Atypical PKCzeta is involved in RhoA-dependent mitogenic signaling by the P2Y(12) receptor in C6 cells

When nucleotide hydrolysis is prevented, agonists of the P2Y(12) receptor enhance the proliferation of C6 glioma cells by RhoA-dependent, protein kinase C (PKC)-dependent activation of the extracellular signal-regulated kinase (ERK) pathway [Claes P, Grobben B, Van Kolen K, Roymans D & Slegers H (2001) Br J Pharmacol134, 402-408; Grobben B, Claes P, Van Kolen K, Roymans D, Fransen P, Sys SU & Slegers H (2001) J Neurochem78, 1325-1338]. In this study, we show that ERK1/2 phosphorylation was not affected by transfection of the cells with the Gbetagamma-subunit-scavenging adrenergic receptor kinase peptide [betaARK1-(495-689)] or with Rap1GAPII, indicating that P2Y(12) receptor stimulation enhances ERK1/2 phosphorylation by G(i)alpha subunit-mediated signaling independently of Rap1 activation. Inhibition of the RhoA downstream effector Rho-associated coiled-coil-containing kinase (ROCK) with Y-27632 did not affect the P2Y(12) receptor-induced increase in ERK1/2 phosphorylation but abrogated the mitogenic response. Involvement of growth factor receptor transactivation in the signaling towards ERK phosphorylation could be ruled out by the lack of an effect of PP2, AG1024, AG1296 or SU1498, inhibitors of Src, insulin-like growth factor receptor, platelet-derived growth factor receptor and vascular endothelial growth factor receptor kinase activity, respectively. Experiments with bisindolylmaleimide I and IX indicated the requirement of PKC activity. Classical and novel PKC isoforms could be excluded by treatment of the cells with G?6976 and calphostin C, whereas addition of a myristoylated PKCzeta pseudosubstrate inhibitor completely abolished P2Y(12) receptor-induced ERK1/2 activation. Moreover, coimmunoprecipitation experiments revealed PKCzeta/Raf1 and PKCzeta/ERK association, indicating the involvement of PKCzeta. From the data presented, we can conclude that the P2Y(12) receptor enhances cell proliferation by a G(i)alpha-dependent, RhoA-dependent PKCzeta/Raf1/MEK/ERK pathway that requires activation of ROCK, which is not involved in ERK1/2 signaling.     

Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets

We have previously shown that ADP-induced thromboxane generation in platelets requires signalling events from the G(q)-coupled P2Y1 receptor (platelet ADP receptor coupled to stimulation of phospholipase C) and the G(i)-coupled P2Y12 receptor (platelet ADP receptor coupled to inhibition of adenylate cyclase) in addition to outside-in signalling. While it is also known that extracellular calcium negatively regulates ADP-induced thromboxane A2 generation, the underlying mechanism remains unclear. In the present study we sought to elucidate the signalling mechanisms and regulation by extracellular calcium of ADP-induced thromboxane A2 generation in platelets. ERK (extracllular-signal-regulated kinase) 2 activation occurred when outside-in signalling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished ERK phosphorylation, indicating that both P2Y receptors are required for ADP-induced ERK activation. Inhibitors of Src family kinases or the ERK upstream kinase MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] abrogated ADP-induced ERK phosphorylation and thromboxane A2 generation. Finally ADP- or G(i)+G(z)-induced ERK phosphorylation was blocked in the presence of extracellular calcium. The present studies show that ERK2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and this plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of ERK activation.     

P2Y nucleotide receptor interaction with alpha integrin mediates astrocyte migration

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5'-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y(2) receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y(2) nucleotide receptor (P2Y(2)R) expression with siRNA of P2Y(2)R (P2Y(2)R siRNA). UTP also increased the expression in astrocytes of alpha(V)beta(3/5) integrins that are known to interact directly with the P2Y(2)R to modulate its function. Anti-alpha(V) integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y(2)R/alpha(V) interactions mediate the activation of astrocytes by UTP. P2Y(2)R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-alpha(V) integrin antibody. These results suggest that P2Y(2)Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.     

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

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 G_i/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.     

Cell cycle regulation of astrocytes by extracellular nucleotides and fibroblast growth factor-2

Extracellular ATP enhances the mitogenic activity of fibroblast growth factor-2 (FGF2) in astrocytes, but the molecular mechanism underlying this synergistic interaction is not known. To determine whether the potentiating effect of extracellular ATP involves cell cycle control mechanisms, we have measured the expression of cyclins that are induced in different phases of the cell cycle in primary cultures of rat cortical astrocytes. We found that ATP potentiated the ability of FGF2 to stimulate expression of cyclin D1, a regulator of cell cycle entry, as well as cyclin A, a regulator of DNA replication. Because FGF2 and P2 purinergic receptors are coupled to extracellular signal regulated protein kinase (ERK), a key member of a signaling cascade that regulates proliferation, we also investigated the role of ERK in regulating cyclin expression induced by FGF2 and ATP. We found that the potentiating effect of ATP on cyclin expression was significantly reduced by U0126, an inhibitor of MEK, the upstream activator of ERK. P2 receptor agonist studies revealed that UTP enhanced FGF2-induced cyclin expression and mitogenesis whereas 2-methylthioADP was ineffective. By contrast, 2′,3′-O-(4-benzoyl)-benzoyl-ATP markedly inhibited FGF2-induced mitogenesis. Consistent with opposing effects of P2Y and P2X receptors on mitogenesis, UTP stimulated a transient activation of ERK whereas BzATP stimulated a more sustained ERK signal. These findings suggest that signaling by P2Y receptors, most likely of the purine/pyrimidine subtype, enhance the ability of FGF2 to stimulate entry into a new cell cycle, as well as DNA replication, by an ERK-dependent mechanism, whereas signaling by P2X receptors, possibly the P2X7 subtype, inhibits FGF2-induced mitogenesis in astrocytes. Interactions between P2Y, P2X and polypeptide growth factor signaling pathways may have important implications for CNS development as well as injury and repair.     

P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells

Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.     

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.     

Molecular determinants of P2Y2 nucleotide receptor function

In the mammalian nervous system, P2 nucleotide receptors mediate neurotransmission, release of proinflammatory cytokines, and reactive astrogliosis. Extracellular nucleotides activate multiple P2 receptors in neurons and glial cells, including G protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels. In glial cells, the P2Y2 receptor subtype, distinguished by its ability to be equipotently activated by ATP and UTP, is coupled to pro-inflammatory signaling pathways. In situ hybridization studies with rodent brain slices indicate that P2Y2 receptors are expressed primarily in the hippocampus and cerebellum. Astrocytes express several P2 receptor subtypes, including P2Y2 receptors whose activation stimulates cell proliferation and migration. P2Y2 receptors, via an RGD (Arg-Gly-Asp) motif in their first extracellular loop, bind to alphavbeta3/beta5 integrins, whereupon P2Y2 receptor activation stimulates integrin signaling pathways that regulate cytoskeletal reorganization and cell motility. The C-terminus of the P2Y2 receptor contains two Src-homology-3 (SH3)-binding domains that upon receptor activation, promote association with Src and transactivation of growth factor receptors. Together, our results indicate that P2Y2 receptors complex with both integrins and growth factor receptors to activate multiple signaling pathways. Thus, P2Y2 receptors present novel targets to control reactive astrogliosis in neurodegenerative diseases.     

Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1β-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1β for 24 h induced a 2–3-fold increase in GRK2 and decreased C–C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/− animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.     

Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase.

Extracellular nucleotides acting through specific P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue, the human osteosarcoma cell line SaOS-2 and primary osteoblasts express P2Y1 and P2Y2 receptors, respectively. Their activation by nucleotide agonists (ADP and ATP for P2Y1; ATP and UTP for P2Y2) elevates [Ca2+]i and moderately induces expression of the c-fos proto-oncogene. A synergistic effect on c-fos induction is observed by combining ATP and parathyroid hormone, a key bone cell regulator. Parathyroid hormone elevates intracellular cAMP levels and correspondingly activates a stably integrated reporter gene driven by the Ca2+/cAMP-responsive element of the human c-fos promoter. Nucleotides have little effect on either cAMP levels or this reporter, instead activating luciferase controlled by the full c-fos promoter. This induction is reproduced by a stably integrated serum response element reporter independently of mitogen-activated protein kinase activation and ternary complex factor phosphorylation. This novel example of synergy between the cAMP-dependent protein kinase/CaCRE signaling module and a non-mitogen-activated protein kinase/ternary complex factor pathway that targets the serum response element shows that extracellular ATP, via P2Y receptors, can potentiate strong responses to ubiquitous growth and differentiative factors.     

Signaling from Nucleotide Receptors to Protein Kinase Cascades in Astrocytes

In the CNS, extracellular ATP can function as an excitatory neurotransmitter as well as a trophic factor. These short-term and long-term actions are mediated by nucleotide receptors. Extracellular ATP can also act as a co-mitogen in conjunction with polypeptide growth factors such as basic fibroblast growth factor (FGF2). Cellular proliferation, differentiation and survival are regulated by signaling cascades composed of protein kinases, including extracellular signal regulated protein kinase (ERK) and protein kinase B (also called Akt). Here we summarize recent studies on nucleotide receptor signaling to ERK and Akt in astrocytes and the role of protein kinase cascades in mediating the trophic actions of extracellular ATP, alone or together with FGF2. Because extracellular ATP and FGF2 contribute to the hyperplastic and hypertrophic response of astrocytes to CNS injuries, an understanding of their protein kinase signaling mechanisms may lead to novel therapeutic approaches for neurological conditions that involve gliosis and the generation of reactive astrocytes, such as trauma, stroke, seizure and neurodegenerative and demyelinating disorders.Special issue dedicated to Lawrence F. Eng.     

P2Y receptors play a critical role in epithelial cell communication and migration

Cellular injury induces a complex series of events that involves Ca2+ signaling, cell communication, and migration. One of the first responses following mechanical injury is the propagation of a Ca2+ wave (Klepeis et al. [2001] J Cell Sci 114(Pt 23):4185-4195). The wave is generated by the extracellular release of ATP, which also induces phosphorylation of ERK (Yang et al. [2004] J Cell Biochem 91(5):938-950). ATP and other nucleotides, which bind to and activate specific purinergic receptors were used to mimic injury. Our goal was to determine which of the P2Y purinergic receptors are expressed and stimulated in corneal epithelial cells and which signaling pathways are activated leading to changes in cell migration, an event critical for wound closure. In this study, we demonstrated that the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors were present in corneal epithelial cells. A potency profile was determined by Ca2+ imaging for nucleotide agonists as follows: ATP > or = UTP > ADP > or = UDP. In contrast, negligible responses were seen for beta,gamma-meATP, a general P2X receptor agonist and adenosine, a P1 receptor agonist. Homologous desensitization of the Ca2+ response was observed for the four nucleotides. However, P2Y receptor internalization and degradation was not detected following stimulation with ATP, which is in contrast to EGFR internalization observed in response to EGF. ATP induced cell migration was comparable to that of EGF and was maximal at 1 microM. Cells exposed to ATP, UTP, ADP, and UDP demonstrated a rapid twofold increase in phosphorylation of paxillin at Y31 and Y118, however, there was no activation elicited by beta,gamma-meATP or adenosine. Additional studies demonstrated that wound closure was inhibited by reactive blue 2. These results indicate that P2Y receptors play a critical role in the injury repair process.     

Signaling from P2 nucleotide receptors to protein kinase cascades induced by CNS injury

Gliosis is a hypertrophic and hyperplastic response to many types of central nervous system injury, including trauma, stroke, seizure, as well as neurodegenerative and demyelinating disorders. Reactive astrocytes, a major component of the glial scar, express molecules that can both inhibit and promote axonal regeneration. ATP, which is released upon traumatic injury, hypoxia, and cell death, contributes to the gliotic response by binding to specific cell surface astrocytic P2 nucleotide receptors and evoking characteristic features of gliosis such as increased expression of glial fibrillary acidic protein (GFAP), generation and elongation of astrocytic processes, and cellular proliferation. Here, we review recent studies that demonstrate that (1) metabotropic, P2Y, and ionotropic, P2X, receptors expressed in astrocytes are coupled to protein kinase signaling pathways that regulate cellular proliferation, differentiation, and survival such as ERK and protein kinase B/Akt and (2) these P2 receptor/protein kinase cascades are activated after trauma induced by mechanical strain. We suggest that P2 receptor/protein kinase signaling pathways might provide novel therapeutic targets to regulate the formation of reactive astrocytes and the production of molecules that affect axonal regeneration and neurodegeneration.     

Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes

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.     

P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.     

P2Y2 Nucleotide Receptors Mediate Metalloprotease-dependent Phosphorylation of Epidermal Growth Factor Receptor and ErbB3 in Human Salivary Gland Cells

The G protein-coupled receptor P2Y₂ nucleotide receptor (P2Y₂R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y₂Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y₂R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-α protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y₂R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y₂R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

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.