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.
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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.