Thromboxane A2 Promotes Interleukin-6 Biosynthesis Mediated by an Activation of Cyclic AMP-Response Element-Binding Protein in 1321N1 Human
Department of Cellular Signaling, 21st COE program "CRESCENDO", Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (Y.O., N.N.)
Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (H.K.)
Abstract
1321N1 human astrocytoma cells express thromboxane A2 (TXA2) receptors (TP). However, physiological consequences of TXA2 signaling in glial cells remain unclear. Herein, we show that TXA2 promotes interleukin-6 (IL-6) biosynthesis in glial cells. A TP agonist, 9,11-dideoxy-9,11-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619 [GenBank] ), enhanced IL-6 production in both 1321N1 cells and cultured mouse astrocytes. It has been shown that IL-6 gene expression is regulated by various transcription factors. Among them, we found a significant increase in cyclic AMP-response element-binding protein (CREB) activity with its phosphorylation at Ser133 by U46619 [GenBank] in 1321N1 cells. Although U46619 [GenBank] increased IL-6 promoter activity, a mutation at cyclic AMP-response element (CRE) on the promoter clearly suppressed the effect, suggesting that CRE is involved in U46619 [GenBank] -induced IL-6 expression. Furthermore, both CREB and IL-6 promoter activities were suppressed by SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], a p38 mitogen-activated protein kinase (MAPK) inhibitor, and H89 [N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline], a protein kinase A (PKA) inhibitor, indicating involvements of p38 MAPK and PKA in CREB activation and IL-6 expression. To determine which G-proteins are implicated in the U46619 [GenBank] -induced IL-6 synthesis, the interfering mutants of Gq, G12, or G13 by were overexpressed in 1321N1 cells adenoviral approach. It is noteworthy that the Gq or G13 mutant blocked the IL-6 production by U46619 [GenBank] . The constitutively active mutant of Gq, G12, or G13 enhanced IL-6 production, indicating that Gq and G13 were involved in U46619 [GenBank] -induced IL-6 production. In conclusion, TXA2 enhances the IL-6 biosynthesis via the PKA p38 MAPK/CREB pathway in 1321N1 cells. IL-6 induction depends on Gq and G13 as well. This is the first report showing TP-mediated IL-6 production in glial cells.
The central nervous system (CNS) consists mainly of neurons and glial cells. Neurons play a central role in signal transduction by releasing neurotransmitters, whereas glial cells support the neuronal functions. Glial cells in CNS are generally classified into astrocytes, oligodendrocytes, and microglia. Astrocytes form the blood-brain barrier along with endothelial cells, metabolize neurotransmitters, cause gliosis after brain injury, and regulate extrasynaptic ion and H+ concentrations (Somjen, 1988; Ransom et al., 2003). In addition to these roles, they produce neurotrophic factors to promote neuronal survival (prevention of apoptosis), differentiation, or regeneration. It has been shown that they secrete neurotrophic factors, such as neurotrophin family members (Althaus and Richter-Landsberg, 2000), interleukin-6 (IL-6) (Schwaninger et al., 1997; Van Wagoner and Benveniste, 1999), and glial cell line-derived neurotrophic factor (Lin et al., 1993). We have previously shown that 1321N1 human astrocytoma cells produce nerve growth factor and IL-6 (Obara et al., 2001). These neurotrophic factors have been suggested as potential therapeutic agents for the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.
Thromboxane A2 (TXA2), a metabolite of arachidonic acid produced by cyclooxygenase and TXA2 synthetase, is known to induce many cellular responses, including platelet aggregation and contraction in various smooth muscle types (Huang et al., 2004). Because TXA2 is associated with various diseases, such as thrombogenesis, hypertension, bronchial asthma, and nephrotic syndrome, the antagonist for TXA2 receptor and inhibitor of its synthesis have been suggested as therapeutic targets (Huang et al., 2004).
We found TXA2 receptors (TPs) in 1321N1 human astrocytoma cells and rabbit cultured astrocytes (Nakahata et al., 1989, 1992). 1321N1 human astrocytoma cells express both TP isoforms (TP- and TP-) (Honma et al., 1998), which are alternatively spliced variants from the same gene. They are seven-transmembrane receptors and have been shown to associate with heterotrimeric GTP-binding proteins, such as Gq/11, Gi, G12, G13 and Gh (Offermanns et al., 1994; Nakahata et al., 1995; Allan et al., 1996; Vezza et al., 1999; Gao et al., 2001). In response to TXA2 analog, an elevation of intracellular Ca2+ concentration was observed as a result of phosphatidylinositol-specific phospholipase C activation in 1321N1 cells. TP-mediated extracellular signal-regulated kinase (ERK) activation in differentiated 1321N1 cells depends on the activation of phosphatidylcholine-specific phospholipase C (Kobayashi et al., 2000). It has also been shown that TXA2 analog elevates cyclic AMP concentration in several cell types, such as Schwann cells (Muja et al., 2001), even though there is currently no evidence for the interaction between TPs and Gs. In that report, cyclic AMP-response element-binding protein (CREB) is activated, which is assumed to be by a cyclic AMP-dependent mechanism. Thus, the downstream pathway of TPs is complex and has not yet been clarified in detail.
In screening for compounds that enhance neurotrophic factor production in 1321N1 cells, we found that the TXA2 analog U46619 [GenBank] significantly promoted IL-6 secretion. Herein, we show the mechanism of IL-6 production by TXA2 in detail.
Materials and Methods
U46619 was obtained from Cayman Chemical (Ann Arbor, MI). Prostaglandin E2, Ro20-1724, isoproterenol, carbachol, dibutyryl cyclic AMP (dbcAMP), H89, wortmannin, and KN62 were purchased from Sigma-Aldrich (St. Louis, MO). IL-6 enzyme-linked immunosorbent assay (ELISA) kit was from Amersham Biosciences Inc. (Piscataway, NJ). RNA extraction kit was from Nippon Gene Co., Ltd. (Toyama, Japan), and reverse transcription-polymerase chain reaction (RT-PCR) kit was from Toyobo Engineering (Osaka, Japan). Phorbol 12-myristate 13-acetate (PMA), SB203580, and forskolin were from Wako Pure Chemicals (Tokyo, Japan). SQ29548 was from Bristol-Myers Squibb (Tokyo, Japan). KT5720 was from Calbiochem (La Jolla, CA). CRE-luciferase reporter plasmid was from BD Biosciences Clontech (Palo Alto, CA). FuGENE 6 transfection reagent was from Roche Diagnostics (Indianapolis, IN). PD98059, anti-phospho-ERK antibody, anti-phospho-p38 mitogen-activated protein kinase (MAPK) antibody, anti-p38 MAPK antibody, anti-phospho-CREB antibody, anti-CREB antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were from Cell Signaling (Beverly, MA). Anti-ERK2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-glial fibrillary acidic protein (GFAP) antibody was from Progen (Heidelberg, Germany). Fluorescein isothiocyanate-conjugated anti-mouse antibody was from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD). Cholera toxin was from List Biological Laboratories Inc. (Campbell, CA).
Cell Culture. 1321N1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (Cell Culture Laboratory, Cleveland, OH), 50 units/ml penicillin, and 50 e/ml streptomycin in a 5% CO2 incubator at 37°C. Mouse astrocytes were prepared from neonatal mouse brains and then cultivated in DMEM supplemented with 10% fetal calf serum, penicillin, and streptomycin (Muller et al., 1984).
IL-6 Sandwich ELISA. IL-6 ELISA was performed as the protocol of IL-6 ELISA kit. In brief, 1321N1 cells were seeded onto six-well plates (1 x 106 cells/well) and cultivated overnight in the absence of fetal calf serum. The cells were cultured for 24 h in the presence of U46619 [GenBank] , and then the conditioned media were collected after centrifugation to remove contaminating cells. These conditioned media were regarded as samples for ELISA and then added to 96-well plates precoated with IL-6 antibody. After incubation, IL-6 antibody conjugated with biotin was added; then, avidin-horseradish peroxidase was added.
Semiquantitative RT-PCR. Total RNA from 1321N1 cells was extracted by using a total RNA extraction kit, and semiquantitative RT-PCR was carried out by using a RT-PCR kit. IL-6 mRNA expression was examined as described previously (Obara et al., 2001). For analysis of human IL-6 mRNA, the sense primer (5'-AAA TTC GGT ACA TCC TCG AC-3' of human IL-6 cDNA) and the antisense primer (5'-CAG GAA CTG GAT CAG GAC TT-3'), which were complementary to conserved regions of cDNA from human IL-6, were used. For analysis of mouse IL-6 mRNA, the sense primer (5'-CAA GAG ACT TCC ATC CAG TTG C-3') and the antisense primer (5'-TTG CCG AGT AGA TCT CAA AGT GAC-3') were used. The human IL-6 cDNA of 295 base pairs was amplified from 26 to 30 cycles (95°C for 60 s, 56°C for 60 s, and 74°C for 60 s), and mouse IL-6 cDNA of 614 base pairs was amplified 28 cycles (94°C for 30 s, 57°C for 60 s, and 72°C for 60 s). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) transcripts of 700 base pairs were used as an internal control. The number of cycles that yielded a quantitative amount of product was determined in preliminary experiments. The densities of the bands corresponding to IL-6 and G3PDH were analyzed by densitometry (NIH Image software), and the data were expressed as a ratio to G3PDH.
Immunostaining. Mouse astrocytes were fixed with 4% paraformaldehyde and stained with anti-GFAP antibody (1:10 dilution) and fluorescein isothiocyanate-anti-mouse antibody (1:80 dilution).
Plasmids, Transfection, and Reporter Gene Assay. Plasmids containing IL-6 promoter (wild type or CRE mutated) linked to luciferase gene (Funakoshi et al., 1999; Tokunou et al., 2001) were kindly given by Dr. Toshihiro Ichiki (Kyushu University, Fukuoka, Japan). Human mitogen- and stress-activated protein kinase 1 (MSK1) wild-type and the kinase-dead mutant (MSK1 D195A/D565A) (Arthur et al., 2004) were from Dr. Soren Impey (Vollum Institute, Oregon Health Sciences University, Portland, OR). A plasmid encoding PKI was from Dr. Philip Stork (Vollum Institute, Oregon Health Sciences University). Plasmids encoding constitutively active mutants of G-proteins (GqQ209L, G12Q231L, and G13Q226L) were kindly provided by the laboratory of John Scott (Vollum Institute, Oregon Health Sciences University). For transfection, 1321N1 cells were seeded onto 24-well plates at 1 x 105 (cells/well). On the next day, plasmids (0.6-1.2 e/well) and FuGENE 6 (0.75-1.8 e/well) were mixed gently in 25 e of DMEM and added to culture plates. For luciferase assay, media were replaced by serum-free media 24 h after transfection. The cells were incubated with the indicated drugs at 37°C for 6 h on the following day. Luciferase assay was performed by using dual luciferase assay kit (Promega, Madison, WI) according to its instruction. Renilla reniformis luciferase (phRL-TK, Promega) activity was measured for normalizing the transfection efficiency. The luciferase activity was measured using a luminometer (GENE LIGHT 55; Microtech Nition, Funabashi, Japan).
cAMP Assay. Intracellular cAMP concentration was measured as follows. 1321N1 cells on a 24-well plate were labeled with 2 e藽i/ml [3H]adenine (23 Ci/mmol; Amersham Biosciences) in the growth medium for 3 h. The labeled cells were washed twice with DMEM-HEPES (20 mM, pH 7.4). The cells were incubated for 10 min with U46619 [GenBank] (or with SQ29548) in DMEM-HEPES containing phosphodiesterase inhibitor Ro20-1724 (100 e). After aspirating the incubation buffer, the reactions were stopped by adding 0.4 ml of 2.5% HClO4. Acid extracts were mixed with 1/10 volume of 4.2 M KOH to neutralize and deposit potassium perchlorate. [3H]cAMP in the clear supernatant was separated by Dowex 50W-X8 (Bio-Rad, Hercules, CA)/alumina (Merck Biosciences, Darmstadt, Germany) double columns and determined by liquid scintillation counting. An increase in [3H]cAMP was expressed as the -fold over the control (no drug).
Adenoviral Infection. Adenoviruses encoding carboxyl-terminal regions of Gq (Gq-ct, amino acids 305-359), G12 (G12-ct, amino acids 325-379), and G13 (G13-ct, amino acids 322-378) were created in our laboratory. The expressions of those genes are regulated by CMV promoter, and their products putatively block G-protein coupling with their corresponding receptors (Arai et al., 2003). Each adenoviral vector contains both G-protein-ct and GFP that is upstream of the G-protein-ct, and each entity is under the control of CMV promoter. 1321N1 cells were infected with the indicated virus at 75 m.o.i. for 1 day. The medium was then replaced with serum-free medium on the next day, and the cells were cultured overnight. At the time of drug incubation, more than 90% of the cells were GFP-positive.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting. Samples used for immunoblotting were prepared as follows. 1321N1 cells were seeded onto six-well plates at a density of 1 x 106 (cells/well). The cells were cultured overnight in serum-free DMEM, and then drugs were added for various periods of time. The incubation medium was aspirated after the reaction, and the cells were dissolved in Laemmli sample buffer (final concentration: 75 mM Tris-HCl, 2% SDS, 15% glycerol, 3% 2-mercaptoethanol, pH 6.8) and boiled at 95°C for 5 min.
Electrophoresis was performed on 11% acrylamide gels. Proteins were transferred electrically from the gel onto a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA) by the semidry blotting method. The blots were blocked for 40 min with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween 20 and incubated with primary antibodies [anti-phospho-ERK (Thr202/Tyr204) antibody, anti-ERK2 antibody, anti-phospho-p38 MAPK (Thr180/Tyr182) antibody, anti-p38 MAPK antibody, anti-phospho-CREB (Ser133) antibody, or anti-CREB antibody (1:1000 dilution)] overnight at 4°C. The blots were washed several times and then incubated at 25°C for 2 h with a 1:3000 dilution of secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG antibody). Blots were developed using an enhanced chemiluminescence assay kit (Amersham Biosciences) and visualized by chemiluminescence on Hyperfilm ECL. The densities of the bands corresponding to CREB, ERK, and p38 MAPK were analyzed by densitometry (NIH Image), and the data were expressed as a ratio to their total proteins.
Statistical Methods. Data were expressed as the mean values ± S.E.M, and the significant differences were analyzed by Student's t test or Tukey's method for multiple comparisons.
Results
U46619 Enhances IL-6 Production in 1321N1 Cells. We investigated the biosynthesis of IL-6. 1321N1 cells were stimulated with the TP agonist, U46619 [GenBank] , and then we examined IL-6 secretion by sandwich-ELISA (Fig. 1A). U46619 [GenBank] significantly increased IL-6 secretion from 1321N1 cells in a concentration-dependent manner that was inhibited by TP antagonist SQ29548. Adenylyl cyclase activator, forskolin, and cAMP analog dbcAMP also promoted IL-6 secretion by 1.6- and 2.9-fold, respectively (data not shown). U46619 [GenBank] (10 e) also enhanced IL-6 gene expression (5.8-fold), and this effect was abolished by 1 e SQ29548 again (Fig. 1B). Carbachol (10 e) did not affect IL-6 gene expression. Forskolin and a PKC activator, PMA, enhanced IL-6 expression 4.5- and 7.0-fold in the same condition, respectively (Fig. 1B). U46619 [GenBank] and SQ29548 have low affinity to other prostanoid receptors, such as the prostaglandin E2 receptor subtype EP4 (Kiriyama et al., 1997), and it is also known that prostaglandin E2 induces IL-6 release in U373 MG human astrocytoma cells (Fiebich et al., 2001). To avoid the possibility that the effect of U46619 [GenBank] on IL-6 induction is mediated by EPs, we tested prostaglandin E2. U46619 [GenBank] -induced IL-6 expression was completely blocked by SQ29548 (3% U46619 [GenBank] alone), whereas most of the effect of prostaglandin E2 was not blocked by SQ29548 (82% prostaglandin E2 alone) (Fig. 1C). Cholera toxin as a positive control enhanced IL-6 expression 13.2-fold (Fig. 1C). This result indicates that the effect of U46619 [GenBank] is mediated by TPs. To examine the IL-6 promoter activity, the plasmid encoding wild-type IL-6 promoter (-1039 +1) linked to luciferase gene was transfected into 1321N1 cells and luciferase activity was measured (Fig. 1D). U46619 [GenBank] (10 e) as well as 10 e forskolin significantly enhanced IL-6 promoter activity. Furthermore, GFAP-positive cultured mouse astrocytes (Fig. 2A) were stimulated with U46619 [GenBank] to determine whether IL-6 enhancement is 1321N1 cell-specific or not. As expected, 10 e U46619 [GenBank] similarly promoted IL-6 gene expression (2.7-fold) in astrocytes as dbcAMP (2.1-fold), and the effect of U46619 [GenBank] was mostly reversed by SQ29548 (12% U46619 [GenBank] alone) (Fig. 2B). Taken together, these results suggest that U46619 [GenBank] enhances the secretion of IL-6 from 1321N1 cells accompanied by an increase in IL-6 gene expression.
CRE Regulates the IL-6 Gene Expression by U46619 [GenBank] in 1321N1 Cells. IL-6 gene expression is regulated by multiple transcription factors, including CREB, activator protein-1 (AP-1), nuclear factor-B (NF-B), CCAAT enhancer-binding protein-, and glucocorticoid receptor in complex ways (Obara and Nakahata, 2002). Because CREB activation by U46619 [GenBank] has been reported (Muja et al., 2001), we determined whether CREB is involved in the U46619 [GenBank] -induced IL-6 gene expression. CRE activity was measured by reporter gene assay. pCRE-luciferase plasmid was transfected into 1321N1 cells, and then newly induced luciferase activity was regarded as the CREB-transcriptional activity. U46619 [GenBank] (10 e) and 10 e forskolin significantly increased CRE activity (Fig. 3A). To subsequently confirm the specificity of CRE in enhanced IL-6 gene expression, we used the IL-6 promoter-luciferase plasmid harboring a mutant CRE in the IL-6 promoter region (from ATGACGTCA to ATCGATCCA). The effect of U46619 [GenBank] on IL-6 promoter activity was completely abolished by mutation of CRE as in the case of forskolin (Fig. 3B). These results indicate that CRE activation is essential for U46619 [GenBank] -induced IL-6 gene expression in 1321N1 cells.
PKA and p38 MAPK/MSK Pathways Are Involved in CREB Activation and IL-6 Gene Expression by U46619 [GenBank] in 1321N1 Cells. It has been shown that CREB activity is regulated by phosphorylation at Ser133, where various kinases have been proposed to phosphorylate, including protein kinase A (PKA), Akt (or protein kinase B), calmodulin-dependent kinases (CaMKs), ribosomal S6 kinases (RSKs), and MSKs (Lonze and Ginty, 2002). Akt is a downstream effector of phosphoinositide 3-kinase, and RSKs and MSKs can be activated by ERK1/2, ERK5, or p38 MAPK. To investigate the U46619 [GenBank] signaling to CREB in detail, pharmacological tools were used to identify the CREB kinase. 1321N1 cells were pretreated with 10 e H89 (PKA inhibitor), 30 e PD98059 (MEK inhibitor), 30 e SB203580 (p38 MAPK inhibitor), 1 e wortmannin (phosphoinositide 3-kinase inhibitor), or 10 e KN62 (CaMK inhibitor) for 15 min, and then they were stimulated with 10 e U46619 [GenBank] for 6 h (Fig. 4A). The U46619 [GenBank] -induced CRE activation was significantly blocked by H89 and SB203580. Carbachol (10 or 100 e) did not increase the CRE activity. Because H89 has been reported to inhibit MSK as well as PKA (Davies et al., 2000), another PKA inhibitor, KT5720, was tested. KT5720 (2 e) also inhibited the CRE activity significantly (Fig. 4B). Increase in CRE activity by U46619 [GenBank] was also blocked by 2 e SQ29548 and a lower concentration of 3 e SB203580 (Fig. 4C). Furthermore, MSK 1 wild-type and the kinase-dead mutant (D195A and D565A) were overexpressed in 1321N1 cells, and CRE activity was measured (Fig. 4D). The MSK1 kinase-dead mutant partially but significantly attenuated the ability of U46619 [GenBank] to increase CRE activity. CREB family members, including CREB, activating transcription factor 1, and CRE modulator, binds to CRE. To investigate whether CREB is involved in this process, CREB phosphorylation at Ser133 was monitored. This phosphorylation site can be regarded as an index of CREB activation. The CREB phosphorylation by U46619 [GenBank] via TPs was observed (Fig. 5A), confirming that CREB is activated by U46619 [GenBank] . This phosphorylation was completely blocked by 10 e H89, 30 e SB203580, and 2 e KT5720 again (Fig. 5B). These results suggest that PKA and p38 MAPK/MSK pathways mediate CREB activation by U46619 [GenBank] . The effects of these kinase inhibitors on IL-6 gene expression were investigated next. H89 (10 e) and 30 e SB203580 decreased IL-6 promoter activity accelerated by 10 e U46619 [GenBank] (Fig. 6A), similar to the inhibition of CRE activity shown in Fig. 4A. The effect of U46619 [GenBank] was also significantly inhibited by 2 e SQ29548 and 3 e SB203580, overexpressing a physiological PKA pseudosubstrate peptide, PKI (Fig. 6B). Furthermore, when 1321N1 cells were pretreated with 30 e SB203580, IL-6 gene expression induced by 10 e U46619 [GenBank] was largely suppressed (21% U46619 [GenBank] alone), as determined by RT-PCR (Fig. 6C). Because the involvement of p38 MAPK in TP-mediated IL-6 expression was suggested, we examined whether p38 MAPK was activated by U46619 [GenBank] . When 1321N1 cells were incubated with 10 e U46619 [GenBank] for 5 to 20 min, transient phosphorylation of ERK1/2 was observed (2.2-fold at 5 min, 1.9-fold at 10 min, and 0.97-fold at 20 min) (Fig. 7). In addition to ERKs, p38 MAPK phosphorylation was induced by 10 e U46619 [GenBank] for 5 to 20 min (1.8-fold at 5 min, 2.1-fold at 10 min, and 2.8-fold at 20 min). CREB phosphorylation at Ser133 in the same lysates was also monitored. As expected, U46619 [GenBank] induced CREB phosphorylation correlated with p38 MAPK phosphorylation (5.6-fold at 5 min, 9.1-fold at 10 min, and 2.9-fold at 20 min) (Fig. 7). Gs coupling to TPs has not currently been reported, although an intracellular cAMP concentration by TP agonist is elevated in many cell types. We measured cAMP level after U46619 [GenBank] treatment (Fig. 8). The -adrenergic receptor agonist isoproterenol (10 e) strongly elevated the cAMP level. Compared with isoproterenol, the effect of U46619 [GenBank] on cAMP accumulation was much weaker, but the significant cAMP increase via TPs was observed. Taken together, these results suggest that PKA and p38 MAPK/MSK pathways are involved in CREB activation and IL-6 gene expression induced by U46619 [GenBank] in 1321N1 cells.
Gq and G13 Mediate TXA2 Signaling to IL-6 Expression. TPs couple with various kinds of G-proteins, such as Gq, G12, and G13 (Huang et al., 2004). We have shown that TPs associate with at least Gq and G12 in 1321N1 cells (Honma et al., 1998). To determine which G-proteins are involved in the signaling pathway leading to IL-6 production, we took an adenoviral approach to interfere with endogenous G-protein functions. The products from adenoviruses that encode the C-terminal regions of Gq, G12, or G13 inhibited the coupling among these G-proteins and their associated receptors (Arai et al., 2003). GFP gene was introduced to monitor the infection efficiency of those G-protein-interfering mutants. 1321N1 cells infected with those viruses were stimulated with 10 e U46619 [GenBank] for 4 h, and then RT-PCR was carried out. IL-6 gene expression was promoted with U46619 [GenBank] treatment in 1321N1 cells infected with control virus, and the enhancement was blocked by overexpressing C-terminal regions of Gq or G13 [45 and 53% U46619 [GenBank] (GFP), respectively] but not by G12 (Fig. 9A). Furthermore, to determine whether these G-proteins can affect IL-6 production, the cells were transfected with constitutively active mutants (GqQ209L, G12Q231L, and G13Q226L). GqQ209L and G13Q226L promoted IL-6 gene expression 15- and 7.9-fold, respectively, determined by RT-PCR, whereas G12Q231L marginally promoted it (2.6-fold) (Fig. 9B). In addition, these active mutants could significantly enhance both CRE and IL-6 promoter activities (Fig. 9C). Taken together, the results suggest that Gq and G13 are necessary and sufficient for U46619 [GenBank] -induced IL-6 gene expression.
Discussion
In the present study, we showed that U46619 [GenBank] promoted IL-6 biosynthesis in 1321N1 human astrocytoma cells and GFAP-positive mouse astrocytes. In addition, U46619 [GenBank] increased CREB activity that was accompanied by its phosphorylation at Ser133 by PKA and p38 MAPK/MSK, and this CREB activation is essential for IL-6 induction in 1321N1 cells (Fig. 10). This is the first report showing TP-mediated IL-6 production in glial cells.
The amount of arachidonic acid metabolites are increased dramatically after brain injury, viral infection, or inflammation. These metabolites, including TXA2, are assumed to play important roles in the CNS under such pathologic conditions. In fact, astrocytes and microglia have the ability to release TXA2 after activation by bacterial lipopolysaccharide and ATP (Pearce et al., 1989; Bruner and Murphy, 1993; Minghetti and Levi, 1995). Thus, the elevation of adenine nucleotide concentration or microglial activation after CNS injury or infection may cause the secretion of TXA2, leading to activation of TPs on glial cells via autocrine or juxacrine mechanisms. Therefore, a clarification of the physiological roles of TPs in astrocytes will help in identifying therapeutic targets in various CNS disorders.
IL-6 is a pleiotropic factor involved in the regulation of various inflammatory and endocrine functions in both central and periphery nervous systems. In the CNS, although various cell types can produce IL-6, astrocytes play a dominant role in IL-6 production (Gruol and Nelson, 1997). Under normal physiological conditions, IL-6 expression levels in CNS remain low, whereas IL-6 is up-regulated upon stress, diseases, inflammation, or injury. To date, it has been shown that numerous physiological receptor agonists or pharmacological reagents influence IL-6 production (Sehgal, 1992; Van Wagoner and Benveniste, 1999). Cytokines and proinflammatory factors, including lipopolysaccharide, IL-1, tumor necrosis factor-, and prostaglandin E2 are known as IL-6 inducers and play important roles in CNS diseases. In addition, various kinds of neurotransmitters and neuropeptides, including catecholamines, adenosine, serotonin, histamine, substance P, calcitonin, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating polypeptide, also influence IL-6 production. Abnormal physiological condition often changes the induction of IL-6, such as hypoxia/reoxygenation, generation of reactive oxygen species, viral infections, and irradiation. It is not surprising that compounds that increase cyclic AMP concentration, such as cholera toxin, cyclic AMP analogs, and forskolin, also lead to an induction of IL-6 in astrocytes (Norris et al., 1994). Furthermore, phorbol ester and Ca2+ ionophore, A23187 [GenBank] , induced IL-6 production and both of these reagents caused synergistic production, suggesting that PKC and Ca2+ signaling are responsible for IL-6 production in rat astrocytes (Norris et al., 1994). Fiebich et al. (1995) showed that ceramide induced IL-6 gene expression in U373-MG human astrocytoma cells. In terms of transcription factors, IL-6 promoter contains various consensus sequences of transcription factors, indicating that multiple transcription factors influence its gene expression. For example, CREB, AP-1, NF-B, CCAAT enhancer-binding protein- (or NF-IL6), and glucocorticoid receptor are known as regulators, and stimuli leading to the activation of these transcription factors are assumed to modulate IL-6 gene expression (Sehgal, 1992; Obara and Nakahata, 2002).
In the present study, it was demonstrated that U46619 [GenBank] increased the CRE activity by reporter gene assay, accompanied by the phosphorylation of CREB at Ser133, which is a key regulatory site for its transcription activity (Figs. 3A, 4, 5, and 7). Furthermore, this CREB activation is required for IL-6 gene expression (Fig. 3B). To date, it has been shown that the phosphorylation of this site occurs via multiple kinases, including PKA, CaMK, Akt, RSKs, and MSKs (Lonze and Ginty, 2002). RSKs and MSKs are known as downstream effectors of ERK1/2, ERK5, and p38 MAPK (Lonze and Ginty, 2002). In our study, to clarify the mechanism leading to CREB activation by U46619 [GenBank] , various pharmacological tools were tested. U46619 [GenBank] -induced CREB activation was significantly blocked by H89, KT5720, and SB203580 (Fig. 4). In addition, MSK1 kinase-dead mutant blocked CRE activity (Fig. 4D), suggesting the involvements of both PKA and p38 MAPK/MSK pathways. Although the mechanism remains unclear, cyclic AMP elevation and PKA activation have been shown in response to TP agonist (Muja et al., 2001; Miggin and Kinsella, 2002). On the other hand, p38 MAPK activation by TXA2 has been reported in platelets and isolated rat mesenteric resistance arteries (Bolla et al., 2002; Minuz et al., 2002). In the present study, we confirmed the elevation of cAMP and activation of p38 MAPK by U46619 [GenBank] in 1321N1 cells (Figs. 7 and 8). Taken together, this study strongly indicates that PKA and p38 MAPK participate in CREB activation by TXA2. However, as mentioned above, IL-6 gene expression is regulated by multiple transcription factors. Although it was demonstrated in this study that CREB activity was critical for IL-6 induction, we do not deny the possibility that other transcription factors, such as AP-1 and NF-B, activated by U46619 [GenBank] (Huang et al., 2004), may work cooperatively with CREB.
We demonstrated previously that TPs coupled with the subunits of at least two heterotrimeric GTP-binding proteins, Gq and G12, in 1321N1 cells (Honma et al., 1998). Phosphoinositide hydrolysis is promoted through stimulation with TP agonists, which causes increase in intracellular Ca2+ concentration and PKC activation (Nakahata et al., 1989). In dbcAMP-treated 1321N1 cells, phosphatidylcholine-specific phospholipase C, rather than phosphatidylinositol-specific phospholipase C, is predominantly activated by TP agonist, which causes subsequent ERK activation (Kobayashi et al., 2000). Because carbachol, an agonist of M3 receptor that couples to Gq, neither increased CRE activity nor induced IL-6 expression in this study, it was assumed that the other signaling pathway, such as G12 or G13, other than Gq-mediated PLC pathway might be essential for U46619 [GenBank] -induced IL-6 expression. In fact, although all of the constitutively active G-protein mutants (Gq, G12, and G13) could activate CRE and IL-6 promoter, interfering mutants Gq-ct and G13-ct, but not G12-ct, blocked IL-6 gene expression by TP stimulation (Fig. 6), indicating that TPs use Gq and G13 pathways for IL-6 production. Even though G12 and G13 belong to the same heterotrimeric G-protein family, the functional difference between those two G-proteins remain unclear. Nevertheless, in our study, G13 but not G12 was essential for TP signaling. Identification of direct downstream effector of G13 leading to CRE activation and IL-6 gene expression is necessary.
In vascular smooth muscle cells, thrombin induces IL-6 expression through the CRE, as in the case of TXA2 (Tokunou et al., 2001). In that report, involvements of ERK, p38 MAPK, and epidermal growth factor receptor (EGFR) are also implicated in thrombin-induced IL-6 production. EGFR transactivation that lies upstream of ERK and p38 MAPK is essential for this thrombin-induced IL-6 induction. EGFR transactivation by TXA2, which was probably mediated by either EGF-like ligand release by metalloproteinases or direct EGFR trans-phosphorylation by unidentified tyrosine kinases, has been also reported (Gao et al., 2001; Miggin and Kinsella, 2001). Thus, it is possible that this cross-talk between G-protein-coupled receptors and receptor tyrosine kinases is involved in p38 MAPK and CREB activation leading to IL-6 gene expression.
It has been shown that IL-6 acts as a neurotrophic factor as well as cytokine, and numerous studies have demonstrated both beneficial and unfavorable effects of IL-6 in CNS (Van Wagoner and Benveniste, 1999). A variety of in vitro and in vivo studies provide evidence for the involvement of IL-6 in neuronal survival, differentiation, and regeneration. IL-6 directly affects neurons, whereas it also affects glial cells; i.e., IL-6 promotes proliferation of astrocytes and production of nerve growth factor by autocrine mechanism. In contrast to the benefits above, IL-6 sometimes cause destructive effects. For example, in IL-6 transgenic mouse models, a high level of IL-6 led to neurodegeneration, blood-brain barrier breakdown, abnormal angiogenesis, increased level of complement proteins, and impaired learning. In those mice, it has been demonstrated that i.c.v. injection of lipopolysaccharide resulted in higher level productions of proinflammatory factors, such as tumor necrosis factor- and IL-1. IL-6 overproduction in the CNS may trigger unnecessary inflammatory responses and confusion of neuronal network. Thus, both beneficial and detrimental effects of IL-6 in the CNS have to be considered for the further study.
In conclusion, we showed that TXA2 analog induced a production of IL-6 mediated via PKA p38 MAPK/CREB pathway through Gq and G13 in 1321N1 cells. This observation indicates one of physiological roles of TPs in astrocytes and raises a possibility of TP as a therapeutic target in neuronal diseases.
Acknowledgements
We thank Dr. Toshihiro Ichiki (Kyushu University) for providing IL-6 promoter-luciferase plasmid, Dr. Philip Stork (Vollum Institute, Oregon Health Sciences University) for providing PKI DNA construct, Dr. John Scott (Vollum Institute) for providing G-protein DNA constructs, and Dr. Soren Impey (Vollum Institute) for MSK DNA constructs. We appreciate Dr. Quentin Low (Vollum Institute) for critical reading of the manuscript and scientific discussion. We also thank Manami Saika, Shigeyoshi Honma, and Hiroshi Kobayashi (Tohoku University) for assistance in the experiments.
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Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (H.K.)
Abstract
1321N1 human astrocytoma cells express thromboxane A2 (TXA2) receptors (TP). However, physiological consequences of TXA2 signaling in glial cells remain unclear. Herein, we show that TXA2 promotes interleukin-6 (IL-6) biosynthesis in glial cells. A TP agonist, 9,11-dideoxy-9,11-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619 [GenBank] ), enhanced IL-6 production in both 1321N1 cells and cultured mouse astrocytes. It has been shown that IL-6 gene expression is regulated by various transcription factors. Among them, we found a significant increase in cyclic AMP-response element-binding protein (CREB) activity with its phosphorylation at Ser133 by U46619 [GenBank] in 1321N1 cells. Although U46619 [GenBank] increased IL-6 promoter activity, a mutation at cyclic AMP-response element (CRE) on the promoter clearly suppressed the effect, suggesting that CRE is involved in U46619 [GenBank] -induced IL-6 expression. Furthermore, both CREB and IL-6 promoter activities were suppressed by SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], a p38 mitogen-activated protein kinase (MAPK) inhibitor, and H89 [N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline], a protein kinase A (PKA) inhibitor, indicating involvements of p38 MAPK and PKA in CREB activation and IL-6 expression. To determine which G-proteins are implicated in the U46619 [GenBank] -induced IL-6 synthesis, the interfering mutants of Gq, G12, or G13 by were overexpressed in 1321N1 cells adenoviral approach. It is noteworthy that the Gq or G13 mutant blocked the IL-6 production by U46619 [GenBank] . The constitutively active mutant of Gq, G12, or G13 enhanced IL-6 production, indicating that Gq and G13 were involved in U46619 [GenBank] -induced IL-6 production. In conclusion, TXA2 enhances the IL-6 biosynthesis via the PKA p38 MAPK/CREB pathway in 1321N1 cells. IL-6 induction depends on Gq and G13 as well. This is the first report showing TP-mediated IL-6 production in glial cells.
The central nervous system (CNS) consists mainly of neurons and glial cells. Neurons play a central role in signal transduction by releasing neurotransmitters, whereas glial cells support the neuronal functions. Glial cells in CNS are generally classified into astrocytes, oligodendrocytes, and microglia. Astrocytes form the blood-brain barrier along with endothelial cells, metabolize neurotransmitters, cause gliosis after brain injury, and regulate extrasynaptic ion and H+ concentrations (Somjen, 1988; Ransom et al., 2003). In addition to these roles, they produce neurotrophic factors to promote neuronal survival (prevention of apoptosis), differentiation, or regeneration. It has been shown that they secrete neurotrophic factors, such as neurotrophin family members (Althaus and Richter-Landsberg, 2000), interleukin-6 (IL-6) (Schwaninger et al., 1997; Van Wagoner and Benveniste, 1999), and glial cell line-derived neurotrophic factor (Lin et al., 1993). We have previously shown that 1321N1 human astrocytoma cells produce nerve growth factor and IL-6 (Obara et al., 2001). These neurotrophic factors have been suggested as potential therapeutic agents for the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.
Thromboxane A2 (TXA2), a metabolite of arachidonic acid produced by cyclooxygenase and TXA2 synthetase, is known to induce many cellular responses, including platelet aggregation and contraction in various smooth muscle types (Huang et al., 2004). Because TXA2 is associated with various diseases, such as thrombogenesis, hypertension, bronchial asthma, and nephrotic syndrome, the antagonist for TXA2 receptor and inhibitor of its synthesis have been suggested as therapeutic targets (Huang et al., 2004).
We found TXA2 receptors (TPs) in 1321N1 human astrocytoma cells and rabbit cultured astrocytes (Nakahata et al., 1989, 1992). 1321N1 human astrocytoma cells express both TP isoforms (TP- and TP-) (Honma et al., 1998), which are alternatively spliced variants from the same gene. They are seven-transmembrane receptors and have been shown to associate with heterotrimeric GTP-binding proteins, such as Gq/11, Gi, G12, G13 and Gh (Offermanns et al., 1994; Nakahata et al., 1995; Allan et al., 1996; Vezza et al., 1999; Gao et al., 2001). In response to TXA2 analog, an elevation of intracellular Ca2+ concentration was observed as a result of phosphatidylinositol-specific phospholipase C activation in 1321N1 cells. TP-mediated extracellular signal-regulated kinase (ERK) activation in differentiated 1321N1 cells depends on the activation of phosphatidylcholine-specific phospholipase C (Kobayashi et al., 2000). It has also been shown that TXA2 analog elevates cyclic AMP concentration in several cell types, such as Schwann cells (Muja et al., 2001), even though there is currently no evidence for the interaction between TPs and Gs. In that report, cyclic AMP-response element-binding protein (CREB) is activated, which is assumed to be by a cyclic AMP-dependent mechanism. Thus, the downstream pathway of TPs is complex and has not yet been clarified in detail.
In screening for compounds that enhance neurotrophic factor production in 1321N1 cells, we found that the TXA2 analog U46619 [GenBank] significantly promoted IL-6 secretion. Herein, we show the mechanism of IL-6 production by TXA2 in detail.
Materials and Methods
U46619 was obtained from Cayman Chemical (Ann Arbor, MI). Prostaglandin E2, Ro20-1724, isoproterenol, carbachol, dibutyryl cyclic AMP (dbcAMP), H89, wortmannin, and KN62 were purchased from Sigma-Aldrich (St. Louis, MO). IL-6 enzyme-linked immunosorbent assay (ELISA) kit was from Amersham Biosciences Inc. (Piscataway, NJ). RNA extraction kit was from Nippon Gene Co., Ltd. (Toyama, Japan), and reverse transcription-polymerase chain reaction (RT-PCR) kit was from Toyobo Engineering (Osaka, Japan). Phorbol 12-myristate 13-acetate (PMA), SB203580, and forskolin were from Wako Pure Chemicals (Tokyo, Japan). SQ29548 was from Bristol-Myers Squibb (Tokyo, Japan). KT5720 was from Calbiochem (La Jolla, CA). CRE-luciferase reporter plasmid was from BD Biosciences Clontech (Palo Alto, CA). FuGENE 6 transfection reagent was from Roche Diagnostics (Indianapolis, IN). PD98059, anti-phospho-ERK antibody, anti-phospho-p38 mitogen-activated protein kinase (MAPK) antibody, anti-p38 MAPK antibody, anti-phospho-CREB antibody, anti-CREB antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were from Cell Signaling (Beverly, MA). Anti-ERK2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-glial fibrillary acidic protein (GFAP) antibody was from Progen (Heidelberg, Germany). Fluorescein isothiocyanate-conjugated anti-mouse antibody was from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD). Cholera toxin was from List Biological Laboratories Inc. (Campbell, CA).
Cell Culture. 1321N1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (Cell Culture Laboratory, Cleveland, OH), 50 units/ml penicillin, and 50 e/ml streptomycin in a 5% CO2 incubator at 37°C. Mouse astrocytes were prepared from neonatal mouse brains and then cultivated in DMEM supplemented with 10% fetal calf serum, penicillin, and streptomycin (Muller et al., 1984).
IL-6 Sandwich ELISA. IL-6 ELISA was performed as the protocol of IL-6 ELISA kit. In brief, 1321N1 cells were seeded onto six-well plates (1 x 106 cells/well) and cultivated overnight in the absence of fetal calf serum. The cells were cultured for 24 h in the presence of U46619 [GenBank] , and then the conditioned media were collected after centrifugation to remove contaminating cells. These conditioned media were regarded as samples for ELISA and then added to 96-well plates precoated with IL-6 antibody. After incubation, IL-6 antibody conjugated with biotin was added; then, avidin-horseradish peroxidase was added.
Semiquantitative RT-PCR. Total RNA from 1321N1 cells was extracted by using a total RNA extraction kit, and semiquantitative RT-PCR was carried out by using a RT-PCR kit. IL-6 mRNA expression was examined as described previously (Obara et al., 2001). For analysis of human IL-6 mRNA, the sense primer (5'-AAA TTC GGT ACA TCC TCG AC-3' of human IL-6 cDNA) and the antisense primer (5'-CAG GAA CTG GAT CAG GAC TT-3'), which were complementary to conserved regions of cDNA from human IL-6, were used. For analysis of mouse IL-6 mRNA, the sense primer (5'-CAA GAG ACT TCC ATC CAG TTG C-3') and the antisense primer (5'-TTG CCG AGT AGA TCT CAA AGT GAC-3') were used. The human IL-6 cDNA of 295 base pairs was amplified from 26 to 30 cycles (95°C for 60 s, 56°C for 60 s, and 74°C for 60 s), and mouse IL-6 cDNA of 614 base pairs was amplified 28 cycles (94°C for 30 s, 57°C for 60 s, and 72°C for 60 s). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) transcripts of 700 base pairs were used as an internal control. The number of cycles that yielded a quantitative amount of product was determined in preliminary experiments. The densities of the bands corresponding to IL-6 and G3PDH were analyzed by densitometry (NIH Image software), and the data were expressed as a ratio to G3PDH.
Immunostaining. Mouse astrocytes were fixed with 4% paraformaldehyde and stained with anti-GFAP antibody (1:10 dilution) and fluorescein isothiocyanate-anti-mouse antibody (1:80 dilution).
Plasmids, Transfection, and Reporter Gene Assay. Plasmids containing IL-6 promoter (wild type or CRE mutated) linked to luciferase gene (Funakoshi et al., 1999; Tokunou et al., 2001) were kindly given by Dr. Toshihiro Ichiki (Kyushu University, Fukuoka, Japan). Human mitogen- and stress-activated protein kinase 1 (MSK1) wild-type and the kinase-dead mutant (MSK1 D195A/D565A) (Arthur et al., 2004) were from Dr. Soren Impey (Vollum Institute, Oregon Health Sciences University, Portland, OR). A plasmid encoding PKI was from Dr. Philip Stork (Vollum Institute, Oregon Health Sciences University). Plasmids encoding constitutively active mutants of G-proteins (GqQ209L, G12Q231L, and G13Q226L) were kindly provided by the laboratory of John Scott (Vollum Institute, Oregon Health Sciences University). For transfection, 1321N1 cells were seeded onto 24-well plates at 1 x 105 (cells/well). On the next day, plasmids (0.6-1.2 e/well) and FuGENE 6 (0.75-1.8 e/well) were mixed gently in 25 e of DMEM and added to culture plates. For luciferase assay, media were replaced by serum-free media 24 h after transfection. The cells were incubated with the indicated drugs at 37°C for 6 h on the following day. Luciferase assay was performed by using dual luciferase assay kit (Promega, Madison, WI) according to its instruction. Renilla reniformis luciferase (phRL-TK, Promega) activity was measured for normalizing the transfection efficiency. The luciferase activity was measured using a luminometer (GENE LIGHT 55; Microtech Nition, Funabashi, Japan).
cAMP Assay. Intracellular cAMP concentration was measured as follows. 1321N1 cells on a 24-well plate were labeled with 2 e藽i/ml [3H]adenine (23 Ci/mmol; Amersham Biosciences) in the growth medium for 3 h. The labeled cells were washed twice with DMEM-HEPES (20 mM, pH 7.4). The cells were incubated for 10 min with U46619 [GenBank] (or with SQ29548) in DMEM-HEPES containing phosphodiesterase inhibitor Ro20-1724 (100 e). After aspirating the incubation buffer, the reactions were stopped by adding 0.4 ml of 2.5% HClO4. Acid extracts were mixed with 1/10 volume of 4.2 M KOH to neutralize and deposit potassium perchlorate. [3H]cAMP in the clear supernatant was separated by Dowex 50W-X8 (Bio-Rad, Hercules, CA)/alumina (Merck Biosciences, Darmstadt, Germany) double columns and determined by liquid scintillation counting. An increase in [3H]cAMP was expressed as the -fold over the control (no drug).
Adenoviral Infection. Adenoviruses encoding carboxyl-terminal regions of Gq (Gq-ct, amino acids 305-359), G12 (G12-ct, amino acids 325-379), and G13 (G13-ct, amino acids 322-378) were created in our laboratory. The expressions of those genes are regulated by CMV promoter, and their products putatively block G-protein coupling with their corresponding receptors (Arai et al., 2003). Each adenoviral vector contains both G-protein-ct and GFP that is upstream of the G-protein-ct, and each entity is under the control of CMV promoter. 1321N1 cells were infected with the indicated virus at 75 m.o.i. for 1 day. The medium was then replaced with serum-free medium on the next day, and the cells were cultured overnight. At the time of drug incubation, more than 90% of the cells were GFP-positive.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting. Samples used for immunoblotting were prepared as follows. 1321N1 cells were seeded onto six-well plates at a density of 1 x 106 (cells/well). The cells were cultured overnight in serum-free DMEM, and then drugs were added for various periods of time. The incubation medium was aspirated after the reaction, and the cells were dissolved in Laemmli sample buffer (final concentration: 75 mM Tris-HCl, 2% SDS, 15% glycerol, 3% 2-mercaptoethanol, pH 6.8) and boiled at 95°C for 5 min.
Electrophoresis was performed on 11% acrylamide gels. Proteins were transferred electrically from the gel onto a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA) by the semidry blotting method. The blots were blocked for 40 min with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween 20 and incubated with primary antibodies [anti-phospho-ERK (Thr202/Tyr204) antibody, anti-ERK2 antibody, anti-phospho-p38 MAPK (Thr180/Tyr182) antibody, anti-p38 MAPK antibody, anti-phospho-CREB (Ser133) antibody, or anti-CREB antibody (1:1000 dilution)] overnight at 4°C. The blots were washed several times and then incubated at 25°C for 2 h with a 1:3000 dilution of secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG antibody). Blots were developed using an enhanced chemiluminescence assay kit (Amersham Biosciences) and visualized by chemiluminescence on Hyperfilm ECL. The densities of the bands corresponding to CREB, ERK, and p38 MAPK were analyzed by densitometry (NIH Image), and the data were expressed as a ratio to their total proteins.
Statistical Methods. Data were expressed as the mean values ± S.E.M, and the significant differences were analyzed by Student's t test or Tukey's method for multiple comparisons.
Results
U46619 Enhances IL-6 Production in 1321N1 Cells. We investigated the biosynthesis of IL-6. 1321N1 cells were stimulated with the TP agonist, U46619 [GenBank] , and then we examined IL-6 secretion by sandwich-ELISA (Fig. 1A). U46619 [GenBank] significantly increased IL-6 secretion from 1321N1 cells in a concentration-dependent manner that was inhibited by TP antagonist SQ29548. Adenylyl cyclase activator, forskolin, and cAMP analog dbcAMP also promoted IL-6 secretion by 1.6- and 2.9-fold, respectively (data not shown). U46619 [GenBank] (10 e) also enhanced IL-6 gene expression (5.8-fold), and this effect was abolished by 1 e SQ29548 again (Fig. 1B). Carbachol (10 e) did not affect IL-6 gene expression. Forskolin and a PKC activator, PMA, enhanced IL-6 expression 4.5- and 7.0-fold in the same condition, respectively (Fig. 1B). U46619 [GenBank] and SQ29548 have low affinity to other prostanoid receptors, such as the prostaglandin E2 receptor subtype EP4 (Kiriyama et al., 1997), and it is also known that prostaglandin E2 induces IL-6 release in U373 MG human astrocytoma cells (Fiebich et al., 2001). To avoid the possibility that the effect of U46619 [GenBank] on IL-6 induction is mediated by EPs, we tested prostaglandin E2. U46619 [GenBank] -induced IL-6 expression was completely blocked by SQ29548 (3% U46619 [GenBank] alone), whereas most of the effect of prostaglandin E2 was not blocked by SQ29548 (82% prostaglandin E2 alone) (Fig. 1C). Cholera toxin as a positive control enhanced IL-6 expression 13.2-fold (Fig. 1C). This result indicates that the effect of U46619 [GenBank] is mediated by TPs. To examine the IL-6 promoter activity, the plasmid encoding wild-type IL-6 promoter (-1039 +1) linked to luciferase gene was transfected into 1321N1 cells and luciferase activity was measured (Fig. 1D). U46619 [GenBank] (10 e) as well as 10 e forskolin significantly enhanced IL-6 promoter activity. Furthermore, GFAP-positive cultured mouse astrocytes (Fig. 2A) were stimulated with U46619 [GenBank] to determine whether IL-6 enhancement is 1321N1 cell-specific or not. As expected, 10 e U46619 [GenBank] similarly promoted IL-6 gene expression (2.7-fold) in astrocytes as dbcAMP (2.1-fold), and the effect of U46619 [GenBank] was mostly reversed by SQ29548 (12% U46619 [GenBank] alone) (Fig. 2B). Taken together, these results suggest that U46619 [GenBank] enhances the secretion of IL-6 from 1321N1 cells accompanied by an increase in IL-6 gene expression.
CRE Regulates the IL-6 Gene Expression by U46619 [GenBank] in 1321N1 Cells. IL-6 gene expression is regulated by multiple transcription factors, including CREB, activator protein-1 (AP-1), nuclear factor-B (NF-B), CCAAT enhancer-binding protein-, and glucocorticoid receptor in complex ways (Obara and Nakahata, 2002). Because CREB activation by U46619 [GenBank] has been reported (Muja et al., 2001), we determined whether CREB is involved in the U46619 [GenBank] -induced IL-6 gene expression. CRE activity was measured by reporter gene assay. pCRE-luciferase plasmid was transfected into 1321N1 cells, and then newly induced luciferase activity was regarded as the CREB-transcriptional activity. U46619 [GenBank] (10 e) and 10 e forskolin significantly increased CRE activity (Fig. 3A). To subsequently confirm the specificity of CRE in enhanced IL-6 gene expression, we used the IL-6 promoter-luciferase plasmid harboring a mutant CRE in the IL-6 promoter region (from ATGACGTCA to ATCGATCCA). The effect of U46619 [GenBank] on IL-6 promoter activity was completely abolished by mutation of CRE as in the case of forskolin (Fig. 3B). These results indicate that CRE activation is essential for U46619 [GenBank] -induced IL-6 gene expression in 1321N1 cells.
PKA and p38 MAPK/MSK Pathways Are Involved in CREB Activation and IL-6 Gene Expression by U46619 [GenBank] in 1321N1 Cells. It has been shown that CREB activity is regulated by phosphorylation at Ser133, where various kinases have been proposed to phosphorylate, including protein kinase A (PKA), Akt (or protein kinase B), calmodulin-dependent kinases (CaMKs), ribosomal S6 kinases (RSKs), and MSKs (Lonze and Ginty, 2002). Akt is a downstream effector of phosphoinositide 3-kinase, and RSKs and MSKs can be activated by ERK1/2, ERK5, or p38 MAPK. To investigate the U46619 [GenBank] signaling to CREB in detail, pharmacological tools were used to identify the CREB kinase. 1321N1 cells were pretreated with 10 e H89 (PKA inhibitor), 30 e PD98059 (MEK inhibitor), 30 e SB203580 (p38 MAPK inhibitor), 1 e wortmannin (phosphoinositide 3-kinase inhibitor), or 10 e KN62 (CaMK inhibitor) for 15 min, and then they were stimulated with 10 e U46619 [GenBank] for 6 h (Fig. 4A). The U46619 [GenBank] -induced CRE activation was significantly blocked by H89 and SB203580. Carbachol (10 or 100 e) did not increase the CRE activity. Because H89 has been reported to inhibit MSK as well as PKA (Davies et al., 2000), another PKA inhibitor, KT5720, was tested. KT5720 (2 e) also inhibited the CRE activity significantly (Fig. 4B). Increase in CRE activity by U46619 [GenBank] was also blocked by 2 e SQ29548 and a lower concentration of 3 e SB203580 (Fig. 4C). Furthermore, MSK 1 wild-type and the kinase-dead mutant (D195A and D565A) were overexpressed in 1321N1 cells, and CRE activity was measured (Fig. 4D). The MSK1 kinase-dead mutant partially but significantly attenuated the ability of U46619 [GenBank] to increase CRE activity. CREB family members, including CREB, activating transcription factor 1, and CRE modulator, binds to CRE. To investigate whether CREB is involved in this process, CREB phosphorylation at Ser133 was monitored. This phosphorylation site can be regarded as an index of CREB activation. The CREB phosphorylation by U46619 [GenBank] via TPs was observed (Fig. 5A), confirming that CREB is activated by U46619 [GenBank] . This phosphorylation was completely blocked by 10 e H89, 30 e SB203580, and 2 e KT5720 again (Fig. 5B). These results suggest that PKA and p38 MAPK/MSK pathways mediate CREB activation by U46619 [GenBank] . The effects of these kinase inhibitors on IL-6 gene expression were investigated next. H89 (10 e) and 30 e SB203580 decreased IL-6 promoter activity accelerated by 10 e U46619 [GenBank] (Fig. 6A), similar to the inhibition of CRE activity shown in Fig. 4A. The effect of U46619 [GenBank] was also significantly inhibited by 2 e SQ29548 and 3 e SB203580, overexpressing a physiological PKA pseudosubstrate peptide, PKI (Fig. 6B). Furthermore, when 1321N1 cells were pretreated with 30 e SB203580, IL-6 gene expression induced by 10 e U46619 [GenBank] was largely suppressed (21% U46619 [GenBank] alone), as determined by RT-PCR (Fig. 6C). Because the involvement of p38 MAPK in TP-mediated IL-6 expression was suggested, we examined whether p38 MAPK was activated by U46619 [GenBank] . When 1321N1 cells were incubated with 10 e U46619 [GenBank] for 5 to 20 min, transient phosphorylation of ERK1/2 was observed (2.2-fold at 5 min, 1.9-fold at 10 min, and 0.97-fold at 20 min) (Fig. 7). In addition to ERKs, p38 MAPK phosphorylation was induced by 10 e U46619 [GenBank] for 5 to 20 min (1.8-fold at 5 min, 2.1-fold at 10 min, and 2.8-fold at 20 min). CREB phosphorylation at Ser133 in the same lysates was also monitored. As expected, U46619 [GenBank] induced CREB phosphorylation correlated with p38 MAPK phosphorylation (5.6-fold at 5 min, 9.1-fold at 10 min, and 2.9-fold at 20 min) (Fig. 7). Gs coupling to TPs has not currently been reported, although an intracellular cAMP concentration by TP agonist is elevated in many cell types. We measured cAMP level after U46619 [GenBank] treatment (Fig. 8). The -adrenergic receptor agonist isoproterenol (10 e) strongly elevated the cAMP level. Compared with isoproterenol, the effect of U46619 [GenBank] on cAMP accumulation was much weaker, but the significant cAMP increase via TPs was observed. Taken together, these results suggest that PKA and p38 MAPK/MSK pathways are involved in CREB activation and IL-6 gene expression induced by U46619 [GenBank] in 1321N1 cells.
Gq and G13 Mediate TXA2 Signaling to IL-6 Expression. TPs couple with various kinds of G-proteins, such as Gq, G12, and G13 (Huang et al., 2004). We have shown that TPs associate with at least Gq and G12 in 1321N1 cells (Honma et al., 1998). To determine which G-proteins are involved in the signaling pathway leading to IL-6 production, we took an adenoviral approach to interfere with endogenous G-protein functions. The products from adenoviruses that encode the C-terminal regions of Gq, G12, or G13 inhibited the coupling among these G-proteins and their associated receptors (Arai et al., 2003). GFP gene was introduced to monitor the infection efficiency of those G-protein-interfering mutants. 1321N1 cells infected with those viruses were stimulated with 10 e U46619 [GenBank] for 4 h, and then RT-PCR was carried out. IL-6 gene expression was promoted with U46619 [GenBank] treatment in 1321N1 cells infected with control virus, and the enhancement was blocked by overexpressing C-terminal regions of Gq or G13 [45 and 53% U46619 [GenBank] (GFP), respectively] but not by G12 (Fig. 9A). Furthermore, to determine whether these G-proteins can affect IL-6 production, the cells were transfected with constitutively active mutants (GqQ209L, G12Q231L, and G13Q226L). GqQ209L and G13Q226L promoted IL-6 gene expression 15- and 7.9-fold, respectively, determined by RT-PCR, whereas G12Q231L marginally promoted it (2.6-fold) (Fig. 9B). In addition, these active mutants could significantly enhance both CRE and IL-6 promoter activities (Fig. 9C). Taken together, the results suggest that Gq and G13 are necessary and sufficient for U46619 [GenBank] -induced IL-6 gene expression.
Discussion
In the present study, we showed that U46619 [GenBank] promoted IL-6 biosynthesis in 1321N1 human astrocytoma cells and GFAP-positive mouse astrocytes. In addition, U46619 [GenBank] increased CREB activity that was accompanied by its phosphorylation at Ser133 by PKA and p38 MAPK/MSK, and this CREB activation is essential for IL-6 induction in 1321N1 cells (Fig. 10). This is the first report showing TP-mediated IL-6 production in glial cells.
The amount of arachidonic acid metabolites are increased dramatically after brain injury, viral infection, or inflammation. These metabolites, including TXA2, are assumed to play important roles in the CNS under such pathologic conditions. In fact, astrocytes and microglia have the ability to release TXA2 after activation by bacterial lipopolysaccharide and ATP (Pearce et al., 1989; Bruner and Murphy, 1993; Minghetti and Levi, 1995). Thus, the elevation of adenine nucleotide concentration or microglial activation after CNS injury or infection may cause the secretion of TXA2, leading to activation of TPs on glial cells via autocrine or juxacrine mechanisms. Therefore, a clarification of the physiological roles of TPs in astrocytes will help in identifying therapeutic targets in various CNS disorders.
IL-6 is a pleiotropic factor involved in the regulation of various inflammatory and endocrine functions in both central and periphery nervous systems. In the CNS, although various cell types can produce IL-6, astrocytes play a dominant role in IL-6 production (Gruol and Nelson, 1997). Under normal physiological conditions, IL-6 expression levels in CNS remain low, whereas IL-6 is up-regulated upon stress, diseases, inflammation, or injury. To date, it has been shown that numerous physiological receptor agonists or pharmacological reagents influence IL-6 production (Sehgal, 1992; Van Wagoner and Benveniste, 1999). Cytokines and proinflammatory factors, including lipopolysaccharide, IL-1, tumor necrosis factor-, and prostaglandin E2 are known as IL-6 inducers and play important roles in CNS diseases. In addition, various kinds of neurotransmitters and neuropeptides, including catecholamines, adenosine, serotonin, histamine, substance P, calcitonin, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating polypeptide, also influence IL-6 production. Abnormal physiological condition often changes the induction of IL-6, such as hypoxia/reoxygenation, generation of reactive oxygen species, viral infections, and irradiation. It is not surprising that compounds that increase cyclic AMP concentration, such as cholera toxin, cyclic AMP analogs, and forskolin, also lead to an induction of IL-6 in astrocytes (Norris et al., 1994). Furthermore, phorbol ester and Ca2+ ionophore, A23187 [GenBank] , induced IL-6 production and both of these reagents caused synergistic production, suggesting that PKC and Ca2+ signaling are responsible for IL-6 production in rat astrocytes (Norris et al., 1994). Fiebich et al. (1995) showed that ceramide induced IL-6 gene expression in U373-MG human astrocytoma cells. In terms of transcription factors, IL-6 promoter contains various consensus sequences of transcription factors, indicating that multiple transcription factors influence its gene expression. For example, CREB, AP-1, NF-B, CCAAT enhancer-binding protein- (or NF-IL6), and glucocorticoid receptor are known as regulators, and stimuli leading to the activation of these transcription factors are assumed to modulate IL-6 gene expression (Sehgal, 1992; Obara and Nakahata, 2002).
In the present study, it was demonstrated that U46619 [GenBank] increased the CRE activity by reporter gene assay, accompanied by the phosphorylation of CREB at Ser133, which is a key regulatory site for its transcription activity (Figs. 3A, 4, 5, and 7). Furthermore, this CREB activation is required for IL-6 gene expression (Fig. 3B). To date, it has been shown that the phosphorylation of this site occurs via multiple kinases, including PKA, CaMK, Akt, RSKs, and MSKs (Lonze and Ginty, 2002). RSKs and MSKs are known as downstream effectors of ERK1/2, ERK5, and p38 MAPK (Lonze and Ginty, 2002). In our study, to clarify the mechanism leading to CREB activation by U46619 [GenBank] , various pharmacological tools were tested. U46619 [GenBank] -induced CREB activation was significantly blocked by H89, KT5720, and SB203580 (Fig. 4). In addition, MSK1 kinase-dead mutant blocked CRE activity (Fig. 4D), suggesting the involvements of both PKA and p38 MAPK/MSK pathways. Although the mechanism remains unclear, cyclic AMP elevation and PKA activation have been shown in response to TP agonist (Muja et al., 2001; Miggin and Kinsella, 2002). On the other hand, p38 MAPK activation by TXA2 has been reported in platelets and isolated rat mesenteric resistance arteries (Bolla et al., 2002; Minuz et al., 2002). In the present study, we confirmed the elevation of cAMP and activation of p38 MAPK by U46619 [GenBank] in 1321N1 cells (Figs. 7 and 8). Taken together, this study strongly indicates that PKA and p38 MAPK participate in CREB activation by TXA2. However, as mentioned above, IL-6 gene expression is regulated by multiple transcription factors. Although it was demonstrated in this study that CREB activity was critical for IL-6 induction, we do not deny the possibility that other transcription factors, such as AP-1 and NF-B, activated by U46619 [GenBank] (Huang et al., 2004), may work cooperatively with CREB.
We demonstrated previously that TPs coupled with the subunits of at least two heterotrimeric GTP-binding proteins, Gq and G12, in 1321N1 cells (Honma et al., 1998). Phosphoinositide hydrolysis is promoted through stimulation with TP agonists, which causes increase in intracellular Ca2+ concentration and PKC activation (Nakahata et al., 1989). In dbcAMP-treated 1321N1 cells, phosphatidylcholine-specific phospholipase C, rather than phosphatidylinositol-specific phospholipase C, is predominantly activated by TP agonist, which causes subsequent ERK activation (Kobayashi et al., 2000). Because carbachol, an agonist of M3 receptor that couples to Gq, neither increased CRE activity nor induced IL-6 expression in this study, it was assumed that the other signaling pathway, such as G12 or G13, other than Gq-mediated PLC pathway might be essential for U46619 [GenBank] -induced IL-6 expression. In fact, although all of the constitutively active G-protein mutants (Gq, G12, and G13) could activate CRE and IL-6 promoter, interfering mutants Gq-ct and G13-ct, but not G12-ct, blocked IL-6 gene expression by TP stimulation (Fig. 6), indicating that TPs use Gq and G13 pathways for IL-6 production. Even though G12 and G13 belong to the same heterotrimeric G-protein family, the functional difference between those two G-proteins remain unclear. Nevertheless, in our study, G13 but not G12 was essential for TP signaling. Identification of direct downstream effector of G13 leading to CRE activation and IL-6 gene expression is necessary.
In vascular smooth muscle cells, thrombin induces IL-6 expression through the CRE, as in the case of TXA2 (Tokunou et al., 2001). In that report, involvements of ERK, p38 MAPK, and epidermal growth factor receptor (EGFR) are also implicated in thrombin-induced IL-6 production. EGFR transactivation that lies upstream of ERK and p38 MAPK is essential for this thrombin-induced IL-6 induction. EGFR transactivation by TXA2, which was probably mediated by either EGF-like ligand release by metalloproteinases or direct EGFR trans-phosphorylation by unidentified tyrosine kinases, has been also reported (Gao et al., 2001; Miggin and Kinsella, 2001). Thus, it is possible that this cross-talk between G-protein-coupled receptors and receptor tyrosine kinases is involved in p38 MAPK and CREB activation leading to IL-6 gene expression.
It has been shown that IL-6 acts as a neurotrophic factor as well as cytokine, and numerous studies have demonstrated both beneficial and unfavorable effects of IL-6 in CNS (Van Wagoner and Benveniste, 1999). A variety of in vitro and in vivo studies provide evidence for the involvement of IL-6 in neuronal survival, differentiation, and regeneration. IL-6 directly affects neurons, whereas it also affects glial cells; i.e., IL-6 promotes proliferation of astrocytes and production of nerve growth factor by autocrine mechanism. In contrast to the benefits above, IL-6 sometimes cause destructive effects. For example, in IL-6 transgenic mouse models, a high level of IL-6 led to neurodegeneration, blood-brain barrier breakdown, abnormal angiogenesis, increased level of complement proteins, and impaired learning. In those mice, it has been demonstrated that i.c.v. injection of lipopolysaccharide resulted in higher level productions of proinflammatory factors, such as tumor necrosis factor- and IL-1. IL-6 overproduction in the CNS may trigger unnecessary inflammatory responses and confusion of neuronal network. Thus, both beneficial and detrimental effects of IL-6 in the CNS have to be considered for the further study.
In conclusion, we showed that TXA2 analog induced a production of IL-6 mediated via PKA p38 MAPK/CREB pathway through Gq and G13 in 1321N1 cells. This observation indicates one of physiological roles of TPs in astrocytes and raises a possibility of TP as a therapeutic target in neuronal diseases.
Acknowledgements
We thank Dr. Toshihiro Ichiki (Kyushu University) for providing IL-6 promoter-luciferase plasmid, Dr. Philip Stork (Vollum Institute, Oregon Health Sciences University) for providing PKI DNA construct, Dr. John Scott (Vollum Institute) for providing G-protein DNA constructs, and Dr. Soren Impey (Vollum Institute) for MSK DNA constructs. We appreciate Dr. Quentin Low (Vollum Institute) for critical reading of the manuscript and scientific discussion. We also thank Manami Saika, Shigeyoshi Honma, and Hiroshi Kobayashi (Tohoku University) for assistance in the experiments.
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