Endogenous δ-Opioid and ORL1 Receptors Couple to Phosphorylation and Activation of p38 MAPK in NG108-15 Cells and This Is Regulated by Protein Kinase A and Protein Kinase C

The p38 mitogen-activated protein kinase (MAPK) cascade transduces multiple extracellular signals from cell surface to nucleus and is employed in cellular responses to cellular stresses and apoptotic regulation. The involvement of the p38 MAPK cascade in opioid- and opioid receptor-like receptor-1 (ORL1) receptor-mediated signal transduction was examined in NG108-15 neuroblastoma x glioma hybrid cells. Stimulation of endogenous delta-opioid receptor (DOR) or ORL1 resulted in activation of p38 MAPK. It also induced the activation of extracellular signal-regulated kinases (ERKs), another member of the MAPK family, with slower kinetics. Activation of p38 MAPK was abolished by selective antagonists of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant effect on opioid-induced ERK activation, indicating that p38 MAPK activity was not required for ERK activation, though its stimulation preceded ERK activation. Inhibition of protein kinase A (PKA) strongly diminished p38 activation mediated by DOR or ORL1 but had no significant effect on ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation of p38 while inhibiting activation of ERKs. Taken together, our results provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor-mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.     

Phosphorylation of Ser166 in RGS5 by protein kinase C causes loss of RGS function

RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Galpha subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca(2+) responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Galpha subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca(2+) responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.     

Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: roles of its N-terminal domain

Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca²⁺ responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1-71 of RGS2 with the corresponding residues (1-51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1-51 of RGS5 with the residues 1-71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16-55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56-71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1-33 or 34-52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1-55 or 56-71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.     

Increased atypical PKC activity in endurance-trained human skeletal muscle

Exercise training may modulate protein content and enzyme activities in skeletal muscle. However, it is not known whether atypical protein kinase C (aPKC) is affected by training. Thus, we investigated aPKC, extracellular-regulated protein kinase 1/2 (ERK 1/2), and P38 mitogen-activated protein kinase (P38 MAPK) activities and expression in skeletal muscle from untrained and endurance-trained subjects at rest and after 20min of cycle exercise (80% of VO(2peak)). Activities of aPKC (P<0.05) and ERK 1/2 (P=0.06), but not phosphorylation of P38 MAPK, were higher in trained than in sedentary subjects at rest. Exercise increased the activities of ERK 1/2 (P<0.01) and aPKC (P<0.05) and the phosphorylation (Thr180/Tyr182) of P38 MAPK (P<0.01) similarly in muscle from trained and sedentary subjects. Protein expression of the kinases was similar in trained and sedentary muscle. The increased aPKC activity in exercise-trained subjects could be important in explaining the enhanced insulin action in these individuals.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

The role of the Ca2+-sensitive tyrosine kinase Pyk2 and Src in thrombin signalling in rat astrocytes

We have recently demonstrated that multiple signalling pathways are involved in thrombin-induced proliferation in rat astrocytes. Thrombin acts by protease-activated receptor-1 (PAR-1) via mitogen-activated protein kinase activity. Signalling includes both Gi/(betagamma subunits)-phosphatidylinositol 3-kinase and a Gq-phospholipase C/Ca2+/protein kinase C (PKC) pathway. In the present study, we investigated the possible protein tyrosine kinases which might be involved in thrombin signalling cascades. We found that, in astrocytes, thrombin can evoke phosphorylation of proline-rich tyrosine kinase (Pyk2) via PAR-1. This process is dependent on the increase in intracellular Ca2+ and PKC activity. Moreover, in response to thrombin stimulation Pyk2 formed a complex with Src tyrosine kinase and adapter protein growth factor receptor-bound protein 2 (Grb2), which could be coprecipitated. Furthermore, both thrombin-induced Pyk2 phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation can be attenuated by Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. From these data we conclude that PAR-1 uses Ca2+- and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation, which predominantly recruits Grb2 in rat astrocytes.     

Resistance to age-related, normal body weight gain in RGS2 deficient mice

RGS2 (regulator of G protein signaling 2) is known to limit signals mediated via Gq- and Gs-coupled GPCRs (G protein coupled receptors), and it has been implicated in the differentiation of several cells types. The physiology of RGS2 knockout mice (rgs2^−/−) has been studied in some detail, however, a metabolic phenotype has not previously been reported. We observed that old (21-24 month) rgs2^−/− mice weigh much less than wild-type C57BL/6 controls, and exhibit greatly reduced fat deposits, decreased serum lipids, and low leptin levels. Lower weight was evident as early as four weeks and continued throughout life. Younger adult male rgs2^−/− mice (4-8 months) were found to show similar strain-related differences as the aged animals, as well improved glucose clearance and insulin sensitivity, and enhanced beta-adrenergic and glucagon signaling in isolated hepatocytes. In addition, rgs2^−/− pre-adipocytes had reduced levels of differentiation markers (Peroxisome proliferator-activated receptor γ (PPARγ); lipoprotein lipase (Lpl); CCAAT/enhancer binding protein α (CEBPα)) and also rgs2^−/− white adipocytes were small relative to controls, suggesting altered adipogenesis. In wild-type animals, RGS2 mRNA was decreased in brown adipose tissue after cold exposure (7 h at 4 °C) but increased in white adipose tissue in response to a high fat diet, also suggesting a role in lipid storage. No differences between strains were detected with respect to food intake, energy expenditure, GPCR-stimulated lipolysis, or adaptive thermogenesis. In conclusion this study points to RGS2 as being an important regulatory factor in controlling body weight and adipose function.     

Potent inhibition of angiotensin AT1 receptor signaling by RGS8: importance of the C-terminal third exon part of its RGS domain

R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in regulation of many GPCR-mediated responses. Multiple RGS proteins are usually expressed in a cell, and it is difficult to point out which RGS protein species are functionally important in the cell. To evaluate intrinsic potency of these RGS proteins, we compared inhibitory effects of RGS1, RGS2, RGS3, RGS4, RGS5, RGS8 and RGS16 on AT₁ receptor signaling. Intracellular Ca²⁺ responses to angiotensin II were markedly attenuated by transiently expressed RGS2, RGS3 and RGS8, compared to weak inhibition by RGS1, RGS4, RGS5 and RGS16. N-terminally deleted RGS2 (RGS2 domain) lost this potent inhibitory effect, whereas RGS domains of RGS3 and RGS8 showed strong inhibition similar to those of the full-length proteins. To investigate key determinants that specify the differences in potency, we constructed chimeric domains by replacing one or two of three exon parts of RGS8 domain with the corresponding part of RGS5. The chimeric RGS8 domains containing the first or the second exon part of RGS5 showed strong inhibitory effects similar to that of wild type RGS8, but the chimeric domain with the third exon part of RGS5 lost its activity. On the contrary, replacement of the third exon part of RGS5 with the corresponding residues of RGS8 increased the inhibitory effect. The role of the third exon part of RGS8 domain was further confirmed with the chimeric RGS8/RGS4 domains. These results indicate the potent inhibitory activity of RGS8 among R4/B subfamily proteins and importance of the third exon.     

Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C

A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes.     

Ligands of peroxisome proliferator-activated receptor-alpha promote glutamate transporter-1 endocytosis in astrocytes

Astrocytes, a stellate-shape glial population in the central nervous system (CNS), maintain glutamate homeostasis in adult CNS by undergoing glutamate uptake at the synapse through their glutamate transporter-1 (GLT-1). Peroxisome proliferator-activated receptor-α (PPARα) can be activated by endogenous saturated fatty acids to regulate astrocytic lipid metabolism and functions. However, it is unclear if PPARα can exert the regulatory action on GLT-1 expression in astrocytes. This study showed that treatment with palmitic acid (PA) and the other two PPARα agonists (GW 7647 and WY 14,643) caused no change in the morphology of astrocytes, whereas membranous GLT-1 protein levels in astrocytes were significantly decreased by PA and PPARα agonists. Through lentivirus-mediated overexpression of GLT-1 tagged with red fluorescent protein (GLT-1-RFP), we also observed that GLT-1-RFP puncta in the processes of astrocytes were inhibited by the PPARα agonists. This reduction was prevented by the addition of the PPARα antagonist, GW6471. GLT-1-RFP was co-localized to the early endosome marker–EEA1 in astrocytes treated with the PPARα agonists. Moreover, PPARα-induced inhibition in membranous GLT-1 expression was abolished by the addition of dynamin inhibitor (dynasore). Furthermore, the co-treatment of astrocytes with PPARα agonists and dynasore, or with PPARα agonists and protein kinase C (PKC) inhibitor bis-indolylmaleimide 1 (BIS1), prevented the endocytosis of GLT-1-RFP. Based on the results, we conclude that the PPARα agonists increased GLT-1 endocytosis in astrocytes possibly through the PKC signaling pathway. In addition, our findings provide important information of PPARα involvement in the downregulation of astrocytic glutamate uptake via the promoted GLT-1 endocytosis.     

Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.     

p8 Upregulation sensitizes astrocytes to oxidative stress

Here we studied the mechanism of cell sensitization to oxidative stress by analyzing the gene expression profile of serum-deprived astrocytes. Exposure to serum-free medium (i) sensitized astrocytes to oxidative stress, (ii) reduced the expression of several genes involved in protection against oxidative stress, including heme oxygenase 1, and (iii) changed the expression of several genes involved in the control of cell survival, including the stress-regulated protein p8. Our results support that serum deprivation sensitizes astrocytes to oxidative stress via a p38 mitogen-activated protein kinase-dependent p8 upregulation that leads in turn to decreased heme oxygenase 1 expression.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.     

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice,and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross‐talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G‐protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G‐protein activation, plays an important role in embryo implantation by interfering in the cross‐talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β‐estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β‐estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β‐estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.     

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs). Obesity is a metabolic disorder caused by an imbalance between energy uptake and expenditure, resulting in excess stored body fat. Recent studies have shown that GSL levels are increased in obese rodents and that pharmacologically reducing GSL levels by inhibiting GlcCer synthesis improves adipocyte function. However, the molecular mechanism underlying these processes is still not clearly understood. Using Drosophila as a model animal, we report that GlcT-1 expression in the fat body, which is equivalent to mammalian adipose tissue, regulates energy metabolism. Overexpression of GlcT-1 increases stored nutrition (triacylglycerol and carbohydrate) levels. Conversely, reduced expression of GlcT-1 in the fat body causes a reduction of fat storage. This regulation occurs, at least in part, through the activation of p38-ATF2 signaling. Furthermore, we found that GlcCer is the sole GSL of the fat body, indicating that regulation of GlcCer synthesis by GlcT-1 in the fat body is responsible for regulating energy homeostasis. Both GlcT-1 and p38-ATF2 signaling are evolutionarily conserved, leading us to propose an evolutionary perspective in which GlcT-1 appears to be one of the key factors that control fat metabolism.     

Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin-induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up-regulated secretion of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) in primary rat astrocytes in a concentration-dependent manner. However, we found no increase of interleukin (IL)-6, IL-1beta and tumour necrosis factor-alpha secretion. Protease-activated receptor 1 (PAR-1)-induced GRO/CINC-1 release was mainly mediated by c-Jun N-terminal kinase (JNK) activation. Extracellular signal-regulated kinase 1/2 might be partially involved, but not p38 mitogen-activated protein kinase. Further studies demonstrated that PAR-1 activation, as well as application of recombinant GRO/CINC-1, protected astrocytes from C(2)-ceramide-induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB-332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR-1. These results demonstrate for the first time that PAR-1 plays an important role in anti-apoptosis in the brain by regulating the release of chemokine GRO/CINC-1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.     

Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [³²P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.     

Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha

Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.     

Delayed but sustained induction of mitogen-activated protein kinase activity is associated with beta-adrenergic receptor-mediated morphological differentiation of astrocytes

Astroglial beta-adrenergic receptors (beta-ARs) are functionally linked to regulate cellular morphology. In primary cultures, the beta-AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process-bearing, mature stellate cells by 48 h, an effect that can be blocked by the beta-AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP-induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal-regulated kinase; p-ERK) level: an initial decline in p-ERK level followed by a sustained induction at 12-24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p-ERK. A long-lasting activation of p-ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.