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Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but by distinct mechanisms.

Opioid receptor signaling via EGF receptor (EGFR) transactivation and ERK/MAPK phosphorylation initiates diverse cellular responses that are cell type-dependent. In astrocytes, multiple μ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different outcomes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite outgrowth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by μ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFβ1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activation of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibition of TSP1 is reminiscent of the negative effect of μ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation.

Although micro, kappa, and delta 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 micro opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)-induced ERK-mediated astrocyte proliferation. The micro-opioid agonists ([d-ala(2), mephe(4), gly-ol(5)] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via G(i/o) protein, calmodulin (CaM), and beta-arrestin2-dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the micro-opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W-7, and siRNA silencing of beta-arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation-mediated mechanism. This was disclosed by abolishment of micro-opioid-induced ERK phosphorylation with the EGF receptor-specific tyrosine phosphorylation inhibitor, AG1478, and micro-opioid-induced reduction of EGF receptor tyrosine phosphorylation by PTX, and beta-arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long-term (h) treatment of primary astrocytes with [d-ala(2),mephe(4),gly-ol(5)] enkephalin or morphine, attenuated EGF-induced ERK phosphorylation and proliferation (as measured by 5'-bromo-2'-deoxy-uridine labeling). PTX and beta-arrestin2 siRNA but not W-7 reversed the micro-opioid inhibition. Unexpectedly, beta-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 micro-opioid signaling pathways to ERK and their cellular responses.

GTP binding regulatory protein (G protein)-coupled receptors can activate MAPK pathways via G protein-dependent and -independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and beta-arrestin 2 pathways in kappa opioid receptor-induced, extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non-nitrogenous agonist, C(2)-methoxymethyl salvinorin B (MOM-Sal-B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gbetagamma subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of beta-arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM-Sal-B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM-Sal-B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gbetagamma subunits or beta-arrestin 2, suggesting that both G protein-dependent and -independent ERK pathways are required for this outcome.

Acute mu and kappa opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The mu agonist, [D-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas kappa agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant mu opioid receptor that binds CaM poorly or a dominant negative mutant of PKCepsilon were used as a model system to study mu signaling. Evidence was gained to implicate CaM and PKCepsilon in DAMGO stimulation of ERK. DAMGO activation of PKCepsilon and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the mu receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCepsilon. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCzeta, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCzeta. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic mu and kappa opioids.

Astroglia are a principal target of long-term mu antiproliferative actions. The mitogen-activated protein (MAP) kinase known as extracellular signal-regulated kinase (ERK), is a key mediator of cell proliferation. In studies on the mechanism of short- and long-term mu opioid regulation of the ERK signaling pathway, we show that the mu opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), acting via the endogenous mu opioid receptor (MOR), induced sequential epidermal growth factor receptor (EGF) receptor (EGFR) Tyr phosphorylation, Ser phosphorylation, and down-regulation in immortalized rat cortical astrocytes. The short-term action of DAMGO resulted in the stimulation of ERK phosphorylation. 4(3-Chlorophenylamino)-6,7-dimethoxyquinazoline (Tyrphostin AG1478), a selective inhibitor of EGFR Tyr kinase activity, blocked EGFR and ERK activation by short-term DAMGO administration, implicating EGFR transactivation in its stimulation of ERK activity. Inhibitors of matrix metalloproteinases attenuated MOR-mediated ERK phosphorylation, suggesting that shedding of EGF-like ligands from the plasma membrane may be involved in the EGFR transactivation process. Prolonged DAMGO exposure induced EGFR internalization/down-regulation, did not activate ERK, and inhibited exogenous EGF-stimulated ERK phosphorylation. MOR-mediated EGFR down-regulation seems to be MAP kinase-dependent, because it was inhibited by the ERK kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126), and tyrphostin AG1478. The kappa opioid agonist (5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide (U69,593) induced Tyr and Ser phosphorylation of EGFR and activation of ERK. However, long-term application of U69,593 neither down-regulated EGFR nor inhibited EGF-induced ERK activation. Instead, it engendered a sustained activation of ERK. Collectively, our data suggest that long-term application of DAMGO initiates heterologous down-regulation of EGFR via a mechanism involving ERK in astrocytes.

J. Neurochem. (2010) 112, 1431–1441. As embryonic stem cell-derived neural progenitors (NPs) have the potential to be used in cell replacement therapy, an understanding of the signaling mechanisms that regulate their terminal differentiation is imperative. In previous studies, we discovered the presence of functional μ opioid receptors (MOR) and κ opioid receptors (KOR) in mouse embryonic stem cells and NPs. Here, MOR and KOR immunoreactivity was detected in NP-derived oligodendrocytes during three stages of their maturation in vitro. Moreover, we examined the modulation of retinoic acid-induced NP differentiation to astrocytes and neurons by μ, [D-ala2, mephe4, gly-ol5] enkephalin, or κ, U69, 593, opioids. Both opioid agonists inhibited NP-derived neurogenesis and astrogenesis via their corresponding receptors as determined by immunocytochemistry. By administering selective inhibitors, we found that opioid inhibition of NP-derived astrogenesis was driven via extracellular-signal regulated kinase (ERK), while the p38 mitogen-activated protein kinase pathway was implicated in opioid attenuation of neurogenesis. In addition, μ and κ opioids stimulated oligodendrogenesis from NP-derived NG2+ oligodendrocyte progenitors via both ERK and p38 signaling pathways. Accordingly, both opioids induced ERK phosphorylation in NG2+ cells. These results indicate that small molecules, such as MOR and KOR agonists may play a modulatory role in NP terminal differentiation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

GTP binding regulatory protein (G protein)-coupled receptors can activate MAPK pathways via G protein-dependent and -independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β-arrestin 2 pathways in kappa opioid receptor-induced, extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non-nitrogenous agonist, C(2)-methoxymethyl salvinorin B (MOM-Sal-B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β-arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM-Sal-B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM-Sal-B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β-arrestin 2, suggesting that both G protein-dependent and -independent ERK pathways are required for this outcome. [ABSTRACT FROM AUTHOR]

Acute μ and κ opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The μ agonist, [D-ala²,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas κ agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CAM), a mutant μ opioid receptor that binds CaM poorly or a dominant negative mutant of PKCϵ were used as a model system to study μ signaling. Evidence was gained to implicate CAM and PKCϵ in DAMGO stimulation of ERK. DAMGO activation of PKCϵ and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CAM is released from the p receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCϵ. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCζ, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCζ. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic μ and κ opioids. [ABSTRACT FROM AUTHOR]