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Keywords:

  • differentiation;
  • Gα subunits;
  • neurite outgrowth;
  • neuronal survival;
  • signal transducer and activator of transcription 5B;
  • δ-opioid receptor

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure statement
  8. References
Thumbnail image of graphical abstract

It remains unclear how opioid receptors (δ, μ, κ) are implicated in mechanisms controlling differentiation, cell proliferation, and survival. Opioid receptors are coupled to Gi/Go proteins and recent findings have shown that opioid receptors can form a multicomponent signaling complex, consisting of members of G protein and the signal transducer and activator of transcription (STAT)5B. We thus wondered whether activation of the opioid receptors could direct differentiation and neurite outgrowth through a molecular pathway involving STAT5B and other signaling intermediates. We demonstrate that prolonged δ-opioid receptor (δ-OR) activation with opioid agonists induces STAT5B phosphorylation in Neuro-2A cells. Moreover, [D-Ser2, Leu5, Thr6]-enkephalin-activation of δ-OR triggers neurite outgrowth and neuronal survival; these effects are blocked by the selective antagonist naltrindole, by treatment with pertussis toxin, and after expression of a dominant negative mutant of STAT5B (DN-STAT5B), suggesting that the signaling pathway participating in this mechanism involves Gi/o proteins and p-STAT5B. Additional studies have shown that while [D-Ser2, Leu5, Thr6]-enkephalin exposure of neuroblastoma cells induces a marked increase in the differentiation marker proteins, βIII-tubulin (Tuj-1), synaptophysin, and neural cell adhesion molecule, over-expression of the DN-STAT5B attenuated significantly their expression levels. Taken together, our findings demonstrate that δ-OR activation leads to a number of neurotropic events via a Gαi/o-linked and STAT5B-dependent manner.

We propose a novel signalling pathway for δ-opioid receptor (δ-ΟR)-mediated neurotropic events. STAT5B interacts with the δ-ΟR and upon prolonged receptor activation phosphorylates STAT5B in a Gi/Go dependent manner leading to increased neuronal survival, neurite outgrowth and differentiation. These findings contribute to a better understanding of the molecular and cellular events following δ-OR activation and suggest a possible neuroprotective role opioids could exert.

Abbreviations used
5-HT

serotonin receptor

AP-1

activator protein 1

CBR

cannabinoid receptor

CREB

cyclic AMP-response element DNA-binding protein

DMEM

Dulbecco's modified Eagle's medium

DSLET

[D-Ser2, Leu5, Thr6]-enkephalin

EGTA

ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

FBS

fetal bovine serum

GPCR

G protein-coupled receptor

NCAM

neural cell adhesion molecule

PBS

phosphate-buffered saline

PMSF

phenylmethylsulfonil fluoride

PTX

Bordetella pertussis toxin

PVDF membrane

polyvinylidene difluoride membrane

RA

retinoic acid

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

STAT

signal transducer and activator of transcription

δ-ΟR

δ-opioid receptor

μ-ΟR

μ-opioid receptor

Opioid receptors (μ, δ, and κ) are members of the G-protein coupled receptor (GPCR) superfamily which couple to Gi/Go proteins to modulate a variety of physiological responses in the nervous system through activation of a diverse array of effector systems ranging from adenylyl cyclase and phospholipase C, to mitogen-activated protein kinase (Waldhoer et al. 2004). Apart from such effects, opioid administration causes activation of several transcription factors including cyclic-AMP-response element binding protein, activator protein 1, nuclear factor-kB, and members of the signal transducers and activators of transcription (STATs) (Mazarakou and Georgoussi 2005; Ho et al. 2009; Georganta et al. 2010). Such parallel manifestations of opioid receptors suggest that these receptors are involved in different signaling circuits that lead to alterations in the expression of target genes in a pleiotropic fashion. Recent findings support the notion that opioid receptors regulate a number of cellular functions that play an important role in cell proliferation, gliogenesis, and neurogenesis (Narita et al. 2006; Chen et al. 2008).

Neurite outgrowth is a key process during neuronal migration and differentiation. Complex intracellular signaling mechanisms are implicated in the initiation of neurite protrusion and subsequent elongation. The regulation of neurite outgrowth is tightly controlled because of its critical physiological function. Several Gi/o-coupled GPCRs have been shown to play an important role in controlling neurite outgrowth. For example, the D2 dopamine receptors regulate neurite outgrowth in cortical neurons (Reinoso et al. 1996), whereas the serotonin-1B receptors are known to enhance neurite outgrowth in thalamic neurons (Lotto et al. 1999). These receptors are coupled to Giα and Goα proteins with the latter being the most abundant protein in the neuronal growth cones that can induce neurite outgrowth (Strittmatter et al. 1990, 1994). Recent findings have shown that activation of the serotonin receptor leads to neurite outgrowth and neuronal survival via a signaling pathway involving Gαi-Rap-Src-STAT3 (Fricker et al. 2005), whereas the Gαi/o-coupled cannabinoid 1 receptor (CB1R) triggers neurite outgrowth in Neuro-2A cells through activation of a signaling network consisting of Src kinase and the STAT3 transcription factor (He et al. 2005). These studies indicate that a variety of signaling mechanisms, including soluble or membrane-bound guidance cues can signal to effector molecules to mediate cytoskeletal rearrangement, which is a crucial process during neuronal differentiation (Govek et al. 2005). Although several players have been shown to participate, it remains unclear how extracellular signals initiated upon activation of opioid receptors are converted into changes in cytoskeletal rearrangements and how these signals are regulated.

STATs constitute a family of transcription factors that mediate a wide variety of biological functions in the central and peripheral nervous systems. Seven transcription factors are known STAT1-4, STAT5A/5B, and STAT6, which are phosphorylated mainly by JAKs; phosphorylated STATs dimerize and translocate to the nucleus, where they bind to specific DNA sequences inducing transcription of target genes (Lim and Cao 2006). Although STATs are mainly regulated by cytokines, their activity can also be modulated by GPCRs and G proteins. We have previously demonstrated that STAT5A/5B interact directly with μ- and δ-opioid receptors and are phosphorylated by c-Src kinase upon opioid receptor stimulation (Mazarakou and Georgoussi 2005; Georganta et al. 2010). Moreover, we have shown that STAT5B interacts directly with selective members of the Giα/Goα family as well as Gβγ subunits and that the C-terminus of the δ-opioid receptor serves as a platform for the formation of a multicomponent signaling complex consisting of Gα, Gβγ subunit, c-Src, and STAT5B (Georganta et al. 2010; Georgoussi et al. 2012). This protein complex is mediated in an agonist-dependent manner raising several questions as to the downstream signaling events that may be subsequently activated as a result of these interactions as well as to the functional output of other signaling cascades initiated.

In this study, we investigate whether this dynamic protein complex is implicated in mechanisms through which δ-OR activation may regulate neurogenesis, i.e, cell survival, neurite outgrowth, and differentiation using two neuroblastoma cell lines. We demonstrate that activation of the δ-OR, (i) leads to increased cell survival even under starving conditions, and (ii) triggers neurite outgrowth and neuronal differentiation of Neuro-2A cells, via a signaling pathway involving Gαi/o proteins and phosphorylated STAT5B. These studies reveal a novel mechanism of cell biological events mediated by the activated δ-OR and phosphorylated STAT5B.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure statement
  8. References

Constructs and reagents

The human His-tagged Υ699F point mutated STAT5B (in the pCMV vector) was kindly provided by Dr C.M. Silva, University of Virginia, Charlottesville, VA, USA. The cDNA YFP-CAAX construct (in the pCS2 + vector) which encodes the 10 C-terminal amino acids of human H-Ras (GCMSCKCVLS), including the CAAX motif, was kindly provided by Dr H.P. Spaink, Leiden University, Netherlands. [D-Ser2, Leu5, Thr6]-enkephalin (DSLET) was obtained from Tocris Bioscience (Cookson, MI, USA) and turboFECT in vitro transfection reagent from Fermentas Life Sciences (Thermo Fisher Scientific, Waltham, MA, USA). Protein A agarose beads and antibodies against STAT5 and p-STAT5 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protease inhibitors were from Roche (Roche Diagnostics, Basel, Switzerland), while pertussis toxin (PTX), phosphatase inhibitor cocktail, the opioid antagonist naltrindole, and all other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).

Cell cultures and transient transfections

Neuro-2A mouse neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium containing 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS) under 5% CO2 at 37°C. SH-SY5Y cells were grown in basal medium RPMI-1640 (Sigma-Aldrich) and maintained as described previously by Mazarakou and Georgoussi 2005. Neuro-2A cells were transiently transfected using turboFECT in vitro transfection reagent according to the manufacturer's instructions. For the PTX ribosylation experiments, Neuro2A cells were transfected with the human flag-tagged δ-opioid receptor (δ-ΟR) and treated with PTX (100 ng/mL) for 18 h as described previously by Morou and Georgoussi 2005.

Immunoprecipitation assays

Neuro-2A or SH-SY5Y cells, endogenously expressing STAT5B, serum starved for 6 h were treated or not, with 1 μM DSLET, or 1 μM morphine for different times and rinsed in phosphate-buffered saline buffer containing 0.1 mM phenylmethylsulfonil fluoride (PMSF) and 0.1 mM Na3VO4. Cells were lysed in buffer containing 1% Igepal, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EGTA, 25 mM NaF, 0.25% sodium deoxycholate, 0.2 mΜ Na3VO4, and 1 mM PMSF together with protease (Roche) and phosphatase (Sigma) inhibitor cocktail. Approximately 500 μg of the clarified cell lysates were incubated with the STAT5 (2 μg) antibody overnight at 4°C. Immunoprecipitates were recovered on protein A agarose beads, washed extensively and separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Immunoprecipitation of cell lysate proteins was verified by immunoblotting using the appropriate antibodies. Neuro-2A cells were from ATCC, (ATCC® CCL131™).

Western blotting

After protein determination cell lysates and the immunoprecipitated protein complexes were subjected to western blot analysis, as described by Fourla et al. (2012) using the following antibodies: polyclonal-phospho-STAT5 and anti-STAT5 (Santa Cruz Biotechnology), polyclonal anti-flag (Sigma-Aldrich) and monoclonal anti-6xHis (BD Pharmingen, Franklin Lakes, NJ, USA). For the identification of neuronal differentiation, western blot and immunofluorescence staining were performed using the rabbit polyclonal antibodies neural cell adhesion molecule (NCAM), synaptophysin (Gaitanou et al. 1997) and the monoclonal anti-βIII tubulin (Tuj-1) from Covance. Immunoreactive bands were detected by enhanced chemiluminescence (Thermo Scientific - Pierce Protein Biology Products), according to the manufacturer's instructions, using the luminescent image analyzer LAS-4000 (Fujifilm, Tokyo, Japan). Where necessary, membranes were stripped and reprobed with the appropriate antibodies as described above.

Survival assay

SH-SY5Y cells endogenously expressing δ-OR or Neuro-2A cells were transiently transfected with flag-δ-OR (1 μg) and plated in complete media on six-well plates. To investigate the implication of STAT5B in cell survival, some Neuro-2A cell samples were co-transfected with the cDNA encoding the His-STAT5B(Y699F), (DN-STAT5B) construct (1 μg), which cannot be phosphorylated. Next day, cells in half the plates, were placed in starving media (media without FBS), and treated with 1 μM of DSLET alone or DSLET plus 10 μM naltrindole. Control cells were not subjected to any opioid ligand treatment. After 24 h, the number of live cells was counted by the Trypan blue exclusion method. More specifically, a total number of 400 000 cells from each culture were incubated with 0.1% trypan blue, and the number of viable (unstained) versus non-viable cells (blue stained) was measured using a hemocytometer under light microscope. A total number of 100 cells from each case were counted. Control cells not subjected to drug treatment were taken as 100%.

Neurite outgrowth assay

Neuro-2A cells were plated in six-well plates and were transiently transfected with 0.2 μg of the YFP-CAAX construct and 0.8 μg of either δ-OR cDNA or pcDNA3, in the presence or absence of the DN-STAT5B, as described above. Next day, the media were substituted by Dulbecco's modified Eagle's medium without FBS, and cells were treated with either 1 μM DSLET, or 10 μΜ naltrindole, or 1 μΜ DSLET plus 10 μΜ naltrindole for 16–20 h at 37°C. Vehicle-treated cells were used as controls. Fluorescent images were taken using a Zeiss Axiovert 25 microscope (Zeiss, Oberkochen, Germany) at 40× magnification. For examining the effect of PTX on neurite outgrowth, cells were pre-treated with 100 ng/mL PTX for 2 h at 37°C prior to the addition of the opioid receptor ligands. The cells displaying neurite outgrowth were those that had cellular projections of length two times greater than the cell diameter (~ 1.25 μm for Neuro-2A cells). For each culture condition, randomly chosen regions of the plate containing approximately 100 cells were scored under the microscope. All assays were carried out at least in triplicate and the indicated values are represented as mean ± SEM.

Identification of neuronal differentiation

Neuro-2A cells were plated in 60-mm plates and allowed to reach 60–70% confluency. Transient transfections were performed using 2 μg of each pcDNA3 or δ-ΟR, in the presence or absence of the DN-STAT5B for 16–24 h as described above. The media were then removed and cells were subjected in serum free media and treated with 1 μΜ DSLET for 16–20 h at 37°C. Cell monolayers were rinsed in phosphate-buffered saline and lysed in buffer containing 1% Igepal, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EGTA, 25 mM NaF, 0.25% sodium deoxycholate, 0.2 mΜ Na3VO4, and 1 mM PMSF supplemented with protease (Roche) and phosphatase (Sigma) inhibitor cocktail. Soluble proteins separated by centrifugation at 3000 rpm were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10% w/v) and immunoblotted with the Tuj-1, NCAM and synaptophysin antisera as described by Swevers et al. 2005. Alternatively, to induce neuronal differentiation Neuro-2A cells expressing the δ-OR and/or the DN-STAT5B, were subjected to DSLET for 16–20 h with 20 μM retinoic acid (RA) in the presence of 2% FBS, as described by Riboni et al. 1995. The degree of cell differentiation was assessed by immunofluorescence microscopy after staining with the neuronal marker βIII-tubulin (Tuj-1).

Immunofluorescence staining

For the subcellular localization and distribution of NCAM and synaptophysin proteins, Neuro-2A cells expressing the δ-ΟR and or the DN-STAT5B plasmid were grown on poly-l-lysine-coated cover-slips in 12-well plates and treated for 16 h with 1 μΜ DSLET in serum free media as described above. Cells were fixed in 4% paraformaldehyde, for 10 min, permeabilized with Triton X-100 for 5 min at 24°C and incubated overnight at 4°C with the anti-NCAM and or anti-synaptophysin polyclonal antibodies (1 : 100) followed by 1 h incubation with the fluorescein-conjugated secondary antibody Alexa fluor 488 goat anti-rabbit (1 : 500) (Life Technologies, Carlsbad, CA, USA) and TO-PRO-3 (1 : 1000) an optimal fluorescent dye for nuclear counter-staining. Image analysis was carried out using a Nikon Eclipse E600 confocal microscope (Nikon Corporation, Tokyo, Japan).

Statistical analysis

Statistical analysis was performed using the Student's t-test. The data are represented as mean ± SEM. For western blot analysis, all experiments were repeated at least three times and bands were quantified by densitometric analysis and expressed as mean ± SEM. Representative experiments are shown and statistical significance is described in each figure legend. The level of significance for all analyses testing was set at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure statement
  8. References

Stimulation of the δ-opioid receptor leads to cell survival through activation of STAT5B in Neuro-2A and SH-SY5Y cells

Ample experimental evidence supports the concept that the δ-OR plays important role in gliogenesis, cell proliferation, and neurogenesis (Narita et al. 2006), indicating that a variety of signaling mechanisms can signal to effector molecules to mediate intracellular rearrangements. We have previously shown that activation of the δ-OR in HEK293 cells with selective opioid agonists lead to an increase in the phosphorylation status of STAT5B via a δ-ΟR-STAT5B-G protein complex (Georganta et al. 2010); however, the biological consequences of this activation are still unclear. We thus wondered whether STAT5B activation by δ-OR signaling could be implicated in neuronal survival and differentiation. To answer this question, we first examined the levels of STAT5B phosphorylation in two neuroblastoma cell lines, Neuro-2A cells transiently transfected with the δ-OR, used as a model of neuronal differentation and SH-SY5Y cells endogenously expressing the δ-OR receptor, both expressing endogenously the transcription factor STAT5B after prolonged (1–6 h) or acute opioid administration. Western blot analysis of immunoprecipitated STAT5B with a p-STAT5 antibody showed that treatment of δ-OR transfected-Neuro-2A and SH-SY5Y cells endogenously expressing this receptor with DSLET, phosphorylated STAT5B as shown by an increase in the levels of phosphorylated STAT5B after 5 or 15 min agonist exposure (Fig. 1a and b, respectively). STAT5B phosphorylation in SH-SY5Y cells is abolished by the presence of the selective δ-OR antagonist naltrindole, indicating that the effect is δ-OR mediated (Fig. 1b, lane 5). On the other hand, expression of a phosphorylation deficient mutant of STAT5B (Y699F) that functions as a dominant negative mutant of STAT5B (DN-STAT5B) (Bernaciak et al. 2009), in Neuro-2A cells blocked the DSLET-mediated STAT5B activation (Fig. 1a compares lane 3 with 5). In addition, prolonged treatment of Neuro-2A and SH-SY5Y cells with DSLET and morphine, respectively, resulted also in STAT5B phosphorylation (Fig. 1c lane 3 and d lanes 2–4). These observations suggest that STAT5B could be phosphorylated by δ-OR activation after acute or prolonged DSLET or morphine administration in Neuro-2A and SH-SY5Y cells.

image

Figure 1. Stimulation of δ-opioid receptor leads to signal transducers and activators of transcription (STAT)5B phosphorylation in Neuro-2A and SH-SY5Y cells. (a) Neuro-2A cells transiently transfected with the flag-tagged human δ-OR (lanes 2-5) and the His-STAT5B (Y699F) (lanes 4–5) were stimulated with 1 μM [D-Ser2, Leu5, Thr6]-enkephalin (DSLET) (lanes 3, 5) for 5 min. (b) SH-SY5Y cells endogenously expressing δ-OR and STAT5B were stimulated with 1 μM DSLET (lanes 3–4) for the indicated times, or were pre-treated, prior to DSLET stimulation, with 10 μM naltrindole for 30 min (lane 5). (c) Neuro-2A cells were stimulated in the presence (lane 3) or absence (lane 2) of 1 μM DLSET for 4 h. (d) SH-SY5Y cells were stimulated with 1 μM morphine (lanes 2–4) for the indicated times. Lanes 1 in (a–d) represent unstimulated cell lysates. All the samples were immunoprecipitated with a STAT5 antibody and the precipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Upper panels show the phosphorylated STAT5B visualized by immunoblotting with a p-STAT5 antibody (1 : 500). Lower panels show the protein loading controls after stripping and reprobing the same membranes with a specific STAT5 antibody (1 : 1000). The expression of the δ-opioid receptor (δ-ΟR) and the DN-STAT5B was visualized using the anti-flag (1 : 1000) and His (1 : 500) antibodies, respectively. All the immunoblots shown are representative of four independent experiments. Data of densitometric analyses of STAT5B shown below the immunoblots represent the mean ± SEM values from four separate experiments. *p < 0.05 and **p < 0.005 significantly higher than the unstimulated cells, by Student's t-test.

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We next examined whether δ-OR activation could lead to increased neuronal survival and used both SH-SY5Y cells endogenously expressing the δ-OR and Neuro-2A transiently transfected to express the human δ-OR. The cells were plated in either complete or in starving media and treated with DSLET or DSLET plus naltrindole. After 24 h, the number of live cells was visualized and counted under a microscope in the presence of Trypan blue. As shown in Fig. 2a, a higher percentage of cell survival was detected in the presence of DSLET, for both proliferating SH-SY5Y cells grown in the presence of fetal bovine serum, and SH-SY5Y cells that have been given a signal to differentiate, upon serum removal. This effect was reversed by the presence of naltrindole, suggesting that the detected increased rate of SH-SY5Y cell survival is mediated by the activated δ-OR. To confirm these observations and further deduce whether STAT5B phosphorylation by δ-OR is implicated in the increased neuronal survival similar experiments were also performed in Neuro-2A cell expressing the δ-ΟR. As shown in Fig. 2b, DSLET exposure of Neuro-2A increased the rate of both non-differentiated and differentiated living cells.

image

Figure 2. Effect of δ-opioid receptor activation on cell survival. (a) SH-SY5Y cells, or (b) Neuro-2A transiently transfected with flag-δ-OR and/or DN-signal transducers and activators of transcription (STAT)5B, in complete media (left graphs) or in starving media (right graphs) were treated with 1 μΜ [D-Ser2, Leu5, Thr6]-enkephalin (DSLET) or DSLET in the presence of 10 μΜ naltrindole for 20 h as described in 'Materials and methods'. The number of live cells was visualized after Trypan blue treatment and counted. Controls not subjected to drug treatment were taken as 100%. Values represent mean ± SEM; statistically significant differences from control cells are indicated *< 0.05, **< 0.005, and ***< 0.0005, Student's t-test.

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This effect was specific as it was antagonized by co-treatment with the selective δ-OR antagonist naltrindole (Fig. 2b), which did not show any effect on cell survival, suggesting that δ-ΟR activation plays an important role in the survival of neuronal cells even under starvating conditions. Next, we examined whether phosphorylated STAT5B is implicated in cell survival mediated by δ-ΟR. As shown in Fig. 2b, expression of the dominant negative DN-STAT5B construct both under starving cell conditions, or not, significantly reduced DSLET-mediated Neuro-2A cell survival, suggesting that this detected increase in the survival rate induced upon δ-ΟR activation is mediated via a signaling pathway participating phosphorylated STAT5B.

δ-opioid receptor activation triggers neurite outgrowth of Neuro-2A cells via Gi/Go proteins and STAT5B

Recent studies have shown that activation of the Gαi/o-coupled serotonin (5-HT1) and CB1 receptors induce neurite outgrowth of Neuro-2A cells via a signaling pathway consisting of Gαi/o proteins, Src kinase, and the STAT3 transcription factor (Fricker et al. 2005; He et al. 2006). Given that opioid receptors could be involved in cell growth and differentiation via similar mechanisms, we investigated the extent by which activation of δ-OR can lead to changes in neurite outgrowth. For that reason, Neuro-2A cells were transiently transfected with δ-OR or empty vector and treated with DSLET. To facilitate neurite visualization the membrane-yellow fluorescent protein (YFP-CAAX) plasmid was also co-transfected. As shown in Fig. 3, treatment of Neuro-2A cells with DSLET leads to a significant increase in the length of the neurites (compare images b, of Fig. 3a with the respective images f of Fig. 3b). Treatment of cells with naltrindole completely blocked this effect (Fig. 3a and b, images c and g, respectively), suggesting that δ-OR activation of Neuro-2A cells promotes neurite outgrowth. This increase in the length of neurites was in accordance with the enhanced number of cells with neurites detected (Fig. 3c).

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Figure 3. Activation of δ-opioid receptor leads to neurite outgrowth of Neuro-2A cells in a Gi/Go-dependent manner. Neuro-2A cells were transfected with a YFP-CAAX construct that targets the membrane (upper panels), or not (middle and lower panels), pcDNA3, or the δ-OR. Panel (a) is presented images of control Neuro-2A cells transfected with pcDNA3 a, images bd represent cells expressing the δ-opioid receptor (δ-ΟR) alone b, or after 10 μM nalrindole exposure for 16–20 h at 37°C (images c). To examine the involvement of Gαi/o subunits in neurite outgrowth, Neuro-2A cells were treated with 100 ng/mL pertussis toxin (PTX) 2 h prior to treatment with 1 μM [D-Ser2, Leu5, Thr6]-enkephalin (DSLET) (images d). (b) Represents Neuro-2A cells treated with 1 μM DSLET; images f–h represent cells expressing the δ-OR after DSLET administration (images f), DSLET plus naltrindole treatment (images g) and PTX plus DSLET administration (images h) as described in (a); images e represents mock transfected cells treated with 1 μM DSLET. (c) The length of the neurites is presented as percentage of outgrowth. Neurites were scored as described in 'Materials and methods'. Fluorescent or simple images of cells were taken and neurites were quantitated. Cells with neurites that are greater than two times the cell diameter were scored as positive. Values represent mean ± SEM; statistically significant differences from control cells are indicated, ***< 0.0005, Student's t-test.

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Knowing that STAT5B interacts with members of the Gαi/o and that phosphorylation of STAT5B is abolished upon PTX treatment (Georganta et al. 2010), we further investigated the involvement of Gαi/o proteins and measured the length of neurites in Neuro2A cells expressing δ-OR before and after PTX treatment prior to receptor activation with DSLET. Treatment with PTX completely blocked DSLET-mediated increase in neurite length (Fig. 3b, images h upper, middle, and lower panels). Similarly, the percentage of cells with neurites was decreased after PTX treatment (Fig. 3c). No alteration in neurite outgrowth was detected in either mock transfected cells (Fig. 3a, b images a and e), or in cells treated with PTX alone (Fig. 3a, all three images d). These data suggest that Gαi/o proteins are implicated in the δ-OR-induced neurite outgrowth.

A number of previous studies have examined the involvement of STAT3 in Gαi/o-mediated cellular proliferation, differentiation (Corre et al. 1999; Ram et al. 2000) and neurite outgrowth (Fricker et al. 2005; He et al. 2005). On the basis of these observations and our own studies demonstrating that opioid receptors phosphorylate STAT5A/B (Mazarakou and Georgoussi 2005; Georganta et al. 2010), we examined whether STAT5B is involved in δ-OR-mediated neurite outgrowth. For this reason, Neuro-2A cells were transfected with the phosphorylation deficient mutant Y699F of STAT5B (DN-STAT5B), that functions as a dominant negative STAT5B, to block the levels of activated STAT5B prior to agonist activation. As shown in Fig. 4a, the expression of DN-STAT5B significantly decreased DSLET-mediated neurite outgrowth (compare images c and d with g and h). In a similar manner, the number of cells with neurites was significantly reduced in the presence of the DN-STAT5B after DSLET administration of Neuro-2A cells (Fig. 4b). No significant increase in the length of neurites was detected in similarly treated cells expressing or not the DN-STAT5B (images a, b and e, f respectively) in the absence of DSLET. These results suggest that phosphorylation of STAT5B by δ-OR plays a critical role in δ-ΟR signaling that leads to neurite outgrowth.

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Figure 4. Involvement of signal transducers and activators of transcription (STAT)5B in δ-opioid-receptor-mediated neurite outgrowth. (a) Neuro-2A cells expressing the flag-δ-OR were transfected or not with the DN-STAT5B construct without any treatment images a, b, and e, f, respectively, or were treated with 1 μM [D-Ser2, Leu5, Thr6]-enkephalin (DSLET) for 16–20 h in the absence or presence of the DN-STAT5B images c, d and g, h, respectively. (b) Neurites of the cells were scored as described in 'Materials and methods'. Cells with neurites more than twice the cell body were scored as positive. Values represent mean ± SEM; statistically significant differences from control cells are indicated. ***< 0.0005, Student's t-test.

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δ-opioid receptor activation induces Neuro-2A cell differentiation via phosphorylation of STAT5B

To further confirm that activation of δ-OR induces Neuro-2A cell differentiation, we examined the effect of δ-OR activation by DSLET under induced differentiation mediated by the presence of RA. For that reason, Neuro-2A cells were transiently transfected with the flag-tagged δ-OR alone or in combination with the DN-STAT5B mutant. One day after transfection, cells were treated with DSLET for 16–20 h in media containing 2% FBS in the presence of 20 μM RA, following immunolabeling with antibodies recognizing the neuronal differentiation marker βIII-tubulin (Tuj-1) and the flag-tagged δ-OR. As demonstrated in Fig. 5a, DSLET-activation of Neuro-2A cells expressing the δ-OR, showed an increased neurite length as compared with cells co-expressing the DN-STAT5B (Fig. 5a, compare images f and j). This increase in neurites is not detected in untreated δ-ΟR expressing cells (Fig. 5a, image b. In addition, western blot analysis of the Neuro-2A cell lysates indicated a significant increase in the βIII-tubulin (Tuj-1) upon DSLET administration as compared with the control untreated cells, this βIII-tubulin (Tuj-1) increase was attenuated by the presence of the DN-STAT5B mutant (Fig. 5b compares lane 1 with lanes 2 and 3, respectively). These results suggest that activation of δ-ΟR leads to neuronal differentiation via STAT5B phosphorylation pathway.

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Figure 5. Activation of δ-opioid receptor induces the expression of the neuronal differentiation marker βIII-tubulin (Tuj-1) in Neuro-2A cells. Neuro-2A cells were transiently transfected with the flag-δ-OR alone or in combination with the dominant negative DN-signal transducers and activators of transcription (STAT)5B expression plasmids, as indicated. 24 h post-transfection, cells were stimulated or not with 1 μM [D-Ser2, Leu5, Thr6]-enkephalin (DSLET) and induced to differentiate by treatment with 20 μM retinoic acid (RA) in the presence of 2% fetal bovine serum (FBS). After 16–20 h, cells were (a) immunofluorescently labeled for δ-OR detection using an anti-flag FITC and the neuronal differentiation marker βIII-tubulin (Tuj-1 antibody) as described in 'Materials and methods'. Blue represents nuclei stained with the TO-PRO-3, scale bar: 40 μm. (b) Left panel, cell lysates from each culture condition were subjected to western blotting using the Tuj-1 (1 : 800) and the a-tubulin antibodies (1 : 1000). Right panel, quantitative densitometric analysis on the levels of tubulin βIII (Tuj-1). Values represent mean ± SEM; results are representative of an experiment repeated four times. ***< 0.0005, Student's t-test.

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To further explore whether δ-OR-mediated STAT5B phosphorylation can drive neuronal differentiation in Neuro-2A cells upon DSLET administration, we also tested the expression levels of two additional neuronal differentiation markers such as the NCAM and synaptophysin. NCAM is a member of the Ig superfamily of adhesion molecules that plays an important role in directing and regulating the efficiency of neurite outgrowth in the developing nervous system (Maness and Schachner 2007), whereas, the synaptic vesicle marker synaptophysin characterizes mature neurons (Calakos and Scheller 1994). NCAM and synaptophysin protein levels were examined in the presence or not of the dominant negative DN-STAT5B before and after DSLET administration of δ-ΟR expressing Neuro-2A cells differentiated upon serum removal. As shown in Fig. 6a and c, a marked induction of the NCAM levels were detected by western blotting and immunofluorescence labeling of Neuro-2A cells after δ-ΟR activation, as compared with the non-stimulated control conditions (Fig. 6a compares lane 2 with 1, and Fig. 6c compares images a and b). This DSLET mediated increase in NCAM was attenuated in cells expressing the dominant negative mutant of STAT5B as determined by immunoblotting and fluorescence labeling experiments (Fig. 6a, lane 3 and Fig. 6c, image c). To define that indeed DSLET-activated δ-OR drives Neuro-2A to a neuronal phenotype, Neuro-2A cells were cultured in 2% FBS medium containing 20 μΜ RA in the presence of DSLET for 16–20 h and the levels of NCAM were measured. As shown in Fig. 6b, a marked increase in the NCAM protein levels were observed upon DSLET administration of Neuro-2A cells, as compared with the non-stimulated control cells, an effect that is attenuated upon co-expression of the DN-STAT5B mutant (Fig. 6b compare lane 2 with lanes 1 and 3, respectively). These results suggest that this increase in NCAM levels is induced by δ-ΟR activation via a mechanism participating phosphorylated STAT5B, indicating a novel biological effect of δ-ΟR, leading to neurite outgrowth. In a similar manner, an increase in the expression levels of synaptophysin was noted in Neuro-2A cells upon δ-OR activation with DSLET (Fig. 6d). Fluorescence labeling using a synaptophysin antibody confirmed this result (Fig. 6e, images a and b). In contrast, in Neuro-2A cells expressing the DN-STAT5B plasmid, synaptophysin induction was attenuated markedly upon DSLET administration (Fig. 6d compares lane 2 with 3, and Fig. 6e compares images b with c), suggesting that δ-OR activation and subsequent STAT5B phosphorylation induces neuronal differentiation in Neuro-2A cells possibly via synthesis of synaptic vesicle proteins.

image

Figure 6. Activation of the δ-opioid receptor leads to increased neural cell adhesion molecule (NCAM) and synaptophysin levels in Neuro-2A via a signal transducers and activators of transcription (STAT)5B pathway. Western blot analysis of cell lysates from Neuro-2A cells serum starved (a) or treated with 20 μΜ retinoic acid in the presence of 2% fetal bovine serum (FBS) (b) were examined for the NCAM levels upon [D-Ser2, Leu5, Thr6]-enkephalin (DSLET) and/or DN-STAT5B expression. (d) represents western blotting of synaptophysin levels of serum deprived Neuro-2A cell lysates treated as indicated. Tubulin and actin were used as quantitative markers for equal protein loading. Values represent mean ± SEM; results are representative of an experiment repeated four times. Statistically significant differences from control or treated cells are indicated, *< 0.05, **< 0.005, ***< 0.0005, by Student's t-test. Immunofluorescence microscopy analysis of the effect of DSLET on NCAM (c) and synaptophysin (e) levels were detected by fluorescence labeling as described in 'Materials and methods'. Lanes 1, 2, and 3 (panels a, b, d) as well as images a, b and c in panels c, e represent lysates from Neuro-2A cells deprived from serum either untreated a, or upon 1 μM DSLET administration in the absence b, or presence c, of the dominant negative mutant of STAT5B (DN-STAT5B), respectively. Red represents nuclei stained with the TO-PRO-3 fluorescent dye. Scale bar: 2 μm Results are representative of one experiment repeated at least three times.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure statement
  8. References

Substantial effort is invested toward understanding how opioids induce dynamic changes in gene expression that control synaptic plasticity and other cellular responses in the nervous system. These transcriptional alterations could explain in part, the phenomena of cellular adaptation caused by opioid administration, and reveal pathways for several physiological responses such as neuronal growth and differentiation. A number of different studies have shown that administration of opioids induces activation of various transcription factors ranging from cyclic AMP-response element-binding protein, activator protein AP-1, members of the MAP kinase pathway to NF-kB (Tso and Wong 2003). Other observations have demonstrated that activation of the μ- and δ-opioid receptors lead to the phosphorylation of members of the STAT family of transcription factors such as STAT3 (Lo and Wong 2004; Yuen et al. 2004) and STAT5A/B (Mazarakou and Georgoussi 2005; Georganta et al. 2010). All these suggest that opioid receptors are involved in a variety of different signaling networks that contribute to changes in the expression of specific target genes.

It has been previously reported that endogenous opioid peptides and alkaloid agonists can affect the neuronal development by promoting neuronal survival, neuronal and glial proliferation, migration, and spine formation (Meriney et al. 1991; Hauser and Mangoura 1998). Recent observations have shown that the endogenous opioid system modulates neurogenesis in the adult hippocampus (Kolodziej et al. 2008). Others reported that opioid receptors play an important role in cell proliferation, gliogenesis, and neurogenesis (Hauser and Mangoura 1998; Narita et al. 2006; Sargeant et al. 2008). Although several players have been shown to participate, it remains unclear how extracellular signals that trigger the activation of the opioid receptors mediate changes in cytoskeletal rearrangements and how these signals are regulated.

We have recently demonstrated that STAT5B (i) associates with the μ-δ opioid receptors within the YXXL motif on their C-terminal tails, (ii) interacts with selective Gα and Gβγ subunits constitutively or upon δ-OR activation, (iii) is phosphorylated upon δ-OR activation, and, finally, (iv) forms a multi-component signaling complex using as a platform the C-terminal tail of δ-ΟR consisting of Gα, Gβγ subunits and c-Src kinase, implicated in STAT5B transcriptional responses (Mazarakou and Georgoussi, 2005; Georgoussi et al., 2006; Georgoussi 2008; Georganta et al. 2010; Georgoussi et al. 2012). In this study, efforts were made to delineate the biological relevance and define the functional significance of opioid-induced STAT5B activation in neuronal cells. Our observations indicate that prolonged activation of the δ-OR with DSLET leads to increased cell survival, which can be blocked by a δ-OR-selective antagonist, suggesting that DSLET displays protective effects on neuronal survival after trophic factor deprivation. The presence of a dominant negative mutant of STAT5B abrogates the protective effect of DSLET. These results suggest that phosphorylation of STAT5B by DSLET-activated δ-ΟR triggers neuronal survival through a signaling pathway involving p-STAT5B in SH-SY5Y and Neuro-2A cells.

Previous observations have shown that activation of μ-OR promotes neuronal survival in a Gi/Go-mediated-PI-3K-dependent signaling pathway (Iglesias et al. 2003). Furthermore, activation of both μ- and δ-opioid receptors prevents apoptosis in SH-SY5Y and NG108-15 cells, respectively, via a phosphatidylinositol-3-kinase pathway implicating Akt (Iglesias et al. 2003; Heiss et al. 2009), whereas in PC12 cells activation of δ-OR and κ-OR following serum deprivation prevents early apoptosis (Dermitzaki et al. 2000). It has also been shown that activation of δ-OR protects various neuronal networks and reduces neuronal injury in hypoxic and ischemic conditions (Zhang et al. 2002; Narita et al. 2006; Johnson and Turner 2010). All these suggest that opioids exert a distinct functional role in neuroprotection.

Neurite outgrowth is a cellular phenomenon that characterizes neuronal differentiation and regeneration in the organism. The pathways regulating neurite outgrowth in culture are likely to play a role in the terminal differentiation of neurons in vivo. During neurite outgrowth, the actin and microtubule cytoskeletal networks work in a coordinated fashion to generate and stabilize the growing neurites (Bromberg et al. 2008). The actin cytoskeleton reorganizes to allow formation of the growth cone and the microtubules realign into bundles to stabilize the growing neurite. Gi/Go members are abundantly expressed in brain and are enriched at neuronal growth cones (Bromberg et al. 2008). The role of Gi/Go-coupled receptor signaling in the regulation of neurite outgrowth is elucidated.

The δ-ΟR couples with members of the Gi/Go family to regulate a variety of physiological responses. Indeed, we have previously demonstrated the ability of δ-OR and μ-OR to couple with a specific subset of Giα/Goα to mediate various cellular responses (Georgoussi et al. 1993; Georgoussi and Zioudrou 1993; Georgoussi et al. 1995, 1997). Other observations have also shown that treatment of HEK293 cells with PTX blocks DSLET-mediated STAT5B phosphorylation, implicating the involvement of PTX-sensitive Gαi/o proteins in the mechanism leading to STAT5B phosphorylation (Georganta et al. 2010). Moreover, it was shown that STAT5B selectively couples with Gαi3 and Gαo with a certain specificity and potency upon δ-ΟR activation with DSLET, DPDPE, and/or morphine (Georganta et al. 2010). Interestingly, in this study, we indicate that activation of δ-OR by DSLET leads to increased neurite outgrowth of Neuro-2A cells, an effect that can be blocked by co-administration of naltrindole, a δ-OR-selective antagonist. PTX treatment of Neuro-2A cells markedly attenuates DSLET-mediated neurite outgrowth demonstrating the crucial role in which Gi/Go proteins play in this process. Another finding of this study is that the expression of a DN-STAT5B mutant that blocks STAT5B phosphorylation attenuates δ-OR-mediated neurite outgrowth. The fact that a selective STAT5B-driven coupling with a specific subset of G proteins exists, and, moreover, that expression of a DN-STAT5B mutant or PTX treatment can abolish DSLET-mediated neurite outgrowth, suggests a yet undiscovered mechanism through which δ-OR activation promotes neurite outgrowth in a Gi/Go-linked, STAT5B-dependent manner, demonstrating that STAT5B phosphorylation may be a key regulatory downstream component implicated in this biological effect.

To evaluate whether the morphological changes induced by δ-OR treatment involve increases of expression of neuronal-specific molecules, we detected the levels of tubulin βIII, an isoform that specifically localizes in neurons (Ferreira and Caceres 1992). Immunofluorescence studies correlated with western blot analysis demonstrate that DSLET acts on Neuro-2A cells by increasing elongation of individual neurites, an effect that is abolished by expression of the DN-STAT5B mutant. To further establish whether activation of δ-OR indeed directs neurotropic events mediated by STAT5B phosphorylation, the levels of NCAM and synaptophysin were detected in Neuro-2A cells after DSLET activation. NCAM is a cell surface glycoprotein widely expressed during the embryonic development mediating adhesion between neural cells and stimulating neurite outgrowth (Ditlevsen et al. 2008), whereas synaptophysin is a neuroendocrine marker involved in the regulation of synaptic vesicle fusion and neurotransmitter release, which is identified as a direct binding partner of the μ-OR implicated in receptor's trafficking and signaling (Liang et al. 2007). Our results demonstrate a significant increase in the levels of these neuronal differentiation protein markers, suggesting that activation of δ-ΟR by DSLET, triggers Neuro-2A cell differentiation. This DSLET-mediated increase was markedly attenuated when phosphorylation of STAT5B was blocked by the expression of the DN-STAT5B mutant, demonstrating the involvement of phosphorylated STAT5B in this effect. Collectively, our results demonstrate a key regulatory role of the δ-OR-STAT5B interplay and suggest an important physiological relevance of STAT5B in the development and protection of the nervous system.

In a similar manner, it has been previously shown that activation of the Gαi/o-coupled CB1 and 5-HT1 receptors leads to neurite outgrowth via a signaling pathway involving Gαi/o proteins, c-Src kinase, and the STAT3 transcription factor (Fricker et al. 2005; He et al. 2005). Previous studies have shown that the collapse of growth cones is mediated by G proteins in a PTX sensitive-dependent manner (Igarashi et al. 1993); whereas the expression of a constitutively active Gαo mutant in PC12 and N1E-115 neuroblastoma cells increases both the number and length of neurites per cell (Strittmatter et al. 1994). In addition, STAT3 and STAT5 members have been linked to neuroprotection mediated by trophic factors and cytokines after ischemic brain or nerve injury and shown to promote neuronal survival by inducing the expression of neuroprotective genes (Dziennis and Alkayed 2008). STAT5 activation is required for the anti-apoptotic effects of erythropoietin in SH-SY5Y cells and contributes to erythropoietin-mediated neuroprotection against hippocampal neuronal death after transient global cerebral ischemia (Um and Lodish 2006; Zhang et al. 2007), whereas STAT3 may play a role in neuronal differentiation in response to nerve growth factor (Ng et al. 2006).

Collectively, the present results reveal a novel biological role of STAT5B as an important component in δ-ΟR signaling and function in Neuro-2A cells. It would be of interest to identify more proteins that are regulated by STAT5B activation during neurite outgrowth and detect if they are also involved in neuronal development. This would provide evidence on how opiates are implicated in neural plasticity and how STAT5B-regulated genes may modulate cytoskeletal dynamics to induce neurite outgrowth.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure statement
  8. References

The technical assistance of Mrs Konstantina Kapolou and Stavroula-Maria Vrana is highly acknowledged. This study was supported by the European Union grant «Normolife» (LSHC-CT2006-037733) to Z.G.

Disclosure statement

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure statement
  8. References

The authors do not have financial or other conflicts of interest associated with this work.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure statement
  8. References