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. Author manuscript; available in PMC: 2011 Dec 14.
Published in final edited form as: J Neurochem. 2009 May 8;110(2):662–674. doi: 10.1111/j.1471-4159.2009.06156.x

Inhibition of EGF-induced ERK/MAP kinase-mediated astrocyte proliferation by µ opioids: integration of G protein and β-arrestin 2-dependent pathways

Mayumi Miyatake 1, Tal J Rubinstein 1, Gregory P McLennan 1, Mariana M Belcheva 1, Carmine J Coscia 1
PMCID: PMC3236703  NIHMSID: NIHMS328579  PMID: 19457093

Abstract

Although µ, κ, and δ opioids activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase, the mechanisms involved in their signaling pathways and the cellular responses that ensue differ. Here we focused on the mechanisms by which µ opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)-induced ERK-mediated astrocyte proliferation. The µ-opioid agonists ([d-ala2, mephe4, gly-ol5] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via Gi/o protein, calmodulin (CaM), and β-arrestin2-dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the µ-opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W-7, and siRNA silencing of β-arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation-mediated mechanism. This was disclosed by abolishment of µ-opioid-induced ERK phosphorylation with the EGF receptor-specific tyrosine phosphorylation inhibitor, AG1478, and µ-opioid-induced reduction of EGF receptor tyrosine phosphorylation by PTX, and β-arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long-term (h) treatment of primary astrocytes with [d-ala2, mephe4, gly-ol5] enkephalin or morphine, attenuated EGF-induced ERK phosphorylation and proliferation (as measured by 5′-bromo-2′-deoxy-uridine labeling). PTX and β-arrestin2 siRNA but not W-7 reversed the µ-opioid inhibition. Unexpectedly, β-arrestin-2 siRNA diminished both EGF-induced ERK activation and primary astrocyte proliferation suggesting that this adaptor protein plays a novel role in EGF signaling as well as in the opioid receptor phase of this pathway. The results lend insight into the integration of the different µ-opioid signaling pathways to ERK and their cellular responses.

Keywords: astrocytes, extracellular signal-regulated kinase/mitogen-activated protein kinase, G proteins, opioid receptors, opioids, β-arrestins

The MAP kinases play central role in the regulation of cellular processes as diverse as proliferation, differentiation, and cell–cell communication (Raman et al. 2007). Initially, MAP kinase pathways were discovered to be driven by growth factors, but subsequently they were also found to entail cross-talk between G protein-coupled receptor (GPCR) and growth factor signaling. The multiple mechanisms of extracellular signal-regulated kinase (ERK)/MAP kinase activation are cell type specific and the cellular environment can influence GPCR agonist and antagonist regulation of this signaling pathway. Therefore, it is important to characterize and distinguish the signaling pathways leading to ERK and their integration in cellular responses such as proliferation in discrete cells.

Astrocytes are the most abundant cells in human brain. They are dynamic partners with neurons in synaptogenesis and with adult neural progenitor cells in neurogenesis as shown in vitro and in vivo (Christopherson et al. 2005; Nishida and Okabe 2007; Jiao et al. 2008). Astrocyte proliferation is important in brain development, and astrogliosis has been found to be associated with brain or spinal cord injury, neurodegenerative, and other diseases, such as autism, muscular dystrophy, HIV infection, and dementia in drug users and Alzheimer’s disease (Bell et al. 1998; Terai et al. 2001; Yang et al. 2007; Buffo et al. 2008; Fatemi et al. 2008). Astrocyte dysfunction impacting their proliferation has been found in some neurodegenerative diseases such as amyotrophic lateral sclerosis (Lepore et al. 2008). The proliferation of astrocytes may also be triggered for the purpose of structural reorganization as proposed for spinal cord injury (Xu et al. 2007). Astrogliosis may be beneficial or it may be a maladaptive feature ensuing under pathophysiological conditions.

In our initial studies on opioid signaling in immortalized rat cortical astrocytes, we found that both µ- and κ-opioids acting via their receptors rapidly (min) stimulated ERK phosphorylation (Belcheva et al. 2003). µ-Opioid receptor (MOR) and κ-opioid receptor (KOR) signaling via ERK/MAP kinase differ in their temporal patterns, cellular responses, and signaling components upstream of ERK (Belcheva et al. 2001, 2003, 2005). Sustained (h) KOR activation of ERK led to the stimulation of astrocyte proliferation (McLennan et al. 2008). In contrast, one outcome of long-term MOR signaling entailed inhibition of the epidermal growth factor (EGF)-induced ERK activation by the selective µ-opioid agonist enkephalin analog, [D-ala2,mephe4,gly-ol5] enkephalin (DAMGO). Studies on the inhibitory mechanism suggested that it was triggered by the acute activation of ERK that then initiated a feedback inhibition loop. We also learned that EGF receptor (EGFR) serine phosphorylation and internalization occurs via this ERK-dependent putative feedback loop mechanism and thereby may be involved in the attenuation of the exogenous EGF-driven signaling pathway upon long-term DAMGO exposure (Belcheva et al. 2003).

In a study of the GPCR phase of this pathway, we obtained evidence for a CaM-dependent, G-protein independent µ-opioid signaling pathway to ERK via EGFR transactivation wherein CaM binds to the third intracellular loop of MOR (Belcheva et al. 2001, 2005). Inhibition of this pathway using a mutant K273A MOR which has reduced affinity for CaM compared with the wild type receptor along with other data, suggested that CaM binds to MOR and displaces G protein. Use of K273A MOR, CaM antisense or pharmacological inhibitors of CaM signaling, only partially attenuated ERK activation indicating that other MOR pathways to ERK exist.

The existence of β-arrestin-dependent signaling to ERK has been demonstrated for many GPCRs in different cell types (Ahn et al. 2004; Barnes et al. 2005; Gesty-Palmer et al. 2006; Shenoy et al. 2006; McLennan et al. 2008). MOR interactions with β-arrestin in vitro and in vivo have also been documented and its signaling to ERK may be mediated by β-arrestin 1 or 2 depending upon the ligand, state of receptor heterodimerization, and the time of agonist exposure among other factors (Bohn et al. 1999; Macey et al. 2006; Groer et al. 2007; Rozenfeld and Devi 2007; Tidgewell et al. 2008; Zheng et al. 2008).

The question that we address here is which of these three pathways, CaM, G protein, and β-arrestin occur in primary astrocytes and are involved in opioid inhibition of exogenous EGF-induced ERK activation and subsequent astrocyte proliferation. We compared the action of morphine with that of DAMGO, because in some cellular environments, this narcotic does not initiate rapid MOR internalization (Keith et al. 1996). The role of morphine has implications for the pathology of opiate abuse and there is in vitro as well as in vivo evidence for an inhibitory role of morphine in astrocyte proliferation (Stiene-Martin et al. 1991, 2001; for a review see Sargeant et al. 2008). Therefore, the impact of both short- and long-term exposure of these two agonists on MOR signaling to EGFR and ERK via CaM, G protein, and β-arrestin and on the cellular response of inhibition of EGF-induced proliferation was investigated using immortalized and primary astrocyte cultures.

Experimental procedures

Reagents

Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) with the following exceptions: CTAP, DAMGO, morphine, and norbinaltorphimine were obtained from NIDA Drug Supply (Research Triangle, NC, USA); Protein G plus/Protein A-Agarose beads, EGF, AG1478, and W-7 were obtained from Calbiochem (San Diego, CA, USA); trypsin-EDTA solution was obtained from Gibco (Carlsbad, CA, USA); PTX was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA); Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from ATCC (Manassas, VA, USA); anti-phospho-ERK1/2 (p-ERK; directed against phospho Thr202/Tyr204) antibody (Ab), anti-EGFR Abs for immunoprecipitation and immunoblotting, and antiphosphoTyr Ab were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-glial fibrillary acidic protein (GFAP) Ab was obtained from ImmunoStar, Inc. (Catalog #:22522; Hudson, WI, USA); anti-β-arrestin2 and anti-ERK Ab were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-TuJ 1 Ab was obtained from Neuromics (Edina, MN, USA); FuGENE 6, 5′-bromo-2′-deoxy-uridine (BrdU), and the Abs for its detection from the BrdU Labeling and Detection Kit I was obtained from Roche (Basel, Switzerland), Alexa Fluor-labeled secondary Abs and horse serum were obtained from Invitrogen (Carlsbad, CA, USA) or Molecular Probes (Eugene, OR, USA); and Vectashield Mounting Medium was obtained from Vector Laboratories, Inc. (Burlingame, CA, USA). All siRNA directed against the β-arrestin2 gene and non-targeting control siRNA were purchased from Thermo Scientific Dharmacon RNA Technologies (Lafayette, CO, USA).

Primary astrocyte cultures

Postnatal day 1 Sprague–Dawley rat pups were killed and their cortical regions were dissected out, minced, suspended in 2.5 mL ice-cold phosphate-buffered saline (PBS), and trypsinized by incubation with an equal volume of 0.05% trypsin-EDTA solution at 37°C for 15 min. The tissue was pelleted (1000 g,, 10 min), resuspended in 5 mL DMEM containing 5% FBS and 5% horse serum, triturated, and plated onto poly-l-lysine-coated (mol. wt: 30 000–70 000) tissue culture flasks as indicated.

After 7 days in culture, flasks were shaken for at least 4 h, after which the unattached cells were removed and fresh culture medium was added (DMEM, 5% FBS + 5% horse serum). For ERK1/2 assays growth medium was replaced with DMEM without serum 24 h prior to ligand treatment. For BrdU incorporation/GFAP staining, cells were grown to 50–60% confluency and growth medium was replaced with DMEM without serum for 28 h. Of the total number of cells in primary cultures, 90% were GFAP positive and <1% were TuJ 1 (neuronal marker) positive.

Type-1 immortalized rat cortical astrocyte cultures

Rat cortical astrocytes (CTX TNA2; ATCC, Manassas, VA, USA) were established from cultures of primary type 1 astrocytes from 1-day-old rat brain frontal cortex (Radany et al. 1992) and grown as described (Belcheva et al. 2003, 2005).

Transient transfections

Immortalized rat astrocytes were transiently transfected with MOR cDNA (pCMV neo-expression vector) using FuGENE 6 transfection reagent following the manufacturer’s instructions and adding 1 µg of cDNA and 3 µL of transfection reagent.

Preparation and transfection of siRNAs

siRNA targeting the rat β-arrestin2 gene was designed and synthesized by Dharmacon RNA Technologies. The following siRNA preparations were used: siGENOME standard SMART pool to rat ARRB2 (Catalog #D-080157-00; target sequences: 5′-GGAGCUACCUUUCGUCCUA-3′, 5′-GAUGAAGGAUGACGACUGU-3′, 5′-GAGAAGACCUGGAUGUACU-3′, and 5′-CAAAGAUCUGUUCAUCGC-3′) and siCONTROL non-targeting siRNA pool (negative control that has been bioinformatically designed and validated to not have any known targets, Catalog #D-001206-13, target sequences: 5′-AUGAACGUGAAUUGCUCAA-3′, 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′, and 5′-UAGCGACUAAACACAUCAA-3′). The siRNA preparations were resuspended in Dharmacon-provided siRNA buffer to a stock concentration of 20 µM. Immortalized astrocytes were transiently transfected with the siRNA preparation using the Amaxa Nucleofector electroporator (Amaxa Biosystems, Gaithers-burg, MD, USA). Briefly, cells were removed from flasks by treatment with 0.05% trypsin and 0.02% EDTA for 1 min at 37°C, washed with media, and incubated for 2 h at 37°C in a 50 mL conical tube. Equal amounts of cells no more than 2.0 × 106 were distributed in tubes, harvested by centrifugation, and resuspended in 100 µL rat astrocyte nucleofactor solution (Amaxa Biosystems). Two µg of MOR cDNA and 1 µM control or target siRNAs were added to each tube with immortalized astrocytes and electroporation was performed using optimal Amaxa Nucleofector program T-20. This program was developed by Amaxa specifically for astrocytes. The transfection efficiency of Amaxa electroporation was much higher than the 9 ± 1% that we obtained with FuGENE 6 as determined by Gal expression measurements. The Amaxa estimate for rat astrocyte transfection was 70% using their T-20 program. To document this, cells were transfected with enhanced green fluorescent protein, along with GFAP and DAPI and their stainings were compared. The majority of GFAP+ cells were labeled with enhanced green fluorescent protein with varying degrees of staining. Immediately following electroporation, cells were transferred to 6-well tissue culture plates or 8-well chambers and cultured overnight at 37°C. Then cells were washed three times with media deprived of serum and cultured for an additional 24 h in fresh growth media. Cells were grown for 24 h in serum-deprived media prior to initiation of ERK1/2 activity or proliferation determinations. In some experiments, FuGENE 6 was used to transfect immortalized and primary astrocytes as described (Belcheva et al. 2003). The efficiency of gene silencing was validated by immunoblotting with corresponding Abs.

ERK1/2 assays

ERK1/2 phosphorylation was measured by immunoblotting as described (Belcheva et al. 2001). Cells were treated first with either inhibitors or antagonists, and then with DAMGO or morphine as described in the figure legends. Cells were then washed with PBS and lysed with buffer containing 20 mM HEPES, 10 mM EGTA, 40 mM β-glycerophosphate, 2.5 mM MgCl2, 2 mM sodium vanadate, 1% Nonidet-40, 1 mM phenylmethylsulfonyl fluoride, 20 µg/mL aprotinin, and 20 µg/mL leupeptin. Cell lysates were centrifuged at 14 000 g for 10 min at 4°C and protein concentration of the supernatants was determined. Samples (10 µg protein/lane) were separated by 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. Proteins were blotted on Immobilon P™ polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). Non-specific sites were blocked with 5% milk in Tris-buffered saline + 0.2% Tween 20 (TBST). Blots were then washed three times with TBST and incubated with anti-pERK1/2 Ab (1 : 2000) for at least 15 h at 4°C. After three washes with TBST, blots were incubated with 1 : 2000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse-IgG for 1 h at 23°C. For assurance of equivalent total ERK1/2 protein per lane, representative blots were stripped (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris–HCl, pH 6.8, 60 min at 50°C) and exposed to anti-ERK1/2 Ab (1 : 1000), followed by 1 : 20000 diluted HRP-conjugated goat anti-rabbit-IgG Bands were visualized using an enhanced chemiluminescence detection system. Band intensities were determined by densitometry using photos taken with a Kodak DC120 digital camera (Kodak ds 1D version 3.0.2; Scientific Imaging Systems, Rochester, NY, USA) and analysing them with NIH ImageJ version 1.41m software (NIH, Bethesda, MD, USA). Data were calculated as pERK/ERK ratios. ERK stimulation in opioid-treated cells was expressed as fold change over basal levels in untreated cells.

EGFR immunoprecipitation and immunoblotting

Astrocytes cultures were serum-starved for 24 h and treated with opioids (1 µM, 1–2 min). Cultures were lysed as described (Belcheva et al. 2003) by using a modified radioimmunoprecipitation assay buffer containing: 50 mM Tris–HCl, pH 7.4, 1% TritonX-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenyl-methylsulfonyl fluoride, 1 µg/mL leupeptin, 1 µg/mL aprotinin, 1 mM Na3VO4, and 1 mM NaF. Cell lysates of 0.8 to 1.0 mg of protein (diluted to 1 µg/µL) were used for EGFR immunoprecipitation by adding 5 µg of a rabbit monoclonal anti-EGFR Ab and incubating overnight at 4°C. This step was followed by addition of a 40-µL suspension of protein G plus/Protein A-Agarose beads per sample and incubation for 3–4 h at 4°C. Beads were washed three times with PBS, resuspended in SDS loading buffer, and boiled for 5 min before 7.5% SDS–polyacrylamide gel electrophoresis. Proteins were blotted as described for ERK and incubated with a mouse monoclonal anti-phospho-Tyr100 Ab (1 : 2000), followed by incubation with HRP-conjugated IgG Membranes were reprobed for detection of EGFR (about 170 kDa) as a loading control by applying a rabbit polyclonal anti-EGFR Ab (1 : 1000) and anti-rabbit IgG linked to HRP Ab. Bands were visualized as described above in the ERK assay.

Immunocytochemical detection of BrdU incorporation and GFAP co-staining

Cells were grown to 50–60% confluency in poly-l-lysine-coated 8-well chamber slides treated with opioids and/or PTX, W-7, and siRNA for 24 h and were labeled with 10 µM BrdU for the final 4 h of treatment. Cells were fixed with 4% paraformaldehyde in PBS for 20 min at 23°C. After 3 × 5 min PBS washes, cells were incubated with 2 N HCl/0.5% Triton X-100 in 0.1× PBS for 1 h at 23°C, washed once for 5 min with PBS, pH 8.4, and 3 × 5 min with regular PBS, and blocked with PBS containing 0.5% bovine serum albumin and 0.1% Tween 20 for 30 min. Cells were incubated with rabbit anti-GFAP Ab (1:1 dilution) in blocking solution overnight at 4°C and washed 3 × 5 min with PBS. Then, cells were treated with mouse anti-BrdU monoclonal Ab, 1 : 10 dilution for 30 min at 37°C, washed 3 × 5 min with PBS, and then incubated with both secondary Abs for 1 h at 23°C [red fluorescence emitting Alexa Fluor 594 goat anti-rabbit IgG, highly cross-adsorbed Ab, 2 mg/mL, diluted 1 : 700 (for GFAP detection) and the green fluorescence emitting fluorescein-conjugated anti-mouse IgG Ab (1 : 10 dilution) from the kit for BrdU detection]. After three PBS washes, slides were mounted using anti-fade VECTASHIELD mounting medium and covered with cover slips. Slides were examined for immunofluorescence with an OLYMPUS AH-3 microscope attached to a Soft Imaging Systems F-view II CCD digital camera (Olympus America, Center Valley, PA, USA) that has simultaneous recording capability of dual fluorescence label images. In all proliferation studies, ≥500 cells were counted for each determination.

Protein assay and statistical analysis

Protein concentrations were determined by the method of Bradford (1976) with bovine serum albumin (1 mg/mL) as standard. Statistical determinations were made by Student’s t-test analysis for two groups or a one-way anova followed by the Newman–Keuls post hoc multiple comparison test (for proliferation data). Data were expressed as the mean ± SEM. All experiments were repeated at least three times. In primary culture studies, brains from at least two different litters were used for each determination.

Results

Both Morphine and DAMGO activate ERK via G protein and β-arrestin2 pathways in MOR-transfected immortalized type 1 astrocytes

As morphine had not been previously tested for its long-term effect on EGF activation of ERK, it was examined and found to mimic the DAMGO action (Fig. 1a). Having previously discovered that a CaM-dependent, G-protein independent MOR signaling pathway to ERK existed in astrocytes, we measured both morphine- and DAMGO-driven ERK activation in the presence of PTX to determine which family of G proteins were involved. As seen in Fig. 1b, both DAMGO- and morphine-induced ERK phosphorylation was attenuated by pre-treatment of MOR-transfected immortalized astrocytes with PTX suggesting the existence of a Gi/o-transduced signaling pathway to ERK driven by both agonists. The 50–60% inhibition was consistent with previous data in which CaM antagonism only partially inhibited ERK phosphorylation (Belcheva et al. 2001, 2005).

Fig. 1.

Fig. 1

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Inhibition of µ-opioid or EGF-induced ERK phosphorylation in immortalized astrocytes. (a) Cells were transfected with MOR cDNA, serum-starved, and treated with 1 µM morphine (Morph) or DAMGO, followed by EGF (50 ng/mL, 3 min) and ERK1/2 phosphorylation was assayed. In these and following figures of ERK assays, gels shown are representative immunoblots showing phosphorylated and total ERK1/2. EGF stimulated ERK1/2 [15 ± 1.6 fold over controls (n = 6)]. ERK1/2 phosphorylation was assayed. EGF stimulation of ERK was 10–15 fold over controls at concentrations of 10, 20, 50, and 100 ng/mL in immortalized astrocytes. *p < 0.05, **p < 0.01 versus control. n = 5 – 6. (b) Cells were transfected with MOR cDNA and serum-starved in the presence of 0.1 µg/mL PTX for 24 h, treated with 1 µm Morph or DAMGO for 5 min *p < 0.05, ***p < 0.001 versus control. †p < 0.05 versus their respective agonist alone. n = 6–10. (c) Cells were transfected with MOR cDNA (1 µg) and control (non-targeting) or β-arrestin2 siRNA (1 µM of each). After 24 h growth in serum-deprived media, cells were treated with 1 µM Morph or 1 µM DAMGO for 5 min and ERK1/2 phosphorylation was assayed. ***p < 0.001 versus control, †p < 0.05, ††p < 0.01 versus their respective agonist in the presence of control siRNA. n = 5–6. (d) Cells were transfected with (non-targeting) or β-arrestin2 siRNA (1 µM). After 24 h growth in serum-deprived media, cells were treated with 10 ng/mL EGF for 3 min and ERK1/2 phosphorylation was assayed. †††p < 0.001 versus EGF-treated controls, n = 4. Con, control.

As β-arrestin-mediated MOR activation of ERK existed that was cell type- and agonist-dependent, immortalized astrocytes were pre-treated with siRNA targeting β-arrestin2. The data in Fig. 1c indicate that morphine-induced ERK activation was abolished and there was a 60% diminution of that by DAMGO. Under these conditions, β-arrestin2 siRNA reduced levels of β-arrestin2 protein content, 40 ± 5% (p < 0.05, n = 4), in comparison with non-targeting control siRNA (McLennan et al. 2008).

Total abolition of morphine activation of ERK by β-arrestin2 siRNA was not consistent with the existence of parallel CaM and PTX-mediated signaling pathways to ERK. This led us to address the issue of the site of action of β-arrestin2 in the signaling pathway. Our earlier studies revealed that MOR and KOR activation of ERK was mediated by EGFR transactivation in human embryonic kidney 293 cells and immortalized astrocytes (Belcheva et al. 2001, 2003, 2005). We also found that morphine- and DAMGO-induced EGFR phosphorylation was blocked in CaM antisense transfected immortalized astrocytes, indicating that CaM action occurred at a step upstream of EGFR transactivation. This was consistent with previous evidence that CaM bound directly to MOR. It was then apparent that β-arrestin could interact with cell surface receptors other than GPCRs, including the insulin growth factor-1 receptor (Lefkowitz et al. 2006; Girnita et al. 2007). Therefore, it is possible that this adaptor protein could be acting in the growth factor receptor phase of the ERK signaling pathway. To test this possibility, EGF activation of ERK was measured using β-arrestin2 siRNA-transfected immortalized astrocytes (Fig. 1d). Silencing β-arrestin2 gene expression attenuated ≥10-fold EGF activation of ERK by 40%, thereby implicating β-arrestin2 in the growth factor phase. It would also be consistent with β-arrestin2 siRNA-induced inhibition of ERK activation stimulated by DAMGO and morphine.

Morphine stimulates EGFR phosphorylation via G protein and β-arrestin2 pathways in MOR-transfected immortalized type 1 astrocytes

To determine whether G-protein- and β-arrestin2-dependent µ pathways also entailed EGFR transactivation similar to the CaM-mediated pathway (Belcheva et al. 2005), experiments were undertaken wherein morphine stimulation of EGFR phosphorylation was measured in immortalized astrocytes transfected with β-arrestin2 siRNA or treated with PTX. EGFR in astrocyte lysates were immunoprecipitated with an anti-EGFR Ab and the precipitate was subjected to immunoblotting using a phosphotyrosine Ab. Abolishment of morphine stimulation of EGFR phosphorylation by β-arrestin2 siRNA suggested that the MOR pathway leading to EGFR transactivation also required β-arrestin2 as did the growth factor phase (Fig. 2a). In addition, we found that PTX diminished morphine-induced EGFR transactivation (Fig. 2b). These results were consistent with our hypothesis that CaM, Gi/o protein, and β-arrestin2 mediated MOR activation of ERK via EGFR transactivation.

Fig. 2.

Fig. 2

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Effects of β-arrestin2 siRNA and PTX on morphine (Morph) and DAMGO stimulation of EGFR phosphorylation in immortalized astrocytes. (a). Cells were transfected with MOR cDNA (1 µg) and control (Con, non-targeting) or β-arrestin2 siRNA (1 µM). After 24 h growth in serum-deprived media, cells were treated with 1 µM Morph or DAMGO for 1 min. EGFR was immunoprecipitated with an EGFR antibody and immunoblotting was performed with a phospho-Tyr antibody. Representative immunoblots show phosphorylated Tyr and total EGFR. **p < 0.01 versus control, ††p < 0.01 versus agonist alone. n = 6. (b) Cells were transfected with MOR cDNA, serum-starved in the presence of 0.1 µg/mL PTX for 24 h, and treated with 1 µM Morph for 1 min. EGFR immunoprecipitation and immunoblotting were performed as in (a). **p < 0.01 versus control, †p < 0.05 versus agonist alone. n = 3–4.

Short- and long-term actions of µ opioids on ERK activation in primary astrocytes

Having obtained evidence for different µ-opioid mechanisms of ERK1/2 activation in immortalized astrocytes, the question arises as to their individual cellular responses. κ-Opioids stimulated and µ inhibited astrocyte proliferation in vitro and in vivo during CNS development and injury (Stiene-Martin et al. 1991; Hauser et al. 1996; Stiene-Martin et al. 2001; Xu et al. 2007; McLennan et al. 2008; for a review see Sargeant et al. 2008). Therefore, we focused on the role of each of these pathways in astrocyte proliferation. The use of primary cultures of astrocytes may serve as a more physiologically relevant model system to study the mechanism of endogenous MOR-mediated effects on cell growth. In primary astrocytes, we detected sufficient endogenous MOR and KOR by immunocytochemical techniques (data not shown) and immunoblotting (Fig. 3a) to forego MOR transfection. KOR staining was heavier than that of MOR in the immunoblots because of the differential potency of the MOR and KOR Abs. In contrast, binding and in situ hybridization studies had revealed that rat forebrain contained more MOR than KOR (Spain et al. 1985; Mansour et al. 1994). Therefore, in primary cultures we anticipated that 1 µM DAMGO would act solely on endogenous MOR but 1 µM morphine might bind to and activate both MOR and endogenous KOR.

Fig. 3.

Fig. 3

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Occurrence of MOR and its mediation of morphine (Morph) stimulation of ERK phosphorylation in primary cortical astrocytes (a). Primary astrocytes (prim. ast.) and rat brain homogenates (rat brain) were lysed and immunoblotting was conducted on 100 µg of protein from each tissue as described for pERK assays. (b) After 24 h growth in serum-deprived media, cells were treated with 1 µM Morph and assayed at the indicated time intervals. *p < 0.05, **p < 0.01, ***p < 0.001 versus control. n = 6. (c) After 24 h growth in serum-deprived media, cells were pre-treated with the MOR antagonist CTAP (1 µM) or the KOR antagonist, norbinaltorphimine (norBNI, 1 µM) for 1 h, then treated with 1 µM Morph for 5 min and assayed. *p < 0.05, ***p < 0.001 versus control. †p < 0.05, †††p < 0.001 versus their respective agonist. n = 4–7.

In a time course study (Fig. 3b), morphine activation of ERK proved to be sustained as seen for the prototypic κ agonist, U69593 (Belcheva et al. 2003; McLennan et al. 2008). To test the possibility that morphine acts on both MOR and KOR, we examined the effects of the µ-specific antagonist, CTAP and the κ-specific antagonist, norbinaltorphimine, on morphine-stimulated ERK phosphorylation in primary cultures (Fig. 3c). Both antagonists attenuated morphine activation of ERK. These results established that morphine was acting through both MOR and KOR in primary astrocytes. In addition, the data afford an explanation for the more potent action of morphine over DAMGO and sustained ERK stimulation in primary astrocytes (Fig. 3b and data not shown).

Additional experiments were conducted to ascertain that EGFR transactivation was involved in morphine stimulation of ERK phosphorylation in primary astrocytes. As seen in Fig. 4a, AG1478, a specific inhibitor of tyrosine phosphorylation of EGFR, abolished morphine activation of ERK. The reduction of basal levels of pERK by AG1478 may reflect ERK activation by residual serum in the serum-deprived cells or by astrocyte-derived endogenous opioid peptides (Hauser et al. 1990; Melner et al. 1990). Morphine and DAMGO stimulation of ERK was also completely abolished by β-arrestin2 siRNA in primary astrocytes consistent with data for immortalized astrocytes (Fig. 4b). Transfection of β-arrestin2 siRNA reduced levels of β-arrestin2 protein content (50 ± 5%, p < 0.05, n = 3) in comparison with non-targeting control siRNA (Fig. 4b inset). As β-arrestin2 siRNA was expected to only partially inhibit ERK activation, the results supported the notion that this adaptor protein acted in the EGF phase of ERK activation. As observed in immortalized astrocytes, long-term (2 h) pre-treatment of primary cultures with morphine inhibited EGF-stimulated ERK phosphorylation (Fig. 4c). Although the 30-min point showed a slight increase, its difference from the control and 5-min morphine treatment results was not statistically significant. This stimulatory trend seen at 30 min may reflect the effect of morphine activation of KOR in primary cultures. Finally, we obtained evidence for the existence of a CaM-dependent ERK activation pathway in primary astrocytes by pre-incubating MOR with the CaM antagonist, W-7, for 30 min followed by treatment with morphine or DAMGO for 5 min (Fig. 4d).

Fig. 4.

Fig. 4

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Effects of short- and long-term morphine (Morph) treatment on ERK phosphorylation in the presence of AG1478, β-arrestin2 siRNA, EGF or W-7 in primary astrocytes. (a) Cells were pre-treated with 0.1 µM AG1478 for 20 min and treated for 5 min with 1 µM morphine. *p < 0.05 versus controls.†††p < 0.01 versus Morph-treated cells. n = 6–9. (b) Cells were transfected with control (non-targeting) or β-arrestin 2 siRNA (1 µM), serum-starved for 24 h, and treated with 1 µM Morph or DAMGO for 5 min and ERK1/2 phosphorylation was assayed. *p < 0.05, **p < 0.01 versus control. †p < 0.05, ††p < 0.01 versus their respective agonist. n = 3. (c) Cells were pre-treated with 1 µM Morph for the indicated time points, treated for 3 min with EGF (50 ng/mL), and ERK1/2 phosphorylation was assayed. EGF stimulated ERK phosphorylation 5.7 ± 0.6 fold over controls (n = 4). †††p < 0.001 versus EGF-treated cells. n = 5–6. (d) Cells were pre-treated with 50 µM W-7 for 30 min, followed by 1 µM Morph or DAMGO for 5 min, and ERK1/2 phosphorylation was assayed. **p < 0.01, ***p < 0.001 versus control. †p < 0.05, †††p < 0.001 versus agonist alone. n = 4. Con, control.

Long-term actions of µ opioids on cell proliferation in primary astrocytes

Primary astrocyte cultures can be heterogeneous in that they contain GFAP-positive type 1 astrocytes that are flat polyhedral-shaped cells and type 2 that are spindle-shaped and possess two or more processes (McLennan et al. 2008). We utilized the BrdU incorporation assay to assess cell growth of both type 1 and 2 cells in primary cortical cultures generated under our conditions and discovered that long-term (1 h) pre-treatment with DAMGO or morphine attenuated EGF-stimulation proliferation of both cell types (Figs. 5–8). Of the total number of cells counted, the ratio of type 1 to type 2 was 2.44 : 1. The ratio of BrdU labeled type 1 to type 2 was 0.74 : 1, indicating that type 2 cells proliferated more rapidly than type 1.

Fig. 5.

Fig. 5

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µ-Opioid inhibition of EGF-induced type 1 and 2 primary astrocyte proliferation. Cells were starved for 28 h, treated for the next 25 h with 1 µM DAMGO or morphine, and administered EGF (10 ng/mL) for the last 24 h. BrdU was added for the last 4 h of treatment. Representative micrographs are shown. n = 3–4. GFAP, glial fibrillary acidic protein.

Fig. 8.

Fig. 8

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Effect of β-arrestin2 siRNA on µ-opioid inhibition of EGF-induced type 1 and 2 primary astrocyte proliferation. Cells were transfected with control (non-targeting) or β-arrestin2 siRNA (1 µM). After 28 h growth in serum-deprived media, cells were either treated with EGF (10 ng/mL) for 24 h or treated with 1 µM morphine (Morph) for 25 h and EGF (10 ng/mL) was added for the last 24 h. BrdU was added for the final 4 h of treatment. Cell proliferation was measured as % BrdU-labeled astrocytes (% EGF stimulation) in this analysis because of the highly variable basal proliferation rates experienced after transfection of siRNA. Numbers in parenthesis in the bar graph represent % EGF stimulation as fold over control. **p < 0.01, ***p < 0.001 versus EGF alone in control siRNA-treated cells. §p < 0.05 versus EGF + Morph in control siRNA-treated cells. n = 3–4.

We next addressed the question of which opioid pathway to ERK was involved in reducing EGF-stimulated cell proliferation. We found that PTX attenuated the inhibitory effect of morphine and DAMGO on EGF stimulation of proliferation of both types of astrocytes (Fig. 6). Cells were pre-treated with DAMGO or morphine for 1 h followed by EGF + opioids for an additional 24 h. PTX had no effect on EGF stimulation of proliferation in the absence of opioids. In both type 1 and type 2 astrocytes, PTX partially reversed the inhibitory actions of DAMGO and morphine on EGF-induced proliferation. In summary, the data suggest that the opioid G-protein-dependent pathway participated in the inhibition of type 1 and type 2 astrocyte proliferation induced by EGF.

Fig. 6.

Fig. 6

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PTX reversal of µ-opioid inhibition of EGF induced type 1 and 2 primary astrocyte proliferation. Cells were pre-treated with 0.1 µg/mL PTX during serum deprivation for 28 h, treated for the next 25 h with 1 µM DAMGO or morphine (Morph), and administered EGF (10 ng/mL) for the last 24 h. BrdU was added for the last 4 h of treatment. *p < 0.05, **p < 0.01, ***p < 0.001 versus control. †††p < 0.001 versus EGF-treated cells. §p < 0.05, §§p < 0.01, §§§p < 0.001 versus PTX control. n = 4. Con, control.

In contrast, W-7 did not affect the inhibitory actions of the µ agonists on EGF stimulation of type 1 and type 2 astrocyte proliferation (Fig. 7). Although CaM-dependent MOR signaling was mediated by EGFR transactivation (Belcheva et al. 2005) and ERK (Fig. 4d), it did not appear to be involved in inhibition of EGF-induced primary astrocyte proliferation.

Fig. 7.

Fig. 7

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Lack of effect of CaM inhibitor, W-7, on µ-opioid inhibition of EGF-induced type 1 and 2 primary astrocyte proliferation. Cells were pre-treated with 50 µM W-7, starved for 28 h, treated for the next 25 h with 1 µM DAMGO or morphine (Morph), and administered EGF (10 ng/mL) for the last 24 h. **p < 0.01, ***p < 0.001 versus control. ††p < 0.01, †††P < 0.001 versus EGF-treated cells. n = 3. Con, control.

Primary astrocytes were transfected with β-arrestin2 siRNA or non-targeting control siRNA as before (Fig. 4b inset). Upon β-arrestin2 siRNA silencing, EGF-stimulated proliferation was inhibited by 49% (p < 0.01) and 51% (p < 0.001) in type 1 and type 2 astrocytes, respectively (Fig. 8). The percent inhibition values was estimated from % fold over control values shown in parenthesis in Fig. 8 after subtracting out basal levels (1.0). These results were consistent with the findings in Figs. 1d and 4b that suggested that β-arrestin2 reduced EGF-induced ERK activation by blocking the growth factor pathway.

The significant attenuation of the EGF proliferative actions by morphine also appeared to be partially reversed in the presence of β-arrestin2 siRNA (Fig. 8). In Figs. 6 and 7 we compared % BrdU labeling of opioid and EGF-treated cells directly in the presence or absence of PTX or W-7 (denoted by the § sign), as EGF stimulation of proliferation did not change. However, in Fig. 8, EGF stimulation of proliferation was reduced so we estimated the % inhibition of EGF-induced mitogenesis by morphine under control siRNA and β-arrestin2 targeting siRNA conditions. Upon adjusting for the β-arrestin2 targeting siRNA-induced loss in EGF-induced proliferation, morphine inhibition of EGF-induced proliferation was significantly reversed compared with non-targeting siRNA controls (Fig. 8). In type 1 astrocytes the % reduction by morphine in controls changed from 45% to 8% in the presence of β-arrestin2 targeting siRNA. In type 2 astrocytes the extent of reversal of morphine inhibitory effects was also significant. In these cells morphine pre-treatment reduced EGF-induced proliferation by 46% in the presence of control siRNA, whereas morphine + EGF displayed only 21% diminution in the presence of β-arrestin2 targeting siRNA. These results were also consistent with the finding in Fig. 2a, that suggested β-arrestin2 mediated µ-opioid-stimulated EGFR phosphorylation and thereby played a role in the GPCR phase of the pathway. Although we observed an opioid-induced statistically significant reversal of the EGF proliferative activity that was blocked by β-arrestin2 targeting siRNA in the GPCR pathway, the caveat was that the absolute changes in proliferation detected after transfection with siRNA were relatively small. Nevertheless, taken together the data indicated that exogenous EGF proliferative activity was clearly dependent on β-arrestin2.

Discussion

The data reported here support the hypothesis that there are multiple µ-opioid pathways leading to regulation of ERK activation and subsequently to proliferation of astrocytes. Selective EGFR tyrosine phosphorylation inhibitor, AG1478, blocked µ-opioid activation of ERK suggesting that all µ-opioid mechanisms involve EGFR transactivation. Direct evidence for this was obtained by measuring EGFR phosphorylation in the presence of inhibitors of CaM, Gi/o, and β-arrestin2. In previous studies, we showed that µ- (morphine) but not κ- (U69593) induced EGFR phosphorylation was inhibited by CaM antisense in immortalized astrocytes transfected with MOR and KOR, respectively, (Belcheva et al. 2005). Here we found that PTX and β-arrestin2 targeting siRNA attenuated µ-opioid phosphorylation of EGFR. These results indicated that EGFR was at the point of convergence of all three GPCR and EGF pathways that led to ERK activation.

Time course studies of morphine and DAMGO signaling to ERK revealed a transient activation that subsided by 30 min in immortalized astrocytes (Belcheva et al. 2003 and data not shown). Functional selectivity differences between DAMGO and morphine were not observed despite their established differential ability to induce MOR internalization (Keith et al. 1996). In contrast, U69593, stimulated ERK activation for over 2 h (Belcheva et al. 2003). This sustained activation of ERK by U69593 appeared to be necessary for ERK-mediated astrocyte proliferation as two other κ selective agonists, salvinorin A and a derivative, MOM-Sal B, activated ERK transiently and failed to induce immortalized astrocyte proliferation (McLennan et al. 2008). Similarly, neither DAMGO nor morphine displayed proliferative activities and proved to inhibit EGF-stimulated ERK activation and cell proliferation. In primary astrocytes, time course studies on µ activation of ERK were not as clear. Morphine displayed a sustained ERK activation but as shown by antagonist studies, this might be because of its ability to bind to both endogenous MOR and KOR in primary cells (Fig. 3c). However, in most proliferation studies, morphine-induced activity was comparable with that of DAMGO suggesting that its µ activation of ERK was more efficacious than its κ actions.

In the yeast ERK/MAP kinase signalosome which occurs on the scaffold protein Ste5, different temporal patterns of ERK activation are thought to arise as a result of time and order of recruitment of positive and negative modulators (Bashor et al. 2008; Pryciak 2008; Takahashi and Pryciak 2008). Moreover, temporal patterns of ERK activation may be turned on or off depending upon the location of the Ste5 scaffold protein in the cell (Dohlman 2008). Analogous findings have been reported in mammalian cells. Therefore, the complex spatiotemporal aspects of ERK activation in astrocytes discussed above may reflect similar inter-relationships in which feedback loops exist that differentially modulate ERK phosphorylation depending upon its subcellular localization.

To study the integration of the three pathways that MOR utilized in achieving its cellular responses, we chose to examine changes in astrocyte proliferation. A novel finding of this study is the implication of β-arrestin2 in exogenous EGF stimulation of ERK-mediated astrocyte proliferation (Fig. 9). We demonstrated that β-arrestin2 attenuates exogenous EGF-induced ERK phosphorylation and astrocyte proliferation, suggesting yet another new role for this adaptor protein. This attenuation of ERK phosphorylation and proliferation may also explain why β-arrestin2 siRNA displayed such a strong inhibitory effect despite multiple pathways leading to EGFR transactivation. Nevertheless, we cannot rule out the possibility that the exogenous EGF pathway may be β-arrestin2-dependent unlike the growth factor phase of the MOR-driven pathway. Instead the opioid-stimulated pathway may only be β-arrestin2-dependent in the GPCR phase. GPCR-driven transactivation of EGFR has been shown to entail a membrane-anchored source of EGF-like ligands, such as transforming growth factor α or heparin binding EGF, and there is evidence of diversity in their ERK-signaling mechanisms (Daub et al. 1996; Prenzel et al. 1999; McCole et al. 2002; Konishi and Berk 2003; Schafer et al. 2004). In this scenario, the strong inhibitory actions of β-arrestin2 siRNA may be explained by the debilitating effect of the transfection process which reduces the extent of EGFR and ERK phosphorylation and proliferation and thereby additive effects of each of the three pathways are not as detectable. Yet another factor contributing to strong inhibitory effects may be the previously mentioned differential temporal patterns of activation by the CaM, Gi/o, and β-arrestin2 pathways. In this case, monitoring at a single time interval may favor one pathway over another.

Fig. 9.

Fig. 9

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Working model of the mechanisms of µ-opioid inhibition of EGF-induced ERK activation, astrocyte proliferation, and other cellular responses. As the CaM-dependent opioid signaling pathway does not appear to affect proliferation, pERK functions may differ depending upon its localization in the cell.

An intriguing question raised by these findings is the site of interaction of β-arrestin2 within the growth factor pathway. Some signaling components or adaptor proteins involved in EGF-stimulated ERK activation undergo endocytosis and are recruited along with EGFR to endosomes (Nesterov et al. 1994). As MOR belonged to the Class A family of GPCRs, it would be expected to signal from coated pits on the plasma membrane where it interacts with β-arrestin2 which in turn binds to components of the ERK/MAP kinase phosphorylation cascade thereby generating pERK from this site (Oakley et al. 2000; Lefkowitz and Shenoy 2005). This pERK translocates to the nucleus in contrast to pERK derived from Class B GPCRs that remains in the cytosol. The Class A designation of MOR was consistent with the similar signaling capabilities of DAMGO and morphine. We also demonstrated agonist induced KOR interaction with β-arrestin2 at the plasma membrane of human embryonic kidney 293 cells by immunofluorescence confocal microscopy (McLennan et al. 2008). As Class A GPCRs stimulate translocation of pERK into the nucleus, we tested this possibility by immunofluorescence microscopy. Both µ- and κ-opioid agonists increased nuclear pERK immunostaining significantly (p < 0.05) after 5 min of exposure in immortalized astrocytes (data not shown). The presence of pERK in different compartments of the cell indicates that this kinase acts on different substrates and therefore may have different functions.

Another notable finding in this study is that of the three pathways to ERK, the CaM-mediated route does not participate in the inhibition of EGF-induced cell proliferation (Fig. 9). The CaM pathway differs from the other MOR pathways and this difference may occur by its effect on EGFR via membrane-anchored EGF-like ligands as discussed above. The data also imply differential compartmentation of pERK generated. As it is clear that µ opioids can modulate other cellular responses in astrocytes (M. Miyatake, H. Ikeda, M.M. Belcheva and C.J. Coscia, unpublished observations), it will also be of interest to determine whether the CaM pathway plays an intermediary role in other cellular outcomes either exclusively or in an integrated manner.

Acknowledgements

Supported in part by a grant from the National Institutes of Health DA-05412. We thank Drs. John Chibnall and Maureen Donlin for their assistance in the statistical analysis of the data.

Abbreviations used

Ab

antibody

BrdU

5′-bromo-2′-deoxy-uridine

CaM

calmodulin

DAMGO

[d-ala2,mephe4,gly-ol5] enkephalin

DMEM

Dulbecco’s modified Eagle’s medium

EGF

epidermal growth factor

EGFR

EGF receptor

ERK1/2

extracellular signal-regulated kinase

FBS

fetal bovine serum

GFAP

glial fibrillary acidic protein

GPCR

G protein-coupled receptor

HRP

horseradish peroxidase

KOR

κ-opioid receptor

MAP

mitogen-activated protein

MOR

µ-opioid receptor

PBS

phosphate-buffered saline

pERK

phospho-ERK

PTX

pertussis toxin

SDS

sodium dodecyl sulfate

TBST

Tris-buffered saline + 0.2% Tween 20

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