Regulation of hyaluronan production by ß2 adrenergic receptor signaling.

BACKGROUND: Hyaluronan (HA), the main component of the extracellular matrix, is involved in tissue elasticity and cell scaffolding, and in progression of conditions such as cancer, inflammation and wound healing. Signaling by G protein coupled receptor (GPCR) activation increases expression of hyaluronan synthase (HAS) and HA production. The ß2 adrenergic receptor (ß2AR) is a catecholamine-liganded GPCR that is involved in cancer progression and wound healing. Since HA and ß2AR are involved in a common pathology, we investigated whether ß2AR signaling regulates HA production. METHODS: After stimulating ß2AR-expressing cells with a ß agonist, the amount of HA in the culture medium was measured and HAS expression was examined by real-time PCR. A variety of signaling molecule inhibitors were used to identify signaling pathways that alter HAS expression. RESULTS: ß2AR activation increased HA production and enhanced HAS2 expression. The increase in HAS2 expression by ß2AR activation occurred via the Gs - adenylyl cyclase - PKA - CREB signal transduction pathway. CONCLUSIONS: Downstream signal transduction by ß2AR activation increased HA production by enhancing transcription of the HAS2 gene. This study suggests that ß2AR is a GPCR that regulates HA production, and that stimulation with a catecholamine (ß2 agonist) can regulate HA production. GENERAL SIGNIFICANCE: ß2AR may function through regulation of HA production in cancer progression and wound healing.

Regulation of membrane raft recruitment of the bradykinin B2 receptor by close association with the ATP/UTP receptor P2Y2.

Several G protein-coupled receptors are present in lipid rafts. We have shown that most of the P2Y2 receptor (P2Y2R) protein is fractionated into lipid rafts in COS 7 cells. In the same cells, about 25-30% of the bradykinin B2 receptor (B2R) protein is also fractionated into lipid rafts. When both P2Y2R and B2R are co-expressed, the distribution of P2Y2R remained unchanged, but more B2R shifted into the raft fraction. This indicates that the interaction between both receptors recruited B2R into the lipid rafts. After 15 min of UTP stimulation, both receptors almost completely disappeared from the cell surface by endocytosis as observed with a confocal fluorescence microscope. Furthermore, with bradykinin stimulation for 15 min, portions of both receptors disappeared from the cell surface and were endocytosed. As we reported previously with both CHO-K1 cells and HEK 293 cells, continuous stimulation of COS7 cells with GT1b and CSC resulted in the disappearance of both P2Y2R and B2R from the cell membrane surface. Thus, both P2Y2R and B2R migrate into membrane rafts and are endocytosed in parallel with signal crosstalk, clearly indicating that both closely interact on membrane rafts. The P2Y2R N-glycosylation deficient mutant does not migrate to the cell surface. It remains predominantly in the endoplasmic reticulum and is fractionated into raft fractions. In the presence of this glycosylation mutant, most of B2R remains in the endoplasmic reticulum, and is fractionated into the raft fraction. These findings demonstrate that in the membrane rafts of the endoplasmic reticulum, both receptors are already closely associated, and B2R shifts into the rafts by affinity with P2Y2R.

Homodimer formation by the ATP/UTP receptor P2Y2 via disulfide bridges.

Many class C G-protein coupled receptors (GPCRs) function as homo- or heterodimers and several class A GPCRs have also been shown to form a homodimer. We expressed human P2Y2 receptor (P2Y2R) in cultured cells and compared SDS-PAGE patterns under reducing and non-reducing conditions. Under non-reducing conditions, approximately half of the P2Y2Rs were electrophoresed as a dimer. We then produced Cys to Ser mutants at four sites (Cys25, Cys106, Cys183 and Cys278) in the extracellular domains of P2Y2R and examined the effect on dimer formation and receptor activity. All single mutants formed dimers similarly to the wild-type protein, but C25S, C106S and C183S P2Y2R lost activity, while C278S P2Y2R maintained weak activity. Coexpression with wild-type P2Y2R recovered the activity of the C25S mutant. These results show that Cys106 and Cys183 are required for monomer or homodimer activity; Cys25 is required for monomer activity, but it is not needed in one protomer for homodimer activity; and Cys278 can be replaced in the monomer and homodimer. Approximately, half of C25S/C278S double mutants were electrophoresed as a dimer, similarly to the wild-type and single mutants, and dimers with the wild-type protein were active. These results suggest involvement of Cys106 and Cys183 in disulfide bonding between protomers in homodimer formation.

N-glycan-dependent cell-surface expression of the P2Y2 receptor and N-glycan-independent distribution to lipid rafts.

P2Y2 receptor (P2Y2R) is a G-protein-coupled receptor (GPCR) that couples with Gαq/11 and is stimulated by ATP and UTP. P2Y2R is involved in pain, proinflammatory changes, and blood pressure control. Some GPCRs are localized in lipid rafts for interaction with other signaling molecules. In this study, we prepared N-glycan-deficient mutants by mutating the two consensus Asn residues for N-glycosylation to Gln to examine intracellular localization and association with lipid rafts. Western blotting of the wild type (WT) protein and mutants (N9Q, N13Q, N9Q/N13Q) in COS-7 cells showed that both Asn residues were glycosylated in the WT. Fluorescent microscopy analysis showed that WT, N9Q and N13Q were expressed in the endoplasmic reticulum (ER), Golgi body, and cell membrane, but N9Q/N13Q was only found in the ER. WT, N9Q and N13Q moved from the cell surface to endosomes within 15 min after UTP stimulation. WT and the N9Q/N13Q glycosylation-deficient mutant appeared in the detergent insoluble membrane fraction, lipid raft. These findings suggest that P2Y2R is localized in lipid rafts in the ER during biosynthesis, and that N-glycosylation is required for subsequent expression in the cell membrane. In the presence of epoxomicin, a proteasome inhibitor, there was a significant increase in the level of N9Q/N13Q, which suggests that N-glycan-deficient P2Y2R undergoes proteasomal degradation.

Loss of α1,6-Fucosyltransferase Decreases Hippocampal Long Term Potentiation: IMPLICATIONS FOR CORE FUCOSYLATION IN THE REGULATION OF AMPA RECEPTOR HETEROMERIZATION AND CELLULAR SIGNALING.

Core fucosylation is catalyzed by α1,6-fucosyltransferase (FUT8), which transfers a fucose residue to the innermost GlcNAc residue via α1,6-linkage on N-glycans in mammals. We previously reported that Fut8-knock-out (Fut8(-/-)) mice showed a schizophrenia-like phenotype and a decrease in working memory. To understand the underlying molecular mechanism, we analyzed early form long term potentiation (E-LTP), which is closely related to learning and memory in the hippocampus. The scale of E-LTP induced by high frequency stimulation was significantly decreased in Fut8(-/-) mice. Tetraethylammonium-induced LTP showed no significant differences, suggesting that the decline in E-LTP was caused by postsynaptic events. Unexpectedly, the phosphorylation levels of calcium/calmodulin-dependent protein kinase II (CaMKII), an important mediator of learning and memory in postsynapses, were greatly increased in Fut8(-/-) mice. The expression levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the postsynaptic density were enhanced in Fut8(-/-) mice, although there were no significant differences in the total expression levels, implicating that AMPARs without core fucosylation might exist in an active state. The activation of AMPARs was further confirmed by Fura-2 calcium imaging using primary cultured neurons. Taken together, loss of core fucosylation on AMPARs enhanced their heteromerization, which increase sensitivity for postsynaptic depolarization and persistently activate N-methyl-d-aspartate receptors as well as Ca(2+) influx and CaMKII and then impair LTP.

Close association of B2 bradykinin receptors with P2Y2 ATP receptors.

Two G-protein-coupled receptors (GPCRs) that couple with Gαq/11, B2 bradykinin (BK) receptor (B2R) and ATP/UTP receptor P2Y2 (P2Y2R), are ubiquitously expressed and responsible for vascular tone, inflammation, and pain. We analysed the cellular signalling of P2Y2Rs in cells that express B2Rs. B2R desensitization induced by BK or B2R internalization-inducing glycans cross-desensitized the P2Y2R response to ATP/UTP. Fluorescence resonance energy transfer from P2Y2R-AcGFP to B2R-DsRed was detected in the cells and on the cell surfaces, showing the close association of these GPCRs. BK- and ATP-induced cross-internalization of P2Y2R and B2R, respectively, was shown in a ß-galactosidase complementation assay using P2Y2R or B2R fused to the H31R substituted α donor peptide of a ß-galactosidase reporter enzyme (P2Y2R-α or B2R-α) with coexpression of the FYVE domain of endofin, an early endosome protein, fused to the M15 acceptor deletion mutant of ß-galactosidase (the ω peptide, FYVE-ω). Arrestin recruitment to the GPCRs by cross-activation was also shown with the similar way. Coimmunoprecipitation showed that B2R and P2Y2R were closely associated in the cotransfected cells. These results indicate that B2R couples with P2Y2R and that these GPCRs act together to fine-tune cellular responsiveness. The collaboration between these receptors may permit rapid onset and turning off of biological events.

Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation.

Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.

Involvement of ganglioside GT1b in glutamate release from neuroblastoma cells.

Since gangliosides play many important roles in neural systems, we investigated whether gangliosides are involved in glutamate release from neural cells. Differentiated neruro2a cells were treated with gangliosides, including GM3, GM1, GD1a, GD3, GD1b, or GT1b, for 30 min, and glutamate concentration in the culture media was measured using o-phthalaldehyde derivatization. Among the tested gangliosides, GT1b significantly increased the glutamate concentration when compared with untreated cells. Moreover, GT1b increased the glutamate concentration in the culture media of neuroblastoma × dorsal root ganglion neuron hybrid F11 cells. These results suggested that gangliosides are important in regulating extracellular glutamate concentration in the nervous system.

Gangliosides and chondroitin sulfate desensitize and internalize B2 bradykinin receptors.

Prolonged or repeated agonist activation of G-protein-coupled receptors (GPCRs) initiates their desensitization and internalization, rendering them unresponsive to agonist activation. We analyzed how gangliosides and chondroitin sulfate affect B2 bradykinin (BK) receptors (B2Rs). Gangliosides and chondroitin sulfate did not stimulate intracellular Ca(2+) release from B2R-expressing CHO-K1 cells, but repeated exposure desensitized B2Rs to BK stimulation. Microscopic observation of DsRed-fused B2Rs revealed that several gangliosides and chondroitin sulfate C (CSC) effectively internalized B2Rs. Ganglioside-CSC treatment of B2R mutant-expressing cells failed to desensitize and internalize the mutant receptors. As this mutant lacks the first extracellular domain and cannot activate GPCR kinase (GRK), gangliosides and CSC likely initiate B2R desensitization and endocytosis through GRK-mediated B2R phosphorylation.

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons.

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.

Comparative characterization of GPRC5B and GPRC5CLacZ knockin mice; behavioral abnormalities in GPRC5B-deficient mice.

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor.

Intraplantar injection of gangliosides produces nociceptive behavior and hyperalgesia via a glutamate signaling mechanism.

Gangliosides are abundant in neural tissue and play important roles in cell-cell adhesion, signal transduction, and cell differentiation. Gangliosides are divided into 4 groups: asialo-, a-, b-, and c-series gangliosides, based on their biosynthetic pathway. St8sia1 knockout mice, which lack b- and c-series gangliosides, exhibit altered nociceptive responses. The mechanism underlying this defect, however, remains unclear. To address this issue, we first investigated the possibility that gangliosides in peripheral nociceptor endings are involved in nociception. Intraplantar injection of the b-series ganglioside GT1b, but not a-series gangliosides such as GM1, produced nociceptive responses and enhanced low-concentration formalin-induced nociception. N-methyl-d-aspartic acid receptor and type I metabotropic glutamate receptor antagonists inhibited GT1b-induced hyperalgesia, suggesting the involvement of glutamate receptors. Furthermore, microdialysis analysis revealed elevated glutamate content in subdermal tissues due to intraplantar injection of GT1b. Co-injection of glutamate dehydrogenase with GT1b attenuated GT1b-induced hyperalgesia. These findings suggested that GT1b induced extracellular glutamate to accumulate in subdermal tissues, thereafter activating glutamate receptors, which in turn resulted in hyperalgesia and nociception. On the other hand, intraplantar injection of sialidase, which cleaves sialyl residues from glycoconjugates such as gangliosides, attenuated the late phase of 2% formalin-induced nociception. Thus, the antinociceptive effects of sialidase and the nociceptive effects of GT1b indicated that endogenous gangliosides are involved in nociceptive responses. These results suggest that gangliosides play important roles in nociceptive responses originating in peripheral nociceptor endings. Ganglioside GT1b induced extracellular glutamate to accumulate in subdermal tissues, thereafter activating glutamate receptors, which in turn resulted in hyperalgesia and nociception.

Glycosphingolipid synthesis in cerebellar Purkinje neurons: roles in myelin formation and axonal homeostasis.

Glycosphingolipids (GSLs) occur in all mammalian plasma membranes. They are most abundant in neuronal cells and have essential roles in brain development. Glucosylceramide (GlcCer) synthase, which is encoded by the Ugcg gene, is the key enzyme driving the synthesis of most neuronal GSLs. Experiments using conditional Nestin-Cre Ugcg knockout mice have shown that GSL synthesis in vivo is essential, especially for brain maturation. However, the roles of GSL synthesis in mature neurons remain elusive, since Nestin-Cre is expressed in neural precursors as well as in postmitotic neurons. To address this problem, we generated Purkinje cell-specific Ugcg knockout mice using mice that express Cre under the control of the L7 promoter. In these mice, Purkinje cells survived for at least 10-18 weeks after Ugcg deletion. We observed apparent axonal degeneration characterized by the accumulation of axonal transport cargos and aberrant membrane structures. Dendrites, however, were not affected. In addition, loss of GSLs disrupted myelin sheaths, which were characterized by detached paranodal loops. Notably, we observed doubly myelinated axons enveloped by an additional concentric myelin sheath around the original sheath. Our data show that axonal GlcCer-based GSLs are essential for axonal homeostasis and correct myelin sheath formation.

[Regulation of neuronal cell function by glyco-signals].

Gangliosides and proteoglycans with various sugar chains exist abundantly in the brain. They participate in intercellular recognition by revealing the sugar chains on the cell surface, and some of them show neurite-extension activity. Several recognition features that are mediated by the sugar chains are known such as saccharide-saccharide interaction and cell-surface sugar-chain receptor-mediated recognition. Experiments on animals lacking the sugar-chain synthetic system with the technique of gene targeting suggest that phylogenetically "old" sugar chains such as chondroitin sulfate appear necessary for early development of the organism while relatively "new" sugar chains such as gangliosides, which appear with further development of the brain, are necessary for differentiation maturity processes. On the other hand, research using primary cultured neurons showed similar effects of the gangliosides and chondroitin sulfate on cell differentiation. It is possible that these sugar chains share the glyco-receptor-mediated signal transduction system.

Ganglioside/protein kinase signals triggering cytoskeletal actin reorganization.

Exposure of neuronal cells to nanomolar concentrations of oligosaccharide portions of ganglioside GM2 and GT1b stimulates cAMP-dependent protein kinase (PKA) Ca2+/calmodulin-dependent protein kinase II (CaMKII), respectively, in a few seconds suggesting the presence of glyco-receptor-like molecules on the surface of the cells. Both GM2/PKA (GalNAc/PKA) and GT1b/CaMKII signaling cascades induced cytoskeletal actin reorganization through Cdc42 activation leading to filopodia formation within 2 min. Long-term effects of these glyco-signals were facilitation of dendritic differentiation of primary cultured hippocampal neurons and cerebellar Purkinje neurons indicating physiological roles of the signals in neuronal differentiation and maturation.

Imaging of calcineurin activated by long-term depression-inducing synaptic inputs in living neurons of rat visual cortex.

Long-term depression (LTD) of synaptic transmission is induced by low-frequency stimulation (LFS) of afferents lasting for a long time, typically for 10-15 min, in neocortical and hippocampal slices. It is suggested that calcineurin, Ca2+/calmodulin-dependent protein phosphatase, plays a role in the induction of LTD, based on the results that pharmacological or genetic manipulation of calcineurin activity interfered in its induction. However, questions as to why it takes so long to induce LTD and in which compartment of neurons calcineurin is activated remain unanswered. With a fluorescent indicator for calcineurin activity, we visualized the spatiotemporal pattern of its activation in living neurons in layer II/III of visual cortical slices of rats during the LFS of layer IV that induced LTD of synaptic responses. During LFS, the fluorescence intensity gradually increased with a latency of a few minutes in dendrites and soma of neurons, and remained increased during the whole observation period (10-25 min) after LFS. The onset latency of the increase in the soma was slower than that in the distal dendritic region. The LFS-induced rise in fluorescence was not observed in neurons which were loaded with inhibitors of calcineurin, indicating that the intensity of fluorescence reflects calcineurin activity. Control stimulation at 0.05 Hz and theta-burst stimulation did not significantly change the intensity of fluorescence. Only LFS-type inputs effectively activate calcineurin in postsynaptic neurons in an augmenting manner, and such a time-consuming activation of calcineurin may be a reason why long-lasting LFS is necessary for the induction of LTD.