Journal of Neurochemistry, 2005, 93, 549–559

doi:10.1111/j.1471-4159.2005.03079.x

Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C e resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells Klaus Lieb, Lisa Biersack, Anne Waschbisch, Sonja Orlikowski, Ravi Shankar Akundi, Eduardo Candelario-Jalil, Michael Hu¨ll and Bernd L. Fiebich Department of Psychiatry and Psychotherapy, University of Freiburg Medical School, Freiburg, Germany

Abstract Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and timedependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/ antagonists that were used. 5-HT-induced IL-6 synthesis is

inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) e to be involved in 5-HTinduced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HTinduced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC e isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC e for the induction of cytokine synthesis in astrocytic cells. Keywords: cytokine, depression, immunomodulation, neuroinflammation, neurotransmitter, stress. J. Neurochem. (2005) 93, 549–559.

Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which has been shown to be involved in neuroimmunomodulatory processes (Mossner and Lesch 1998). Previous work by others has demonstrated that 5-HT may induce the synthesis of interleukin (IL)-6, a pleiotropic cytokine with immunomodulatory properties showing both deleterious and restoring functions in the CNS (Gruol and Nelson 1997). The induction of IL-6 synthesis by 5-HT has been shown in two different cell culture systems, primary rat hippocampal astrocytes and human vascular smooth muscle cells (Pousset et al. 1996; Ito et al. 2000). In line with a stimulating function of 5-HT on IL-6 synthesis are results showing that the selective 5-HT reuptake inhibitors fluoxetine and citalopram, when given intraperitoneally to rats for 4 weeks, increase the production of IL-6 in rat splenocytes (Kubera et al. 2000). Conversely, IL-6 is able to stimulate the serotonergic system. It has been demonstrated, by the use of in vivo microdialysis and in vivo amperometry, that the i.p.

injection of IL-6 resulted in an elevation of 5-HT concentrations in the rat striatum and an increment of the evoked release of 5-HT (Zhang et al. 2001). Similar results were shown by others who found that IL-6 perfused locally into the rat anterior hypothalamus elicited a rapid and transient increase in extracellular 5-HT levels (Wu et al. 1999). Taken together, these results point to a bidirectional regulation of the serotoninergic system and IL-6. The present study was undertaken to extend previous investigations of an induction of IL-6 expression by 5-HT Received July 12, 2004; revised manuscript received November 5, 2004; accepted November 25, 2004. Address correspondence and reprint requests to Bernd L. Fiebich, PhD, Department of Psychiatry and Psychotherapy, University of Freiburg Medical School, Hauptstrasse 5, D-79104 Freiburg, Germany. E-mail: bernd.fi[email protected] Abbreviations used: BSA, bovine serum albumin; 5-CT, carboxamidotryptamin; 5-HT, serotonin; IL, interleukin; PKC, protein kinase C.

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

549

550 K. Lieb et al.

in primary rat astrocytes (Pousset et al. 1996) by investigating, in detail, the molecular pathways underlying this induction. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inductors, such as IL-1b (Lieb et al. 1996) or substance P (Lieb et al. 1998; Fiebich et al. 2000b), and which expresses functional 5-HT receptors. Our results show, for the first time, a central role of the 5-HT7 receptor linked to p38 MAPK and protein kinase C (PKC) e for the induction of cytokine synthesis in astrocytic cells.

Materials and methods Materials Serotonin was purchased from Sigma (Deissenhofen, Germany). Several antagonists and agonists of 5-HT receptors were used: GR 46611 (agonist of the 5-HT1D receptor; Tocris, Ko¨ln, Germany), 8-hydroxy-2-(di-n-propylamino)tetralin (agonist of the 5-HT1A and 5-HT1D receptor; Tocris), BRL 15572 (antagonist of the 5-HT1D receptor; Tocris), spiperone (antagonist of the 5-HT2A receptor; Tocris, distributed by Biotrend, Ko¨ln, Germany), allylpiperazine and tropisetron (antagonists of the 5-HT3 receptor; Sigma), a-methyl-5-HT (agonist of the 5HT2A receptor; Tocris), pimozide (partially selective on 5-HT7 receptors; Tocris), SB269970 (selective antagonists of 5-HT7 receptor; Tocris) and carboxamidotryptamin (5-CT) (agonist of 5-HT7; Tocris). Inhibitors of two types of MAPKs were used: SB202190, an inhibitor of p38 MAPK, and PD98059, an inhibitor of p42/44 MAPK. GF109203X, Go¨ 6983, RO-31-8425, rottlerin and Go¨ 6976 were used as inhibitors of PKC isoforms. All inhibitors were obtained from Calbiochem (Bad Soden, Germany). Stock solutions (10 mM) were prepared in dimethylsulphoxide and stored at )20C. Further dilutions were carried out in distilled water. None of the inhibitors, if used in the indicated concentrations, affected the viability of the cells as proved by cell viability assays.

Cell culture The human astrocytoma cell line U373 MG was obtained from the American Type Culture Collection (Rockville, MD, USA) and was grown in Minimum Essential Medium (MEM)-Earle’s medium (PAA Laboratories, Coelbe, Germany) containing 5% fetal calf serum (PAN, Aidenbach, Germany), 2 mM L-glutamine, 1 mM sodium pyruvate, 40 units/mL penicillin/streptomycin (all purchased from PAA Laboratories), 0.4% MEM vitamins and 0.4% MEM non-essential amino acids (both purchased from Life Technologies GmbH, Karlsruhe, Germany). Confluent monolayers were passaged routinely by trypsinization. Cells were plated for RNA extraction and western blot analysis in six-well plates and for determination of IL-6 in 24-well plates (Falcon, BD Biosciences, Heidelberg, Germany). Cultures were grown for 5–6 days at 37C in 5% CO2 and the medium was changed the day before treatment. Monocytes used as control for 5-HT receptor expression were prepared as previously described (Fiebich and Chrubasik 2004). RNA extraction and PCR analysis for the serotonin receptors Total RNA was extracted using the guanidine isothiocyanate method (Chomczynski and Sacchi 1987). For RT-PCR, 1 lg total RNA was reverse transcribed using MuMLV-reverse transcriptase (Gibco, Eggenstein, Germany) and random hexamers. Primers for human 5-HT receptors were designed using PrimerSelect Software from DNA Star Inc. (Madison, WI, USA). PCR was then carried out adding Taq polymerase (Promega, Madison, WI, USA) and a pair of primers for human 5-HT receptors (see Table 1). PCR products were then separated electrophoretically on a 2% agarose gel. Preparation of particulate fractions For the determination of PKC activation as determined by its phosphorylation state and cellular localization, membrane extracts of the cells were prepared considering the fact that the active form of PKC is tightly bound to the membrane and can be solubilized only under higher concentrations of detergents and/or sonification (for review see Hug and Sarre 1993). Cells were serum starved overnight before stimulation. After treatment, cells were washed once with ice-cold phosphate-buffered saline and lysed under 20 mM Tris-Cl, pH 7.5,

Table 1 Primer sequences selected and PCR conditions used to detect different serotonin (5-HT) receptors in U373 MG cells and primary human monocytes Product

Sense

Antisense

Annealing (C)

Cycles

Size (bp)

5-HT1A 5-HT1B 5-HT1D 5-HT1E 5-HT2A 5-HT2B 5-HT2C 5-HT3 5-HT4 5-HT6 5-HT7 Beta actin

5¢-GCCGCGTGCGCTCATCTCG-3¢ 5¢-CAGCGCCAAGGACTACATTTACCA-3¢ 5¢-GCCAAGGCCCAGGAGGAGA-3¢ 5¢-CAAGAGGGCCGCGCTGATGAT-3¢ 5¢-ACTCGCCGATGATAACTTTGTCCT-3¢ 5¢-GGCCCCTCCCACTTGTTCT-3¢ 5¢-TGTGCCCCGTCTGGATTTCTTTAG-3¢ 5¢-CCGGCGGCCCCTCTTCTAT-3¢ 5¢-GGCCTTCTACATCCCATTTCTCCT-3¢ 5¢-CCGCCGGCCATGCTGAACG-3¢ 5¢-GCGCTGGCCGACCTCTC-3¢ 5¢-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3¢

5¢-GCGGCGCCATCGTCACCTT-3¢ 5¢-GAAGAAGGGCGGCAGCGAGATAGA-3¢ 5¢-AGATGATAAAGGCCCCCAGAATGA-3¢ 5¢-CTGCCTTCCGTTCCCTGGTGGTGCTA-3¢ 5¢-TGACGGCCATGATGTTTGTGAT-3¢ 5¢-TAGGCGTTGAGGTGGCTTGTT-3¢ 5¢-CTCTTCCTCGGCCGTATTCCTCTT-3¢ 5¢-GCAAAGTAGCCAGGCGATTCTCT-3¢ 5¢-CTTCGGTAGCGCTCATCATCACA-3¢ 5¢-GCCCGACGCCACAAGGACAAAAG-3¢ 5¢-TCTTCCTGGCAGCCTTGTAAATCT-3¢ 5¢-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3¢

64 62 68 64 64 64.5 60 64 60 64.5 64 60

40 40 40 40 40 40 40 40 40 40 40 25

411 460 403 461 359 416 449 448/352 411 342 436 838

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

Serotonin induces IL-6 via 5-HT7 receptors 551

containing 0.32 M sucrose, 2 mM EDTA, 4 mM EGTA, 5 mM dithiothreitol, 0.2 mM orthovanadate, 20 mM sodium fluoride, 1 mM phenylmethylsulphonyl fluoride and a protease inhibitor cocktail (Sigma) containing aprotinin, leupeptin and pepstatin A. Homogenized lysates were subjected to centrifugation at 51 000 g for 1 h at 4C. The resulting pellet, which was considered as the membrane fraction, was resuspended in the lysis buffer from above but also containing 0.1% Triton X-100. The suspension was pulse sonicated three times and the centrifugation step repeated. The resulting supernatant fluid was analysed for PKC phosphorylation state. For long-term storage samples were diluted with 50% glycerol, aliquoted and stored at )70C. Protein concentrations were determined using the protein-binding dye amido black 10B (Sigma). Briefly, samples were mixed with 4 volumes of amido black solution [0.1% (w/v) amido black in 90% methanol/10% acetic acid (v/v)] and centrifuged at full speed for 15 min at 21C. The resulting pellet was washed in 90% methanol/ 10% acetic acid (v/v) and centrifuged again. The final pellet was resuspended in 0.2 M sodium hydroxide and absorbance measured at 610 nm. Protein concentrations were determined against a bovine serum albumin (BSA) standard. Western blot analysis U373 MG human astrocytoma cells were exposed to 5-HT in the presence or absence of pharmacological compounds. Supernatant fluid (50 lL) was incubated with 50 lL sodium dodecyl sulphate sample buffer (sodium dodecyl sulphate buffer containing 100 lM orthovanadate; Laemmli 1970). Cells were washed with phosphatebuffered saline and lysed in sample buffer. Lysates were homogenized through a 26-gauge needle and measured for protein content using the bicinchoninic acid method (BCA protein kit; Pierce, distributed by KFC Chemikalien, Mu¨nchen, Germany). For western blotting, 60 lg of cell protein from each sample for p38 MAPK and p42/44 MAPK, 60 lg of membrane fraction for PKC analysis or 50 lL of supernatant fluid in 50 lL sodium dodecyl sulphate sample buffer for IL-6 detection were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis on a 12% (IL-6, p38 MAPK, p42/44 MAPK) or 7.5% (PKC e) gel under reducing conditions. Proteins were then transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) by semidry blotting. The membrane was blocked overnight at 4C using Rotiblock (Roth, Karlsruhe, Germany) and for another hour at room temperature before incubation with the antibody. The quantity of active p42/44 MAPK, active p38 MAPK, PKC e and IL-6 in each sample was assessed using rabbit polyclonal antibodies. Anti-active p38 MAPK, recognizing both phosphorylated residues T180 and Y182 (NEB, Schwalbach, Germany), was diluted 1 : 500 in TBS-T Tris-buffered saline with 0.1% Tween 20 + 1% BSA, antip42/44 MAPK (Promega, Mannheim, Germany) was diluted 1 : 20 000 in TBS-T + 1% BSA and anti-IL-6 (Endogen; Biozol, Eching, Germany) was diluted 1 : 1000 in TBS-T + 1% BSA. PKC e was probed with a rabbit anti-phospho-PKC e antibody (Upstate Biotechnology, Lake Placid, NY, USA) at a final concentration in TBS-T + 1% BSA of 2 lg/mL. Subsequent detection was performed using the ECL Western blotting system (Amersham, Biosciences, Freiburg, Germany) according to the manufacturer’s instructions. Determination of interleukin-6 by ELISA Cells were pre-incubated with the inhibitors/antagonists for 30 min. Thereafter, cells were treated with 5-HT or 5-HT receptor agonists

for 18 h. Culture supernatant fluids were harvested, centrifuged for 10 min at 10 000 g and levels of IL-6 in the media were measured by ELISA (Pelikine, distributed by HISS, Freiburg, Germany) according to the manufacturer’s instructions. Experiments were carried out in triplicate.

Results

Serotonin induces interleukin-6 protein synthesis in human U373 MG astrocytoma cells Stimulation of U373 MG cells with 5-HT dose-dependently induced the synthesis of IL-6 protein as shown by western blot analysis (Fig. 1a) and ELISA (Fig. 1b) beginning at 1 nM 5-HT with a maximal induction achieved at a concentration of 0.1–1 lM 5-HT. Higher doses did not further increase IL-6 release in U373 MG cells (data not shown). Stimulation of cells with 100 nM 5-HT for various time periods revealed that IL-6 protein synthesis, as determined by western blot analysis, was induced after 2 h of stimulation and peaked at 4 and 48 h (Fig. 1c). Quantitative analysis of IL-6 protein showed that basal IL-6 protein release in unstimulated cells was low and ranged between 5 and 15 ng/mL as measured by ELISA (not shown). Stimulation with 100 nM 5-HT resulted in a > twofold increase of IL-6 release (38.3 ± 8.2 ng/mL for three independent experiments). Expression of serotonin receptors in human U373 MG astrocytoma cells As 5-HT was found to be pro-inflammatory in U373 MG cells, we were interested to discover which 5-HT receptors are expressed in this cell line. We investigated the expression of several 5-HT receptors and used human monocytes as a control. We found that U373 MG cells strongly express 5HT7 receptors and, to a lesser extent, 5-HT1D, 5-HT2A and 5HT3 receptors (Fig. 2). 5-HT4 receptors are only marginally expressed and 5-HT1A, 5-HT1B, 5-HT1E, 5-HT2B, 5-HT2C and 5-HT6 receptors are not expressed in U373 MG cells (not shown). Monocytes expressed all receptors except 5-HT2C and 5-HT6 receptors (Fig. 2, not shown). Serotonin acts on 5-HT7 receptors to induce interleukin-6 release in U373 MG astrocytoma cells The strongly expressed Gi-coupled 5-HT1D receptor is unlikely to be involved in 5-HT-induced IL-6 release as the 5-HT1D agonists 8-hydroxy-2-(di-n-propylamino)tetralin and GR 46611 did not significantly induce IL-6 release at doses of 0.01–1 lM and only slightly induced IL-6 release at the high dose of 10 lM. Furthermore, 5-HT-induced IL-6 release was not affected by the 5-HT1D antagonist BRL 15572 (not shown). Therefore, we further investigated whether the Gs-coupled 5-HT2A and 5-HT7 receptors and the ion channel 5-HT3 are involved in 5-HT-induced IL-6 synthesis. To this

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

552 K. Lieb et al.

(a)

U373

H20

MW

30 kDa

IL-6

5-HT1D

IL-6 [% of 5-HT control]

co 5HT ntro l (1 00 pM 5HT ) (1 5nM HT ) (1 5HT 0 n M (1 ) 00 n 5M HT ) (1 µM )

21 kDa

(b)

Mo

5-HT2A

400,0%

359 bp

**

350,0%

403 bp

300,0%

**

250,0%

**

200,0% 150,0%

5-HT3A

100,0% 50,0% 0,0%

co 5HT ntro l (1 00 p 5HT M) (1 5nM HT ) (1 5HT 0 n M (1 ) 00 n 5M HT ) (1 µM )

448 bp / 352 bp

5-HT7 436 bp

(c)

30 kDa

IL-6 5-HT (100 nM)

0

2

4

8

24

48

72

h

Fig. 1 Dose-dependent (a and b) and time-dependent (c) induction of interleukin (IL)-6 synthesis by serotonin (5-HT) in U373 MG human astrocytoma cells. Cells were either left untreated or treated with the indicated concentrations for 24 h (a) or for the indicated time periods (b) with 1 lM 5-HT. IL-6 synthesis was determined in cell supernatant fluids by western blot (a) or ELISA (b). (b) Significant differences between unstimulated control and 5-HT-treated cells are indicated (**p < 0.01) evaluated by Student’s t-test. (c) The different bands of IL-6 are due to different glycosylation states of IL-6. A typical example of three independent experiments is shown.

end, we pre-treated U373 MG cells with antagonists of the three 5-HT receptors before stimulation with 5-HT. By using the selective 5-HT2A antagonist spiperone, we found that this antagonist only slightly inhibited 5-HT-induced IL-6 release at a high dose of 10 lM (not shown). The 5-HT3 receptor antagonists allylpiperazine and tropisetron had no effect on 5-HT-induced IL-6 levels (not shown). The partially selective 5-HT7 receptor antagonist pimozide dose-dependently inhibited 5-HT-induced IL-6 synthesis starting with a dose of 100 nM and reaching maximal effects at 1 and 10 lM, which totally blocked IL-6 release induced by 5-HT (Fig. 3a). As pimozide binds to other 5-HT receptors, we further used the selective 5-HT7 receptor antagonist SB269970, which potently prevented 5-HT-induced IL-6 release starting at 10 nM with maximal inhibition down to baseline levels at 1 lM (Fig. 3b). In order to prove the possible involvement of 5-HT2A and 5-HT7 receptors as suggested from the antag-

betaActin Fig. 2 Expression of serotonin (5-HT) receptors in human U373 MG astrocytoma cells and primary human monocytes. RNA was isolated from U373 MG cells and primary monocytes, as described in Materials and methods, followed by PCR for the various 5-HT receptor mRNAs (see Table 1). In U373 MG cells, 5-HT7 receptors were strongly expressed and, to a lesser extent, 5-HT1D, 5-HT2A and 5-HT3 receptors. Monocytes (Mo) were used as positive control. Instead of cDNA, H2O was used to verify DNA contamination of Mastermix subjected to PCR. The corresponding PCR products are indicated by arrows. MW, molecular weight marker (100-bp ladder).

onist experiments, we used selective agonists of those two receptors to induce IL-6 release in human astrocytoma cells. The 5-HT2A agonist alpha-methyl-5-HT only marginally induced the release of IL-6 in U373 MG cells at a high dose of 10 lM, suggesting that this receptor is not a major mediator of 5-HT-induced IL-6 release (data not shown). In contrast, the 5-HT7 receptor agonist 5-CT strongly induced IL-6 synthesis at a low concentration of 10 nM with maximal effects at a concentration of 100 nM resulting in IL-6 levels similar to the 5-HT-inducing effects (Figs 1a and b), suggesting that the 5-HT7 receptor is centrally involved in 5-HT-induced IL-6 release (Fig. 3c). This is supported by the finding that the 5-HT7 antagonists pimozide (Fig. 3d) and SB269970 (Fig. 3e), but not the 5-HT2A antagonist spiperone (not shown), dose-dependently inhibited 5-HT/5-CTinduced IL-6 release.

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

Serotonin induces IL-6 via 5-HT7 receptors 553

(a) IL-6 [% of 5-HT control]

140% 120% 100% 80%

**

60%

**

**

40% 20%

)

)

µM 0 (1 m o pi

+

+

+

+

pi

pi

m o

m o

(1 00

(1

nM

µM

)

) nM (1 0

pi

5-

H

m o

T

co

(1

nt

µM

ro

l

)

0%

(b) IL-6 [% of 5-HT control]

120% 100%

**

80%

**

**

**

60% 40%

**

**

**

**

20%

) (1

0

(1

70

70

97

97 SB

26

SB 26

+

+

SB +

(c) 900% IL-6 [% of untreated control]

µM

µM

nM 00 (1 70 97

26

26 SB +

)

)

) nM 0 (1

97

5-

70

H

T

co

(1

nt

µM

ro

)

l

0%

**

800%

**

**

700%

*

600% 500% 400% 300% 200% 100%

10

1

Serotonin-induced interleukin-6 synthesis is blocked by inhibitors of p38 MAPK and protein kinase C but not by an inhibitor of p42/44 MAPK Several inhibitors of second messenger pathways were tested for their ability to inhibit 5-HT-induced IL-6 release. The p38 MAPK inhibitor SB202190 and the PKC inhibitor GF109203X reduced the amount of IL-6 in the supernatant fluids of 5-HT-treated U373 MG cells (Fig. 4). However, IL-6 release was not significantly inhibited by the p42/44 MAPK inhibitor PD98059 (Fig. 4). To further elucidate the PKC isoforms involved in 5-HT-induced IL-6 release, we used a variety of PKC inhibitors exhibiting different PKC inhibition profiles. We found that Go¨ 6976, a potent inhibitor of the classical PKC isoforms (a, bI and c), was totally ineffective and the PKC inhibitor Go¨ 6983 (a, bI, bII, c, d and f) inhibited 5-HT-induced IL-6 synthesis only at the high

5C

T

5C

T

10 T 5C

5C

µM

µM

nM 0

nM 10 T

5C

T

co

1

nt

ro

l

nM

0%

(d)

Fig. 3 Effects of the partially selective 5-HT7 antagonist pimozide (pimo; a and d) and the selective 5-HT7 antagonist SB269970 (b and e) on serotonin- (5-HT; a and b) or carboxamidotryptamin- (5-CT; d and e) induced interleukin (IL)-6 release and of the 5-HT7 agonist 5-CT (c) on IL-6 levels in U373 MG cells. U373 MG human astrocytoma cells were either left untreated or treated with the indicated concentrations of pimozide (a and d) or SB269970 in the absence or presence of 5-HT or 5-CT or 5-CT alone (c). After pre-treatment with the antagonists, cells were stimulated for an additional 18 h with 5-HT or the agonists. IL-6 was determined in cell supernatant fluids by ELISA according to the manufacturer’s protocol. Data are means ± SD from three independent experiments. Significant differences: *p < 0.05, **p < 0.01 evaluated by Student’s t-test.

140%

100%

14

**

12

80%

**

60% 40%

IL-6 [ng/ml]

20%

10 8 **

6

µM ) (1

0

(1

IL-6 [% of 5-HT control]

100%

*

** **

80% 60% 40% 20%

) µM (1

0

(1

70

70 +

SB 26

97

97 +

SB

26

97 26 SB +

µM )

) 00 (1 70

70 97 SB 26 +

nM

) nM (1

0

(1 T 5C

co

nt

ro

µM )

l

0%

µM ) (1 0

µM ) G F +

+

PD

(1 0 +

120%

(1 0

µM )

µM ) SB

+

co nt r

ol

pi m

o

0 (1

o m pi +

pi m

2

+

+

µM )

) 00 (1 o

pi m

5C

o

T

(1

(1

0

nM

) µM

l ro co nt

nM )

0%

(e)

**

4

5H T

IL-6 [% of 5-HT control]

16

120%

Fig. 4 Serotonin (5-HT)-induced interleukin (IL)-6 synthesis is inhibited by the p38 MAPK inhibitor SB202190 and the protein kinase C inhibitor GF109203X but not significantly by an inhibitor of p42/44 MAPK (PD98059). U373 MG human astrocytoma cells were either left untreated or pre-treated with the indicated concentrations of the inhibitors and subsequently stimulated with 5-HT for 24 h. IL-6 protein was measured in cell supernatant fluids by ELISA according to the manufacturer’s protocol. Data are means ± SD from three independent experiments. Significant differences between 5-HT plus inhibitor vs. 5-HT alone: **p < 0.01 evaluated by Student’s t-test.

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

554 K. Lieb et al.

dose of 10 lM (not shown). These data indicate that classical PKCs are probably not involved in 5-HT-mediated IL6 synthesis, suggesting atypical PKCs, probably PKC d and e, being part of the signal transduction pathway leading from the 5-HT7 receptor to IL-6. We used two other inhibitors which have been shown to have some selectivity on PKC d (rottlerin) and e (RO-31-8425). As shown in Fig. 5(a), RO-31-8425 potently prevented 5-HT-induced IL-6 release at a dose of 10 lM, whereas 10 lM of rottlerin was not able to (a) 160% 120%

**

100%

**

80% 60%

Serotonin induces the activation of p38 MAPK in U373 MG astrocytoma cells The activation of p38 MAPK in 5-HT-treated cell cultures was investigated using phospho-specific antibodies recognizing the phosphorylated, and thus activated, form of p38 MAPK. At 5 min after stimulation with 100 nM 5-HT, the anti-p38 MAPK antibody detected a single band at 38 kDa corresponding to the molecular weight of this kinase (Fig. 7a). Phospho-p38 MAPK immunoreactivity was maximal at 10 and 15 min and disappeared after 60 min. In further experiments, we investigated whether 5-HT-induced p38 MAPK activity is mediated by the activation of other kinases such as p42/44 MAPK or PKC. As shown in Fig. 7(b), 5-HT-induced p38 MAPK activity was only prevented by the specific p38 MAPK inhibitor SB202190 but not by PD98059 (a p42/44 MAPK inhibitor) or GF109203X (an inhibitor of PKC), indicating that the p38 MAPK signal transduction pathway is independent of both kinases.

**

40% 20%

+

5-

co nt ro l R H O T -3 (1 1µ 84 M ) 25 + (1 R 00 O -3 nM 1) 84 + 25 R O ( 1 -3 µM 184 ) 25 + (1 ro 0 ttl µM er ) in (1 00 + n ro M ttl ) er in + (1 ro µ ttl M ) er in (1 0 µM )

0%

(b)

160% 140% 120%

**

100%

**

80% 60% 40% 20%

) µM

µM

0 (1

(1

00

F

F +

G

(1

+

G

F G +

G

)

) nM

µM

µM

0

(1

(1 3

ö6

98

(1

ö6 G +

)

)

) nM

00

0 (1 ö6

98

3

98 3

)

µM

)

µM

(1 6

6 97

ö6 G

+

+

+

+

+

G

G

)

nM

6

ö6 G

97

5-

ö6

97

(1

00

µM

l ro nt

(1

C

T

co 140% 120%

**

**

120%

**

80%

**

40% 20%

) µM

) µM

** **

60% 40% 20%

ttl e

rin

er in

)

µM +

SB

(1 0

(1 SB +

(1

)

µM

) nM

) 0 SB

T 5C

+

+

co nt

Fig. 5 Effects of various inhibitors of protein kinase C (PKC) on serotonin- (5-HT) and carboxamidotryptamin- (5-CT) induced interleukin (IL)-6. U373 MG human astrocytoma cells were either left untreated or pre-treated with the indicated concentrations of the PKC inhibitors and subsequently stimulated with 5-HT (a) or 5-CT (b and c) for 24 h. IL-6 protein was measured in cell supernatant fluids by ELISA according to the manufacturer’s protocol. Data are means ± SD from three independent experiments. Significant differences between 5-HT/ 5-CT plus inhibitor vs. 5-HT/5-CT alone: **p < 0.01 evaluated by Student’s t-test.

00

nM

)

ro

l

ro +

+

ro

ttl

in ttl er ro

(1 0

(1

nM 00

0

(1

(1 25 +

-3 184 O

+

R

)

) µM

) µM

) 25

(1

nM (1

-3 184 O

R +

**

80%

0%

+

R

O

-3 184

25

5C

T

00

(1

co

nt

µM

ro

)

l

0%

*

100%

(1

60%

µM

100%

IL-6 [% of carbox control]

IL-6 [% of carbox control]

(c)

)

0%

SB

IL-6 [% of carbox control]

180%

(1

IL-6 [% of 5-HT control]

140%

block 5-HT-induced IL-6 synthesis to baseline levels. In further experiments, we used the same inhibitors in combination with the 5-HT7 agonist 5-CT. As shown in Figs 5(b and c), the PKC inhibition profile in the experiments using 5-CT was similar to that of 5-HT: strong inhibition by RO-31-8425, less potent inhibition by GF109203X, Go¨ 6983 and rottlerin and no inhibition by Go¨ 6976, supporting the involvement of PKC e and 5-HT7 receptors in mediating 5-HT-induced IL-6 synthesis. In line with the data obtained with 5-HT, we found 5-CTinduced IL-6 release to be strongly inhibited by the p38 MAPK inhibitor SB202190, once more suggesting that 5-HT-induced IL-6 synthesis via the 5-HT7 receptor involves p38 MAPK activation (Fig. 6).

Fig. 6 Carboxamidotryptamin- (5-CT) induced interleukin (IL)-6 synthesis is inhibited by the p38 MAPK inhibitor SB202190. U373 MG human astrocytoma cells were either left untreated or pre-treated with the indicated concentrations of SB202190 and subsequently stimulated with 5-CT for 18 h. IL-6 protein was measured in cell supernatant fluids by ELISA according to the manufacturer’s protocol. Data are means ± SD from three independent experiments. Significant differences between serotonin (5-HT) plus inhibitor vs. 5-HT alone: *p < 0.05, **p < 0.01 evaluated by Student’s t-test.

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

Serotonin induces IL-6 via 5-HT7 receptors 555

(a)

(a)

5-HT (100 nM)

95 kDa

46 kDa

phopsho p38 MAPK 0

2.5

5

10

15

30

60

120

(b)

0

min

1

5

2.5

10

(min)

46 kDa

100 nM 5-HT

phospho p38 MAPK

µM

95 kDa

+

G F

10

10

µM +P D

SB

10

µM 0. 1 HT 5-

+

l ro nt co

µM

(b)

Fig. 7 Time-dependent induction of p38 MAPK by serotonin (5-HT; a) and specific inhibition of this induction by the p38 MAPK inhibitor SB202190 (b). U373 MG human astrocytoma cells were either left untreated, treated with 100 nM 5-HT for the indicated time periods (a) or pre-treated with the indicated concentrations of the inhibitors and subsequently stimulated with 5-HT for 10 min (b). Total cell protein was prepared from control and 5-HT-treated cells and then subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotting using polyclonal antibodies that recognize the phosphorylated (and thus active) form of p38 MAPK.

Serotonin induces the activation of protein kinase C e in U373 MG astrocytoma cells The fact that the PKC inhibitor RO-31-8425 inhibited 5-HTand 5-CT-induced IL-6 release suggests an involvement of at least PKC e in the signal transduction pathway induced by 5-HT. We investigated membrane fractions of 5-HT-treated U373 MG cells using a phospho-specific antibody for PKC e. As shown in Fig. 8(a), 5-HT induced PKC e starting at 2.5 min and lasting until 10 min. In order to confirm the involvement of PKC e in 5-HT signalling, we treated the cells with Go¨ 6976, which is not a good inhibitor of the novel protein kinases (Martiny-Baron et al. 1993). As shown in Fig. 8(b), Go¨ 6976 did not inhibit 5-HT-mediated PKC e activation unlike GF109203X, an antagonist of several PKC isoforms including PKC e. Discussion

In this study, we showed that 5-HT acts on 5-HT7 receptors to dose- and time-dependently induce the synthesis of IL-6 in human astrocytoma cells. We also showed that this induction of IL-6 synthesis is mediated by two protein kinases, p38 MAPK and PKC e, but not by p42/44 MAPK (ERK1/2). We used the human astrocytoma cell line U373 MG for the experiments. Although a tumour cell line, the following arguments led us to use this cell line. First, previous investigations by our group and others have shown that astrocytoma cells are a very suitable and reliable cell culture system for studying molecular signal transduction pathways (Norris et al. 1994; Lieb et al. 1997, 1998; Fiebich et al. 2000b); second, previous investigations have shown that

-

-

GF



100 nM 5-HT (2.5 min) Fig. 8 Induction of protein kinase C (PKC) e phosphorylation by serotonin (5-HT) in U373 MG astrocytoma cells. Cells were treated with 5-HT (100 nM) for various periods of time (a) or with 5-HT (100 nM) for 2.5 min in the presence or absence of the PKC inhibitors GF109203X and Go¨ 6976 (b). Membrane fractions were isolated as described in Materials and methods. Protein (60 lg) was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane and immunoblotted against phospho-PKC e using a phospho-specific PKC e antibody as described in Materials and methods.

signal transduction pathways in this cell line are similar to the pathways seen in primary rat astrocytes (e.g. Das and Potter 1995; Fiebich et al. 2001) and third, it has been demonstrated that primary rat hippocampal astrocytes express the IL-6 gene upon stimulation with 5-HT (Pousset et al. 1996) and that human astrocytoma cells express a variety of functional 5-HT receptors as shown here. As already mentioned, our results confirm previous investigations of an induction of the IL-6 gene by 5-HT in primary rat hippocampal astrocytes (Pousset et al. 1996). This induction of IL-6 expression appeared after 1 h of stimulation with 5-HT, which precedes the production of the IL-6 protein which is seen after 2 h as demonstrated here. In contrast to the primary rat astrocytes where the IL-6 gene was induced at picomolar concentrations, we found a significant induction of IL-6 protein synthesis only at a concentration of 1 nM. This may be due either to the higher sensitivity of the RT-PCR as used by Pousset et al. (1996) detecting more subtle inductions as compared with the western blot analysis as used here or to differences in the sensitivity of 5-HT receptors in rat and human astrocytes. We found two peaks of 5-HT-induced IL-6 release, one after 4 h and the second after 48 h. The different peaks might be due to the signal transduction pathways induced, one rapid and transient (e.g. PKC) and the other delayed but persistent (e.g. p38 MAPK). Another possibility might be secondary effects of 5-HT, such as gene products enhancing or inducing IL-6 release.

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

556 K. Lieb et al.

Our findings also confirm the results of an induction of IL-6 synthesis by 5-HT in human vascular smooth muscle cells (Ito et al. 2000). These authors showed that PKC is involved in 5-HT-induced IL-6 synthesis as the PKC inhibitor calphostin C suppressed 5-HT-induced IL-6 synthesis and the effect of 5-HT was abolished in PKC-depleted vascular smooth muscle cells. They did not characterize the PKC isoform involved. In order to further identify the specific isoform of PKC involved in 5HT-induced IL-6 synthesis in U373 MG cells, we used inhibitors of PKC that reveal isoform-selective inhibition profiles (Way et al. 2000). The indolocarbazole Go¨ 6976 is a highly selective inhibitor of classical PKC isoforms (a, b and c) as well as of the novel PKC, PKC l, which has an IC50 of less than 50 nM as determined on purified recombinant PKC isoenzymes (Martiny-Baron et al. 1993; Gschwendt et al. 1996). Furthermore, this inhibitor has no inhibitory capability on the novel PKC isoforms d, e and the atypical isoform PKC f. Go¨ 6976 did not inhibit 5-HT-induced IL-6 synthesis, suggesting that the classical isoforms of PKC, as well as PKC l, are not involved in 5-HT-induced IL-6 release. Additionally, we used the bisindolylmaleimides, Go¨ 6983 and GF109203X, in our experiments. Go¨ 6983 inhibits PKC a, b, c and d at or below an IC50 of 10 nM and PKC f at 60 nM (Gschwendt et al. 1996). However, only at the high dose of 10 lM was inhibition of 5-HT-induced IL-6 observed with Go¨ 6983, thus confirming the absence of the involvement of classical PKC isoforms as well as PKC d and f. However, to the best of our knowledge, the effect of Go¨ 6983 on PKC e has not yet been tested (Way et al. 2000) and thus, the inhibition of 5-HTinduced IL-6 at micromolar doses could be due to crossinhibition with other PKC isoforms including PKC e. GF109203X, on the other hand, shows inhibition of most isoforms of PKC, with classical isoforms in the nanomolar range and the novel (PKC d, e and l) and atypical (PKC f) PKC in the micromolar range (Way et al. 2000). In our experiments, GF109203X showed a significant inhibition only at 10 lM. Thus, comparing the results obtained with the above three inhibitors, we concluded that, among the isoforms that have been tested for pharmacological inhibition by these inhibitors, at least PKC e plays an important role in 5-HT-induced IL-6 synthesis. PKC e has been identified in U373 MG cells and has previously been reported to be involved in substance P-induced IL-6 synthesis (Lieb et al. 2003). Thus, in order to confirm the role of PKC e in 5-HTinduced IL-6 synthesis, we used another PKC inhibitor, RO-31-8425, which has an IC50 of 8 nM for PKC a and bI, 41 nM for PKC bII, 31 nM for PKC c and 39 nM for PKC e (Wilkinson et al. 1993). No significant inhibition was observed in the nanomolar range, whereas RO-31-8425 significantly inhibited 5-HT-induced IL-6 synthesis at 1 lM, with complete inhibition at 10 lM, thus confirming the involvement of PKC e. We furthermore used the inhibitor rottlerin (Gschwendt et al. 1994) which has an IC50 of 3 lM

for PKC d and has been shown not to inhibit other isoforms below 10 lM. Rottlerin shows no inhibition at 1 lM and partial inhibition at 10 lM. As Go¨ 6983, which inhibits PKC d with an IC50 of 10 nM, failed to inhibit 5-HT-induced IL-6 release in the nanomolar range and as rottlerin only weakly prevents 5-HT-mediated IL-6 release, we conclude that PKC d is probably not involved in 5-HT-induced IL-6 synthesis. Several other reports have recently mentioned that rottlerin is a mitochondrial respiratory chain uncoupler (Soltoff 2001) and a potent inhibitor of MAPK-activated protein kinase 2 and p38-regulated/activated kinase (Davies et al. 2000). This would imply that the inhibition of 5-HT-induced IL-6 observed with rottlerin could be a secondary effect due to the inhibition of kinases regulated by the activation of MAPK/PKC. Taken together from the PKC inhibition experiments, we conclude that at least PKC e is a central switch in the induction of the IL-6 gene by 5-HT. This is also evident from the fact that 5-HT mediated a rapid activation and membrane translocation of PKC e within 2.5 min of stimulation (Fig. 8a). Furthermore, GF109203X, but not Go 6976, inhibited the activation and translocation of PKC e (Fig. 8b). However, as selective inhibitors of the novel (PKC g and h) and atypical (PKC s and k) PKC isoforms have not yet been reported, we cannot totally exclude the involvement of other PKC isoforms in 5-HT-induced IL-6 synthesis. Another central molecular pathway is the stress-regulated p38 MAPK, which has been shown to be involved in the regulation of a broad spectrum of stress-related genes (Su and Karin 1996). Similar to the PKC inhibitor, the p38 MAPK inhibitor SB202190 completely blocked 5-HTinduced IL-6 synthesis. 5-HT-induced activation of p38 MAPK was not prevented by the PKC inhibitor GF109203X, suggesting two independent signal transduction pathways involved in 5-HT-mediated IL-6 expression. The possible role of p38 MAPK might not be a direct involvement in the regulation of gene expression but in the regulation of the IL-6 mRNA turnover as previously described for IL-6 (Wang et al. 1999; Wery-Zennaro et al. 2000) and COX-2 mRNA (Dean et al. 1999; Fiebich et al. 2000a). These results might suggest the importance of continuous stimulation of p38 MAPK by 5-HT to maintain sufficient levels of IL-6 mRNA to result in IL-6 protein synthesis. We found no consistent evidence that the transcription factor nuclear factor kappa B is involved in 5-HT-induced IL-6 synthesis (not shown), which is in contrast to a previous study where it was suggested that 5-HT may induce nuclear factor kappa B activation (Ito et al. 2000). Investigations by others have linked the activation of the p42/44 MAPK by 5-HT to mitogenic processes in U373 MG cells (Luo et al. 1996). Although 5-HT has been reported to activate p42/44MAPK in U373 MG cells, inhibition of p42/44 MAPKs did not affect 5-HT-induced IL-6 release, suggesting that these kinases are not involved in the regulation of the IL-6 gene. Therefore, further studies should investigate whether

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

Serotonin induces IL-6 via 5-HT7 receptors 557

5-HT may induce proliferation of astrocytes through this pathway. Several investigators have tried to link the inter-relationship between the serotoninergic system and immune system to the pathogenesis of neuropsychiatric disorders such as Alzheimer’s disease (e.g. Bonaccorso et al. 1998) or affective disorders (Frommberger et al. 1997). However, results are conflicting and it appears to be difficult to directly link the results obtained here to the clinical situation of patients. For example, in contrast to the inductory function of 5-HT on IL-6 synthesis, it has been hypothesized that a lack of tryptophan, as seen in acute depressive states, may correlate with higher levels of IL-6, as seen in the acute depressed state (Frommberger et al. 1997). However, acute lowering of blood tryptophan concentrations by the tryptophan depletion test in healthy volunteers did not influence IL-6 blood concentrations (Ravindran et al. 1999; Harrison et al. 2002). The hope is that the better understanding of the molecular mechanisms involved in the regulation of the serotonergic and cytokine system may shed light on this very conflicting set of data obtained in vitro and in vivo. Protein kinases, such as PKC, might be possible candidates as key molecules in the pathogenesis of affective and other neuropsychiatric disorders making the changes in 5-HT or cytokine levels only secondary events or epiphenomenons (Manji and Lenox 2000). Our data, showing an inhibition of 5-HT-induced IL-6 release by 5-HT7 antagonists, and the fact that the 5-HT7 receptor agonist 5-CT totally mimics the effects of 5-HT strongly support an involvement of 5-HT7 receptors in 5-HTinduced release of IL-6. This is supported by the 5-HT receptor expression analysis showing that 5-HT7 receptors are predominantly expressed in U373 MG cells, which is in line with data obtained from primary astrocytes (Shimizu et al. 1998). Recently, 5-HT7 receptors have been shown to mediate 5-CT-induced cAMP in U373 MG cells, suggesting the importance of 5-HT7 receptors in serotoninergic functions in astrocytes (Mahe et al. 2004). As demonstrated here for 5-CT/5-HT-induced IL-6 release, the 5-HT7 antagonist SB269970 prevented 5-CT-induced cAMP formation at a potency similar to our data (Mahe et al. 2004). We found that the 5-HT2a antagonist spiperone inhibited 5-HT-induced release of IL-6 only at a high concentration of 10 lM. As spiperone is a very selective antagonist of the 5-HT2A receptor with a pKi value of 8.8 (Barnes and Sharp 1999), the involvement of this receptor in 5-HT-induced IL-6 release, as described for smooth muscle cells (Ito et al. 2000), is not very likely. The fact that pimozide has some affinity to the 5-HT1D receptor might suggest an involvement of 5-HT1D receptors in 5-HT-induced IL-6 release. Due to the fact that two agonists of the 5-HT1D receptor, 8-hydroxy-2-(di-n-propylamino)tetralin and GR 46611, failed to induce IL-6 release in U373 MG cells at the expected potency and that the 5-HT1D receptor antagonist BRL 15572 did not affect 5-HT-induced IL-6 release, we conclude that this receptor type is not involved

in 5-HT-induced IL-6 synthesis. The slight induction of IL-6 at the high dose of 10 lM by both agonists might be due to the cross-reactivity to 5-HT7 receptors, which also has been observed for 5-HT7-mediated cAMP formation (Mahe et al. 2004). Although not involved in 5-HT-induced IL-6 synthesis, we describe, for the first time, the presence of 5-HT3 receptors in in vitro astrocyte cultures. The ligand-gated cation channel 5-HT3 receptor occurs mainly in the peripheral nervous system, particularly on nociceptive sensory neurones and on autonomic and enteric neurones, on which 5-HTexerts a strong excitatory effect (Hoyer et al. 1994). A high density of 5-HT3 receptors has also been identified at the following CNS regions: nucleus tractus solitarii, nucleus dorsalis nervi vagi, nucleus caudatus, nucleus accumbens, amygdala, hippocampus, entorhinal cortex, posterior horn ganglia and particularly in the area postrema, a region of the medulla involved in the vomiting reflex (Barnes and Sharp 1999). The role of the 5-HT3 receptor in astrocytic function is, as yet, totally unknown and is therefore an interesting topic for further investigations. The 5-HT7 receptor, the most recently cloned of all 5-HT receptors, has been shown to be distinctly distributed in the brain and to be the target of some psychotropic compounds. Three splice variants have so far been identified and the receptor is known to be coupled to Gs proteins (Barnes and Sharp 1999), which have also been identified in U373 MG cells (Krobert and Levy 2002; Mahe et al. 2004). As 5-CTinduced IL-6 release is not mediated by the cAMP pathway, it might be interesting if different splice variants of the 5-HT7 receptors contribute to the different signalling pathways, such as cAMP and PKC/p38 MAPK in U373 MG cells. Little is known so far about the role of 5-HT7 receptors in psychiatric disorders. However, the fact that some antipsychotics, such as pimozide, bind to this receptor subtype (Roth et al. 1994; Stowe and Barnes 1998) might suggest a crucial role of this receptor in the pathopyhsiology of psychiatric disorders. It has furthermore been shown that 5-HT7 receptors are involved in sleep and thermal regulation (Hagan et al. 2000; Hedlund et al. 2003) underlining the important role of this receptor in CNS function. So far, no published data are available concerning a role of 5-HT7 receptors in inflammatory events, which has been reported here for the first time. We further demonstrate the first evidence for 5-HT7 receptors activating p38 MAPK and PKC e. The activation of p42/44 MAPKs by 5-HT7 receptors in COS-7 and HEK293 cells has been reported by Norum et al. (2003) and a possible involvement of PKC has been suggested in the 5-HT7-mediated modulation of membrane potentials in motoneurones but the PKC isoform involved in these processes was not identified (Inoue et al. 2002). Taken together, we were able to demonstrate, for the first time, that 5-HT-induced IL-6 release is mediated via 5-HT7 receptors with the involvement of p38 MAPK and PKC e, suggesting that 5-HT, probably via the 5-HT7 receptor, might

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

558 K. Lieb et al.

play an important role in neurogenic inflammation occurring during a variety of CNS disorders. Acknowledgements The technical assistance of Ulrike Goetzinger-Berger, Brigitte Guenter and Sandra Hess is gratefully acknowledged.

References Barnes N. M. and Sharp T. (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38, 1083–1152. Bonaccorso S., Lin A., Song C., Verkerk R., Kenis G., Bosmans E., Scharpe S., Vandewoude M., Dossche A. and Maes M. (1998) Serotonin–immune interactions in elderly volunteers and in patients with Alzheimer’s disease (DAT): lower plasma tryptophan availability to the brain in the elderly and increased serum interleukin-6 in DAT. Aging (Milano) 10, 316–323. Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Ann. Biochem. 162, 156–157. Das S. and Potter H. (1995) Expression of the Alzheimer amyloid-promoting factor antichymotrypsin is induced in human astrocytes by IL-1. Neuron 14, 447–456. Davies S. P., Reddy H., Caivano M. and Cohen P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105. Dean J. L., Brook M., Clark A. R. and Saklatvala J. (1999) p38 mitogenactivated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J. Biol. Chem. 274, 264–269. Fiebich B. L. and Chrubasik S. (2004) Effects of an ethanolic salix extract on the release of selected inflammatory mediators in vitro. Phytomedicine 11, 135–138. Fiebich B. L., Mueksch B., Boehringer M. and Hull M. (2000a) Interleukin-1beta induces cyclooxygenase-2 and prostaglandin E(2) synthesis in human neuroblastoma cells: involvement of p38 mitogen-activated protein kinase and nuclear factor-kappaB. J. Neurochem. 75, 2020–2028. Fiebich B. L., Schleicher S., Butcher R. D., Craig A. and Lieb K. (2000b) The neuropeptide substance P activates p38 mitogenactivated protein kinase resulting in IL-6 expression independently from NF-kappa B. J. Immunol. 165, 5606–5611. Fiebich B. L., Schleicher S., Spleiss O., Czygan M. and Hull M. (2001) Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C. J. Neurochem. 79, 950–958. Frommberger U. H., Bauer J., Haselbauer P., Fraulin A., Riemann D. and Berger M. (1997) Interleukin-6-(IL-6) plasma levels in depression and schizophrenia: comparison between the acute state and after remission. Eur. Arch. Psychiat. Clin. Neurosci. 247, 228–233. Gruol D. L. and Nelson T. E. (1997) Physiological and pathological roles of interleukin-6 in the central nervous system. Mol. Neurobiol. 15, 307–339. Gschwendt M., Muller H. J., Kielbassa K., Zang R., Kittstein W., Rincke G. and Marks F. (1994) Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 199, 93–98. Gschwendt M., Dieterich S., Rennecke J., Kittstein W., Mueller H. J. and Johannes F. J. (1996) Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett. 392, 77–80.

Hagan J. J., Price G. W., Jeffrey P. et al. (2000) Characterization of SB269970-A, a selective 5-HT(7) receptor antagonist. Br. J. Pharmacol. 130, 539–548. Harrison B. J., Olver J. S., Norman T. R. and Nathan P. J. (2002) Effects of serotonin and catecholamine depletion on interleukin-6 activation and mood in human volunteers. Hum. Psychopharmacol. 17, 293–297. Hedlund P. B., Danielson P. E., Thomas E. A., Slanina K., Carson M. J. and Sutcliffe J. G. (2003) No hypothermic response to serotonin in 5-HT7 receptor knockout mice. Proc. Natl Acad. Sci. USA 100, 1375–1380. Hoyer D., Clarke D. E., Fozard J. R., Hartig P. R., Martin G. R., Mylecharane E. J., Saxena P. R. and Humphrey P. P. (1994) International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol. Rev. 46, 157–203. Hug H. and Sarre T. F. (1993) Protein kinase C isoenzymes: divergence in signal transduction? Biochem. J. 291, 329–343. Inoue T., Itoh S., Wakisaka S., Ogawa S., Saito M. and Morimoto T. (2002) Involvement of 5-HT7 receptors in serotonergic effects on spike afterpotentials in presumed jaw-closing motoneurons of rats. Brain Res. 954, 202–211. Ito T., Ikeda U., Shimpo M., Yamamoto K. and Shimada K. (2000) Serotonin increases interleukin-6 synthesis in human vascular smooth muscle cells. Circulation 102, 2522–2527. Krobert K. A. and Levy F. O. (2002) The human 5-HT7 serotonin receptor splice variants: constitutive activity and inverse agonist effects. Br. J. Pharmacol. 135, 1563–1571. Kubera M., Simbirtsev A., Mathison R. and Maes M. (2000) Effects of repeated fluoxetine and citalopram administration on cytokine release in C57BL/6 mice. Psychiat. Res. 96, 255–266. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680–685. Lieb K., Kaltschmidt C., Kaltschmidt K., Baeuerle P. A., Berger M., Bauer J. and Fiebich B. L. (1996) Interleukin-1b uses common and distinct signaling pathways for induction of the interleukin-6 and tumor necrosis factor a genes in the human astrocytoma cell line U373. J. Neurochem. 66, 1496–1503. Lieb K., Fiebich B. L., Berger M., Bauer J. and Schulze-Osthoff K. (1997) The neuropeptide substance P activates transcription factor NF-kappa B and kappa B-dependent gene expression in human astrocytoma cells. J. Immunol. 159, 4952–4958. Lieb K., Schaller H., Bauer J., Berger M., Schulze-Osthoff K. and Fiebich B. L. (1998) Substance P and histamine induce interleukin6 expression in human astrocytoma cells by a mechanism involving protein kinase C and nuclear factor-IL-6. J. Neurochem. 70, 1577–1583. Lieb K., Treffurth Y., Hamke M., Akundi R. S., von Kleinsorgen M. and Fiebich B. L. (2003) Valproic acid inhibits substance P-induced activation of protein kinase C epsilon and expression of the substance P receptor. J. Neurochem. 86, 69–76. Luo W., Sharif T. R. and Sharif M. (1996) Substance P-induced mitogenesis in human astrocytoma cells correlates with activation of the mitogen-activated protein kinase signaling pathway. Cancer Res. 56, 4983–4991. Mahe C., Bernhard M., Bobirnac I., Keser C., Loetscher E., Feuerbach D., Dev K. K. and Schoeffter P. (2004) Functional expression of the serotonin 5-HT7 receptor in human glioblastoma cell lines. Br. J. Pharmacol. 143, 404–410. Manji H. K. and Lenox R. H. (2000) Signaling: cellular insights into the pathophysiology of bipolar disorder. Biol. Psychiat. 48, 518– 530. Martiny-Baron G., Kazanietz M. G., Mischak H., Blumberg P. M., Kochs G., Hug H., Marme D. and Schachtele C. (1993) Selective

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

Serotonin induces IL-6 via 5-HT7 receptors 559

inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem. 268, 9194–9197. Mossner R. and Lesch K. P. (1998) Role of serotonin in the immune system and in neuroimmune interactions. Brain Behav. Immun. 12, 249–271. Norris J. G., Tang L. P., Sparacio S. M. and Benveniste E. N. (1994) Signal transduction pathways mediating astrocyte IL-6 induction by IL-1 beta and tumor necrosis factor-alpha. J. Immunol. 152, 841–850. Norum J. H., Hart K. and Levy F. O. (2003) Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and 5-HT7(a). J. Biol. Chem. 278, 3098–3104. Pousset F., Fournier J., Legoux P., Keane P., Shire D. and Soubrie P. (1996) Effect of serotonin on cytokine mRNA expression in rat hippocampal astrocytes. Brain Res. Mol. Brain Res. 38, 54–62. Ravindran A. V., Griffiths J., Merali Z., Knott V. J. and Anisman H. (1999) Influence of acute tryptophan depletion on mood and immune measures in healthy males. Psychoneuroendocrinology 24, 99–113. Roth B. L., Craigo S. C., Choudhary M. S., Uluer A., Monsma F. J. Jr, Shen Y., Meltzer H. Y. and Sibley D. R. (1994) Binding of typical and atypical antipsychotic agents to 5-hydroxytryptamine-6 and 5-hydroxytryptamine-7 receptors. J. Pharmacol. Exp. Ther. 268, 1403–1410. Shimizu M., Nishida A., Zensho H., Miyata M. and Yamawaki S. (1998) Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes. Brain Res. 784, 57–62. Soltoff S. P. (2001) Rottlerin is a mitochondrial uncoupler that decreases cellular ATP levels and indirectly blocks protein kinase

Cdelta tyrosine phosphorylation. J. Biol. Chem. 276, 37 986– 37 992. Stowe R. L. and Barnes N. M. (1998) Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine. Neuropharmacology 37, 1611–1619. Su B. and Karin M. (1996) Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8, 402–411. Wang S. W., Pawlowski J., Wathen S. T., Kinney S. D., Lichenstein H. S. and Manthey C. L. (1999) Cytokine mRNA decay is accelerated by an inhibitor of p38-mitogen-activated protein kinase. Inflamm. Res. 48, 533–538. Way K. J., Chou E. and King G. L. (2000) Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol. Sci. 21, 181–187. Wery-Zennaro S., Zugaza J. L., Letourneur M., Bertoglio J. and Pierre J. (2000) IL-4 regulation of IL-6 production involves Rac/Cdc42- and p38 MAPK-dependent pathways in keratinocytes. Oncogene 19, 1596–1604. Wilkinson S. E., Parker P. J. and Nixon J. S. (1993) Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem. J. 294, 335–337. Wu Y., Shaghaghi E. K., Jacquot C., Pallardy M. and Gardier A. M. (1999) Synergism between interleukin-6 and interleukin-1beta in hypothalamic serotonin release: a reverse in vivo microdialysis study in F344 rats. Eur. Cytokine Netw. 10, 57–64. Zhang J., Terreni L., De Simoni M. G. and Dunn A. J. (2001) Peripheral interleukin-6 administration increases extracellular concentrations of serotonin and the evoked release of serotonin in the rat striatum. Neurochem. Int. 38, 303–308.

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 549–559

Serotonin via 5-HT7 receptors activates p38 ... - Semantic Scholar

5% CO2 and the medium was changed the day before treatment. Monocytes used as .... another hour at room temperature before incubation with the antibody.

258KB Sizes 4 Downloads 145 Views

Recommend Documents

Strong Calcium Entry Activates Mitochondrial ... - Semantic Scholar
Dec 1, 2004 - Physiol. Rev 80:315–360. Oancea E, Meyer T (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals.

MISMATCH REMOVAL VIA COHERENT SPATIAL ... - Semantic Scholar
{jyma2010, zhaoji84, zhouyu.hust}@gmail.com, [email protected]. ABSTRACT ..... image analysis and automated cartography,” Communi- cations of the ...

Aeroengine Prognostics via Local Linear ... - Semantic Scholar
The application of the scheme to gas-turbine engine prognostics is ... measurements in many problems makes application of ... linear trend thus detected in data is used for linear prediction ... that motivated their development: minimizing false.

Web Query Recommendation via Sequential ... - Semantic Scholar
wise approaches on large-scale search logs extracted from a commercial search engine. Results show that the sequence-wise approaches significantly outperform the conventional pair-wise ones in terms of prediction accuracy. In particular, our MVMM app

Web Query Recommendation via Sequential ... - Semantic Scholar
Abstract—Web query recommendation has long been con- sidered a key feature of search engines. Building a good Web query recommendation system, however, is very difficult due to the fundamental challenge of predicting users' search intent, especiall

Collaborative Filtering via Learning Pairwise ... - Semantic Scholar
assumption can give us more accurate pairwise preference ... or transferring knowledge from auxiliary data [10, 15]. However, in real ..... the most popular three items (or trustees in the social network) in the recommended list [18], in order to.

Learning Speed Invariant Gait Template via Thin ... - Semantic Scholar
2 Department of Computer Science .... and deform the circle depending on the subjects. Thus .... The CMU Mobo gait database [5] has 25 subjects with 6 views.

Early Stage Botnet Detection and Containment via ... - Semantic Scholar
this research is to localize weakly connected subgraphs within a graph that models network communications between .... ultimate objective behind such early state detection is botnet containment. The approach that we ...... 71–86. Staniford, S., Pax

Learning a Factor Model via Regularized PCA - Semantic Scholar
Apr 20, 2013 - parameters that best explains out-of-sample data. .... estimation by the ℓ1 norm of the inverse covariance matrix in order to recover a sparse.

Object Instance Search in Videos via Spatio ... - Semantic Scholar
The dimension of local descriptors is reduced to 32 using PCA, and the number of Gaussian mixture components is set to 512. As in [20], a separate set of images .... 7, 4th–6th rows). 2) Parameter Sensitivity: We test the Max-Path search at dif- fe

Early Stage Botnet Detection and Containment via ... - Semantic Scholar
Detecting Botnet Membership with DNSBL Counterintelligence, pp. 131–142. Springer US. Riley, G. F., Sharif, M. I. and Lee, W. (2004) Simulating Internet worms. In Pro- ceedings of the 12th International Workshop on Modeling, Analysis, and Simu- lat

Visual Tracking via Weakly Supervised Learning ... - Semantic Scholar
video surveillance, human machine interfaces and robotics. Much progress has been made in the last two decades. However, designing robust visual tracking ...

Learning Speed Invariant Gait Template via Thin ... - Semantic Scholar
1 College of Computer Science .... To the best of our knowledge, our ... features and static features is general issue to the other computer vision issues. For.

Learning a Factor Model via Regularized PCA - Semantic Scholar
Apr 20, 2013 - To obtain best performance from such a procedure, one ..... Equivalent Data Requirement of STM (%) log(N/M) vs. EM vs. MRH vs. TM. (a). −1.5. −1. −0.5. 0. 0.5 ...... the eigenvalues of matrix C, which can be written as. R. − 1.

DOPAMINE RECEPTORS
Fax: + 44 (0)117 982 6552 e-mail: .... regarding antagonist affinities, although small differences have been reported for the ..... 16144 Westwoods Business Park.

Physics - Semantic Scholar
... Z. El Achheb, H. Bakrim, A. Hourmatallah, N. Benzakour, and A. Jorio, Phys. Stat. Sol. 236, 661 (2003). [27] A. Stachow-Wojcik, W. Mac, A. Twardowski, G. Karczzzewski, E. Janik, T. Wojtowicz, J. Kossut and E. Dynowska, Phys. Stat. Sol (a) 177, 55

Physics - Semantic Scholar
The automation of measuring the IV characteristics of a diode is achieved by ... simultaneously making the programming simpler as compared to the serial or ...

Physics - Semantic Scholar
Cu Ga CrSe was the first gallium- doped chalcogen spinel which has been ... /licenses/by-nc-nd/3.0/>. J o u r n a l o f. Physics. Students http://www.jphysstu.org ...

Physics - Semantic Scholar
semiconductors and magnetic since they show typical semiconductor behaviour and they also reveal pronounced magnetic properties. Te. Mn. Cd x x. −1. , Zinc-blende structure DMS alloys are the most typical. This article is released under the Creativ

vehicle safety - Semantic Scholar
primarily because the manufacturers have not believed such changes to be profitable .... people would prefer the safety of an armored car and be willing to pay.

Reality Checks - Semantic Scholar
recently hired workers eligible for participation in these type of 401(k) plans has been increasing ...... Rather than simply computing an overall percentage of the.

Top Articles - Semantic Scholar
Home | Login | Logout | Access Information | Alerts | Sitemap | Help. Top 100 Documents. BROWSE ... Image Analysis and Interpretation, 1994., Proceedings of the IEEE Southwest Symposium on. Volume , Issue , Date: 21-24 .... Circuits and Systems for V

TURING GAMES - Semantic Scholar
DEPARTMENT OF COMPUTER SCIENCE, COLUMBIA UNIVERSITY, NEW ... Game Theory [9] and Computer Science are both rich fields of mathematics which.