphine-induced mTOR activation. As shown in Fig. 1A lower panel and 1B, morphine did not activate mTOR in HEK293 cells expressing OPRM1P353A at all time points, demonstrating that receptor association with FKBP12 played an important role in morphine-induced mTOR activation and could be the prerequisite for PI3K-mediated mTOR activation. The activation of p70 S6K, one of mTOR downstream effectors, was measured by detecting its phosphorylation at Thr389. In cells expressing wild type OPRM1, phosphorylation of p70 S6K was increased to 2.09 ± 0.51 fold over basal level after 5 min treatment of morphine (Fig. 2A and 2B), whereas the increase of p70 S6K phosphorylation was not observed in cells expressing OPRM1P353A mutant (Fig. 2A and 2C). To further confirm the effects of FKBP12 association with OPRM1 on morphine-induced mTOR activation, the endogenous FKBP12 of OPRM1-expressing cells were knocked down by specific siRNA. As shown in Fig. 2D and 2E, knockdown of endogenous FKBP12 abolished morphine-induced activation of mTOR and p70 S6K, whereas, treatment with scrambled siRNA did not affect the activities of mTOR and p70 S6K activated by morphine.
Next, we investigated whether morphine-induced mTOR activation was a direct consequence of OPRM1 activation. As shown in Fig. 2A and 2B, morphine-induced phosphorylation of mTOR and p70 S6K was almost totally inhibited by pre-incubation with selective OPRM1 antagonist CTAP. Moreover, treatment with nonselective antagonist naloxone alone did not affect the activity of mTOR (Supplementary Fig. 2A and 2B). These results indicated that the effect of morphine was dependent on OPRM1 activation.
To further explore whether morphine-induced activation of mTOR is mediated by direct interplay between OPRM1 and FKBP12, the receptor signaling complex after activation was immunoprecipitated and analyzed. As shown in Fig. 3A and 3B, mTOR existed in the receptor complex in the absence of morphine. After morphine treatment, the amount of total mTOR in receptor complex did not change, but phosphorylated mTOR increased in activated receptor complex in cells expressing wild type OPRM1 (3.05 ± 0.40 folds over basal level). Whereas, in OPRM1P353A-expressing cells in which the interaction of FKBP12 with receptor is absent or in wild type OPRM1-expressing cells with FKBP12 knockdown, mTOR was still present in the receptor complex without morphine treatment, however, morphine treatment did not change the amount of phosphorylated mTOR in receptor complex. Pretreatment with rapamycin which binds to FKBP12 and inhibits mTOR activity also abolished the morphine-induced increase of phosphorylated mTOR in receptor complex.
The increased activation of mTOR in receptor signaling complex were also confirmed by immunofluorescence. As expected, after treatment of OPRM1-expressing cells with 1 μM morphine for 5 min, OPRM1 and phosphorylated mTOR were observed to be colocalized in cluster mainly on cell membrane and in cytoplasm (Fig. 4A). Quantification analysis demonstrated that the colocalization of receptor and phosphorylated mTOR was increased 1.27 ± 0.05 folds over basal level (Fig. 4B). These results were consistent with the association of phosphorylated mTOR and OPRM1 in response to receptor activation demonstrated by immunoprecipitation assay. Such increased colocalization of OPRM1 and phosphorylated mTOR was absent or significantly reduced in cells expressing mutant OPRM1P353A, as well as in OPRM1-expressing cells with FKBP12 level knocked down by siRNA treatment or with rapamycin pretreatment (Fig. 4A and 4B). These data indicated that receptor-associated FKBP12 promoted the phosphorylation of mTOR within receptor complex.
In the present study, we demonstrated that morphine induced time-dependent mTOR activation in HEK293 cells stably expressing OPRM1. Even though PI3K signaling played an important role in mTOR activation, the effect could not be fully blocked by PI3K inhibitor which only inhibited mTOR activation at the late phase. Whereas, mTOR activation by OPRM1 were blocked in cells expressing the mutant receptor incapable of binding to FKBP12 at all time points.
As a GPCR, OPRM1 could activate PI3K/Akt signaling pathway through Gβγ subunits . The OPRM1-induced activation of mTOR has been attributed to the activation of PI3K/Akt by our and others’ studies [3, 4, 16]. The activation of Akt by morphine has been reported to occur after 15 to 30 min treatment of morphine [24, 25]. In our study, we also demonstrated that Akt was not activated at 5 min of morphine treatment, which is consistent with others’ results. Our current study further demonstrated that PI3K inhibitor did not affect the initial mTOR action induced by morphine. These data suggest that the mTOR activation at 5 min of morphine treatment was not mediated through PI3K/Akt signaling.
Our previous data identified FKBP12 as a specific OPRM1 association protein . Conditional knockout of FKBP12 enhances mTOR-Raptor interaction, long-term potentiation, memory, and perseverative and repetitive behavior . Our results showed that basal mTOR activity was increased in cells transfected with FKBP12 siRNA (Fig. 2D and 2E), which is consistent with previous study . However, morphine-induced mTOR activation was absent in these cells, implicating that morphine-induced mTOR activation is not dependent on mTOR-Raptor interaction. Morphine-induced activation of mTORC1 was blocked in cells expressing mutant receptor deficient in interacting with FKBP12, indicating that morphine-induced mTOR activation is dependent on the association of FKBP12 with the receptor.
Further analysis of receptor complex manifested that the association of receptor and mTOR existed without receptor activation but the phosphorylated mTOR in the receptor complex was specifically increased after receptor activation by morphine. However, such increased association of phosphorylated mTOR was diminished in mutant receptor or in wild type receptor with FKBP12 knockdown without affecting the association of mTOR and receptor. Rapamycin also blocked the increase of phosphorylated mTOR associated with the receptor. Such effects were further confirmed by colocalization of OPRM1 with phosphorylated mTOR. These results demonstrate that receptor’s association with FKBP12 played a critical role of FKBP12 in mTOR activation by morphine and was unnecessary for the association between mTOR and receptor. The association between receptor and mTOR has not been reported and merits further investigation.
Blockade of receptor with antagonist naloxone did not affect mTOR activation. Neither the activated mTOR in receptor complex nor increase in the colocalization of activated mTOR and OPRM1 was observed in cells treated with naloxone (Supplementary Fig. 3A and 3B). Moreover, mTOR association with the receptor could not initiate mTOR activation upon morphine treatment when the interaction between FKBP12 and OPRM1 was impaired. Thus, mTOR activation is dependent on receptor activation and FKBP12 association with the receptor. In that the phosphorylation of mTOR by PI3K could also be blocked in the absence of receptor-FKBP12 interaction, we hypothesize that FKBP12 may act as an adaptor to toggle OPRM1 and mTOR and play a role in mediating the effect of PI3K.
Our current study demonstrated the critical role of receptor-FKBP12 association in OPRM1-induced mTOR signaling. PI3K acts in concert with receptor-associated FKBP12 to modulate mTOR activity. Our study uncovered a new intracellular pathway of mTOR activation through OPRM1. As modulation of mTOR signaling by OPRM1 has been shown to be involved in multiple physiological and pathological processes, our findings may further clarify the underlying mechanisms. Therefore, our study could provide a possible target for modulating these processes.
This research was supported by National Natural Science Foundation of China (81173044), International Science & Technology Cooperation Program of China (2011DFA33180) and Shanghai Pujiang Program (11PJ1406200)) and in parts by National Institutes of Health grants (DA007339, DA011806).
Conflict of interest: The authors have no conflict of interest.