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INFLAMMATION AND IMMUNOPHARMACOLOGY
Opioid Receptors Stimulate Akt-Dependent Phosphorylation of c-jun in T Cells
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee
Received June 22, 2005; accepted October 24, 2005.
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Abstract |
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opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala2-D-Leu5]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH2-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10-10 to 10-6 M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.
opioid receptors (DORs) prepares activated mature peripheral T cells for the immunomodulatory effects of exogenous and endogenous opioids. A relatively small fraction of naive quiescent peripheral T cells express DORs (Miller, 1996
; Sharp et al., 2001, 2005). However, activation through the T cell receptor (TCR) markedly up-regulates both the fraction of T cells that express DORs and the number of DORs present on each T cell. These findings are based on studies showing that DORs were induced in vivo by antigenic stimulation with staphylococcal enterotoxin B and in vitro by mitogen treatment or cross-linking the TCR (Miller, 1996; Shahabi et al., 2000; Sharp et al., 2001).
DOR agonists exert diverse immunomodulatory effects on T cell responses, including T cell proliferation, cytokine production, chemotaxis, and thymic T cell selection (Shahabi and Sharp, 1995; Rogers et al., 2000; McCarthy et al., 2004). Recent studies indicate that DOR agonists affect intracellular signaling pathways in T cells involving mitogen-activated protein kinases (MAPKs). For example, [D-Ala2-D-Leu5]-enkephalin (DADLE), a selective DOR agonist, rapidly and dose-dependently attenuated the anti-CD3-
-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (Shahabi et al., 2000). In contrast, DADLE alone was sufficient to stimulate the hyperphosphorylation of activating transcription factor (ATF)-2, which is involved in cytokine gene transcription (Shahabi et al., 2003). DADLE also induced the association of the MAPK c-jun NH2-terminal kinase (JNK) with hyperphosphorylated ATF-2. Apart from immunomodulatory interactions between DORs and certain TCR-dependent signaling cascades, the effects of DADLE alone on JNK suggest that activation of DOR may be sufficient to signal through certain intracellular pathways. Thus, we hypothesized that DADLE might stimulate c-jun phosphorylation in T cells.
JNK is known to phosphorylate transcription factors and an array of other molecules known to regulate cell viability, cellular stress responses, apoptosis, and proliferation (for review, see Manning and Davis, 2003). c-Jun is one of multiple transcriptional proteins [e.g., ATF-2 and nuclear factor of activated T cell (NFAT)] implicated in JNK signaling within T cells. In addition to c-jun, JNK phosphorylates other transcription proteins that form activator protein-1 dimers (Hibi et al., 1993; Kallunki et al., 1996). Phosphorylation of the c-jun NH2-terminal activation domain enhances the activity of activator protein-1, promoting the transcription of T cell cytokine genes such as interleukin-2, granulocyte-macrophage colony stimulating factor (Thomas et al., 1997), 
-interferon (Ye et al., 1996), and tumor necrosis factor-
(Becker et al., 1999).
Recent studies of opioid receptor signaling in transfected COS-7, SH-SY5Y, and NG108-15 cell lines showed that opioid receptors activate JNK, as indicated by the detection of phosphorylated JNK and the transient phosphorylation of c-jun, using in vitro kinase assays (Kam et al., 2003, 2004). Activation of JNK by the µ opioid receptor was dependent on phosphoinositide 3-kinase (PI3K), whereas DOR and 
opioid receptor signaling were not. In addition, Akt, a serine-threonine kinase that is one of the key downstream targets of PI3K (Cantrell, 2001), was not implicated in JNK phosphorylation by opioid receptors in these neuronal cell lines. However, both PI3K and Akt have been shown to facilitate agonist-induced µ opioid receptor desensitization in sensory neurons (Tan et al., 2003). Thus, we determined whether DADLE induced the PI3K and Akt-dependent phosphorylation of c-jun in splenic T cells. We report herein that DADLE induced JNK-dependent c-jun phosphorylation in normal T cells, requiring both PI3K and Akt.
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), a second Akt inhibitor, C31H27In4S (ai; identification no. 5233705; ChemBridge Corporation, San Diego, CA) (Kau et al., 2003), a JNK inhibitor (SP600125) (Bennett et al., 2001), wortmannin, and LY294002 were obtained from Calbiochem (San Diego, CA).
Animals and Cell Culture. BALB/c mice 5 to 7 weeks old were obtained from NCI (Bethesda, MD) and maintained for 1 week in specific pathogen-free facilities (12 h light/12 h dark) at a constant temperature (20°C) with ad libitum access to food and water. They were sacrificed by cervical dislocation under isofluorane anesthesia. All procedures were conducted in accordance with National Institutes of Health guidelines and approved by the Animal Care Committee of the University of Tennessee. Spleens were dispersed through a wire mesh screen, erythrocytes were eliminated with lysing buffer, and then cells were spun, washed, and layered on Ficoll-Hypaque, followed by centrifugation at 400g for 10 min. The interface layer was recovered, washed, and cultured at 2 x 106 cells/ml in RPMI 1640 with penicillin-streptomycin-glutamine, 5% fetal bovine serum, and 5 x 10-5 M 
-mercaptoethanol. Pooled splenocytes were cultured for 48 h in flasks coated with anti-CD3- (80 ng/cm2), rested for 24 h in the absence of anti-CD3-, and then starved for 4 h in the absence of serum. Thereafter, cells were divided into separate aliquots equivalent to the number of samples necessary for all treatment groups. Each of these samples was treated with the specific reagents identified in the figures and under Results. Each study was based on four to six total samples per treatment group.
Membrane Preparation. Cells were washed briefly with PBS, lysed using 500 ml of radioimmunoprecipitation assay buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, and a mixture of protease and phosphatase inhibitors), and then sonicated three times (2-3 s each) on ice. Lysates were centrifuged at 120,000g for 60 min at 4°C. Pellets were resuspended in radioimmunoprecipitation assay buffer containing 0.1% Triton X-100, sonicated briefly, and protein concentrations were determined using the BCA Protein Assay Kit. Membranes were boiled for 5 min, and 5 to 10 mg of protein was loaded on 11% SDS polyacrylamide gels (1.5-mm depth).
Western Blotting. SDS polyacrylamide gels were transferred to nitrocellulose membrane (Whatman Schleicher and Schuell, Keene, NH) over 16 h at 24°C. Membranes were briefly washed with Tris-buffered saline (20 mM Tris-HCl, 137 mM NaCl, 0.05% Tween 20, and 0.05% NP-40, pH 7.7) and then blocked in Tris-buffered saline containing 5% nonfat dry milk for 2 h. They were incubated at room temperature with primary antibody for 90 min. Membranes were washed extensively, incubated with a peroxidase-tagged secondary antibody (Pierce Chemical) for 1 h at room temperature, and chemiluminescence was detected (Super Signal ULTRA; Pierce Chemical).
Immunofluorescence Flow Cytometry. Splenocytes were cultured for 48 h in tissue culture flasks coated with purified anti-mouse-CD3- (80 ng/cm2; BD Biosciences PharMingen, San Diego, CA). Thereafter, cells were rested for 24 h, starved for 15 h in the absence of serum, and then incubated for 15 min with PBS, DADLE (10-6 M), or phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich) plus A23187
[GenBank]
(1 µM; Calbiochem). Cells were centrifuged, incubated with 1 ml of FCM fixation buffer (Santa Cruz Biotechnology, Inc.) for 20 min at 4°C, washed twice with PBS, and incubated with 1 ml of FCM permeabilization buffer (Santa Cruz Biotechnology, Inc.) for 15 min at 4°C. Cells were then washed (2x) with FCM wash buffer (Santa Cruz Biotechnology, Inc.) and stained for 60 min at room temperature with phosphatidylethanolamine-labeled anti-p-c-jun (Santa Cruz Biotechnology, Inc.) and FITC-labeled anti-CD90 (Thy-1; BD Biosciences PharMingen). Cytofluorimetric analyses (104 cells per run) were performed using an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA) equipped with an argon laser and filtered for excitation at 488 nm and emission at 525 to 695 nm. Mouse isotypes (i.e., IgG1-phosphatidylethanolamine and IgG1,-FITC) were used for background subtraction.
Statistical Analyses. One-way analysis of variance (ANOVA) was performed, and post hoc testing was done using Bonferroni test. Differences were considered significant at p < 0.05. Values are expressed as mean ± S.E.M.
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for 48 h, rested for 24 h, and then starved for 4 h in the absence of serum prior to adding a specific DOR agonist. Phosphorylation of c-jun by DADLE was concentration-dependent [10-12 to 10-6 M (D12-D6); n = 3 experiments; F = 5.08, p = 0.002], and phospho-c-jun (p-c-jun) increased approximately 5-fold over basal levels after treatment with 1 µM DADLE. Phosphorylated c-jun also was detected by immunofluorescence flow cytometry, as shown in Fig. 2. Using the same protocol for cell culture and treatment with DADLE, splenocytes were labeled with anti-Thy-1 to identify T cells and with anti-phospho-c-jun after permeabilization. The percentage of double-positive cells (T cell containing phospho-c-jun) significantly increased from 101 ± 3.9% (mean ± S.E.M.) in controls to 153.6 ± 6.1 in cells treated with 1 µM DADLE (D6) and 241.1 ± 15.7 in response to PMA (n = 9; F = 49.9, p < 0.001; p = 0.005 for control versus DADLE). In addition, after stimulation with DADLE, 95% of the cells positive for phospho-c-jun were T cells.
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The compound SP600125 (S) is a potent, specific inhibitor of the JNKs, which mediate phosphorylation of c-jun (Hibi et al., 1993; Bennett et al., 2001). Thus, this agent was used to demonstrate that DADLE-induced c-jun phosphorylation is dependent on JNK. Figure 5 shows that S alone had a small effect on the level of phospho-c-jun, and it largely prevented DADLE-induced phosphorylation. Densitometric analysis confirmed this, demonstrating that DADLE (D6) significantly increased phospho-c-jun, and S + D6 was significantly less (F = 6.67, p = 0.001; D6 versus control, p < 0.001; D6 versus S10 + D6, p = 0.001; D6 versus S30 + D6, p = 0.01).
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; Shah et al., 2005). Moreover, activation of JNK by the µ opioid receptor required PI3K (Kam et al., 2003, 2004). Therefore, we evaluated the potential role of PI3K and Akt in phosphorylation of c-jun by DADLE. In the first experiments, the ability of DADLE to activate PI3K was evaluated indirectly by assessing the phosphorylation of membrane Akt, which initially docks to phosphatidylinositol-3,4,5-P3 at the inner leaflet of the plasma membrane and subsequently becomes phosphorylated by phosphoinositide-dependent kinase 1 (Vanhaesebroeck and Alessi, 2000). Figure 6 shows that DADLE significantly induced phosphorylation of Akt (p-Akt), increasing p-Akt by 300 to 400% of baseline for at least 30 min (F = 3.6, p = 0.013; p = 0.03, 0.006, and 0.001 for upper band of control versus 10, 20, and 30 min, respectively). Experiments shown in Fig. 7 demonstrate that wortmannin (WT; 100 nM), a specific inhibitor of PI3K (Ui et al., 1995; Carpenter and Cantley, 1996), abolished DADLE-induced phosphorylation of c-jun without affecting basal levels of c-jun phosphorylation. Densitometric analysis confirmed this (bottom panel), showing that the phosphorylation of c-jun by DADLE (D6) in the presence of WT/D6 was significantly less than D6 alone (F = 25.69, p < 0.0001; D6 versus control, p < 0.0001; WT + D6 versus D6, p = 0.0001). Figure 8 demonstrates that LY294002 (10-20 µM; L), a specific inhibitor of PI3K, also reduced c-jun phosphorylation by DADLE (D6). Densitometric analysis (bottom panel) showed that phosphorylation of c-jun by D6 in the presence of LY294002 (L10/D6 or L20/D6) was significantly less (**) than D6 alone [F = 10.22, p < 0.0001; D6 versus control, p < 0.0001 (*); L (10 or 20) + D6 versus D6, p < 0.0001]. Thus, experiments with wortmannin and LY294002 demonstrate that DADLE-induced c-jun phosphorylation depends on PI3K.
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The potential role of Akt in c-jun phosphorylation was studied further using two different inhibitors. The first, AI (Hu et al., 2000), a phosphatidylinositol ether analog (EC50 = 5.0 versus 83 µM against Akt compared with PI3K, respectively), dose-dependently blocked the phosphorylation of c-jun by DADLE (D6) (Fig. 9). This occurred within a concentration range with high Akt specificity. Indeed, c-jun phosphorylation was reduced by more than 50% at 10 µM AI, and 20 µM AI caused no greater inhibition (F = 5.65, p = 0.002; D6 versus control, p = 0.0003; AI10 + D6 versus D6, p = 0.038). The second Akt inhibitor, ai (ChemBridge Corporation identification no. 5233705; Kau et al., 2003), is a benzimidazole compound that blocks the phosphorylation and activation of Akt, apparently without affecting PI3K. The representative experiment in Fig. 10 (top panel) showed that ai dose-dependently reduced the phosphorylation of c-jun by DADLE; both 5 and 10 µM ai abolished the response. Densitometric analysis (bottom panel) demonstrated that 5 or 10 µM ai significantly inhibited D6-induced phosphorylation of c-jun (F = 9.48, p < 0.0001; D6 versus control, p < 0.0001; ai5 + D6 versus D6, p < 0.0001; ai10 + D6 versus D6, p < 0.0001). Thus, Akt seems to be required for c-jun phosphorylation by DADLE.
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). In contrast, we observed that PI3K was required for DADLE-induced c-jun phosphorylation in T cells. As such, Akt, a proxy for PI3K activity, was phosphorylated by DADLE, and both wortmannin and LY294002 blocked c-jun phosphorylation. We also found that two inhibitors of Akt, which are chemically diverse in structure, blocked DOR-dependent c-jun phosphorylation. One of these, a first generation compound, inhibited c-jun phosphorylation at concentrations that would affect Akt, but not PI3K. The other antagonist, a second generation compound, is known to inhibit phosphorylation of Akt, without interacting with PI3K (Kau et al., 2003); however, its precise mechanism of action is unknown. And, in general, the specificity of Akt inhibitors has not been studied definitively. This second generation inhibitor was effective at lower concentrations than the first generation compound. Thus, in addition to PI3K, Akt seems to be required for DOR activation of JNK and c-jun.
Although opioid receptors activate JNK and c-jun, the potential role of Akt in signaling from seven-transmembrane G-protein-coupled receptors to JNK, and c-jun is largely undefined. In sensory neurons chronically exposed to the specific µ opioid receptor (MOR) agonist, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin, desensitization of MOR-dependent modulation of voltagegated calcium channels was shown to require persistent activation of both Akt and PI3K (Tan et al., 2003). This, however, seems to be independent of JNK. In contrast, both MOR and DOR have been reported to stimulate JNK phosphorylation, although only MOR required PI3K, and this did not involve Akt (Kam et al., 2003, 2004). Other studies indicate that Akt can regulate protein kinases upstream of JNK, inhibiting the phosphorylation of JNK and protecting against JNK-dependent apoptosis in susceptible cells (for review, see Brazil et al., 2004). Because there are a variety of kinases capable of activating JNK (for review, see Kyriakis and Avruch, 2001), the specific cellular context, including factors such as cell type and compartmentalization of signaling molecules responsive to specific extracellular stimuli, may determine how Akt regulates JNK activity.
The JNKs are one branch of the MAPK family, and they are activated by upstream kinases termed MAPK kinases (MAPKKs; i.e., MKK4). MAPKKs are themselves activated by serine/threonine phosphorylation mediated by MAPKK kinases. Three JNK isoenzymes have been identified: JNK-1, JNK-2, and JNK-3. These are specifically phosphorylated by MKK4 and MKK7, although more than 10 MAPKK kinases activate the JNK pathway (for review, see Manning and Davis, 2003). Thus, a wide variety of stimuli, most commonly related to environmental stressors, converge on the activation of JNKs. Within the T cell, JNK is involved in thymocyte apoptosis and T cell proliferation and differentiation. Specific roles of isoenzymes of JNK in T cell differentiation were demonstrated by the following: jnk1-/- mice exhibited enhanced T helper cell type 2 (Th2) differentiation and nuclear accumulation of the transcription factor termed NFATc (Dong et al., 1998), and JNK-2 was necessary for Th1 differentiation (Yang et al., 1998). In addition, JNK-2 may be involved in proliferation and differentiation of peripheral T cells (Dong et al., 1998; Sabapathy et al., 1999). Thus, both JNK-1 and -2 seem to be involved in the T helper cell-type-specific differentiation.
Recent investigations indicate that N-terminal phosphorylation of c-jun by JNK is not required for T cell proliferation and differentiation (Behrens et al., 2001). However, JNK-2 is required in these processes, implicating another nuclear effector(s). JNK-2 has been shown to regulate the DNA binding and transactivational properties of the NFAT transcriptional complex upon stimulation of peripheral T cells (Behrens et al., 2001). Recent studies have demonstrated that JNK activates NFATc2-dependent transcription, and this is associated with phosphorylation of multiple residues within the NFATc2 regulatory domain (Ortega-Perez et al., 2005). Mutations of T116 and s170 indicate that these residues are critical for NFATc2 transactivation by JNK in Jurkat T cells. In contrast to previous studies indicating that JNKs exclusively inhibited NFAT (i.e., NFATc1 and NFATc3), Ortega-Perez et al. (2005) have clearly demonstrated positive subtype-specific regulation of NFATc2.
The Akt-dependent inhibition of kinases upstream of JNK, which attenuates JNK activity, may be an incomplete picture of Akt interaction with JNK. Analogous to the foregoing observations, one may postulate that Akt phosphorylates and activates a subset of JNK, defined by compartmentalization and/or different isoenzyme subtype. The data in the current study of DADLE-induced c-jun phosphorylation are potentially consistent with such a model of selective JNK activation by Akt. In summary, these experiments demonstrate that DORs stimulate c-jun phosphorylation through JNK, which is dependent on the upstream activity of PI3K and, most probably, Akt.
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ABBREVIATIONS: DOR, opioid receptor; TCR, T cell receptor; MAPK, mitogen-activated protein kinase; DADLE, [D-Ala2-D-Leu5]-enkephalin (D6); ATF, activating transcription factor; JNK, c-jun NH2-terminal kinase; NFAT, nuclear factor of activated T cell; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; AI, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate; ai, C31H27In4S; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one 1,9-pyrazoloanthrone (S); LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (L); PBS, phosphate-buffered saline; A23187
[GenBank]
, calcimycin; PMA, phorbol 12-myristate 13-acetate; p-c-jun, phospho-c-jun; FITC, fluorescein isothiocyanate; ANOVA, analysis of variance; PTX, pertussis toxin; p-Akt, phosphorylation of Akt; WT, wortmannin; MOR, µ opioid receptor; MAPKK, MAPK kinase; FCM, flow cytometry.
1 c-jun, p-c-jun, and total c-jun refer to protein. 
Address correspondence to: Dr. Burt M. Sharp, Department of Pharmacology, University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163. E-mail: bsharp{at}utmem.edu
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