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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Regulation of Lipopolysaccharide-Induced Inducible Nitric-Oxide Synthase Expression through the Nuclear Factor-B Pathway and Interferon-/Tyrosine Kinase 2/Janus Tyrosine Kinase 2-Signal Transducer and Activator of Transcription-1 Signaling Cascades by 2-Naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (THI 53), a New Synthetic Isoquinoline Alkaloid

Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Korea (H.J.K., K.T., J.M.H., Y.J.K., M.K.P., Y.S.L., J.H.L., H.G.S., K.C.C.); and Natural Product Research Institute, Seoul National University, Seoul, Korea (H.S.Y-C.)

Received for publication August 4, 2006
Accepted November 13, 2006.

	Abstract

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The effects of 2-naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (THI 53), on nitric oxide (NO) production and inducible nitric-oxide synthase (iNOS) protein induction by lipopolysaccharide (LPS) were investigated in RAW 264.7 cells and mice. In cells, THI 53 concentration dependently reduced NO production and iNOS protein induction by LPS. In addition, THI 53 inhibited NO production and iNOS protein induction in LPS-treated mice. LPS-mediated iNOS protein induction was inhibited significantly by the specific tyrosine kinase inhibitor -cyano-(3-hydroxy-4-nitro)cinnamonitrile (AG126) as well as by THI 53. In addition, a c-Jun NH₂-terminal kinase (JNK) inhibitor anthra[1,9-cd]pyrazole-6 (2H)-one) (SP600125) but not an extracellular regulated kinase inhibitor [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98029)] or a p38 inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB230580)] reduced the iNOS protein level induced by LPS. Moreover, a Janus kinase 2 (JAK2) inhibitor -cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490) dose-dependently prevented LPS-mediated iNOS protein induction. LPS activated phosphorylations of tyrosine kinases, especially tyrosine kinase 2 (Tyk2) and signal transducer and activator of transcription-1 (STAT-1); these were reduced by THI 53. LPS also phosphorylated the JNK pathway; however, this phosphorylation was unaffected by THI 53. Interestingly, a JNK inhibitor (SP600125) and another tyrosine kinase inhibitor (genistein) significantly inhibited STAT-1 phosphorylation, suggesting that the LPS-activated JNK pathway and a tyrosine kinase pathway (especially Tyk2) may link to the STAT-1 pathway, which is involved in iNOS induction. However, THI 53 regulates LPS-mediated iNOS protein induction by affecting the Tyk2/JAK2-STAT-1 pathway, not the JNK pathway. The inhibition by THI 53 of LPS-induced NO production was recovered by a tyrosine phosphatase inhibitor (Na₃VO₄), which supports the possibility that THI 53 inhibits the LPS-induced inflammatory response through regulation of tyrosine kinase pathways. THI 53 also inhibited LPS-mediated interferon (IFN)- production and nuclear factor-B (NF-B) activation. Thus, THI 53 may regulate LPS-mediated inflammatory response through both the NF-B and IFN-/Tyk2/JAK2-STAT-1 pathways.

The induction of iNOS expression involves the activation of multiple signal transduction pathways, including mitogen-activated protein kinases (MAPKs) such as p38, ERK1/2 or JNK, NF-B, PI3 kinase, and Janus tyrosine kinase (JAK)-signal transducers and activators of transcription (STATs) (Dell'Albani et al., 2001; Liu et al., 2001; Tan et al., 2002). Both in vitro and in vivo studies with tyrosine kinase inhibitors have shown that activation of tyrosine kinases is necessary for a number of the biological responses to LPS, including activation of JNK (Dong et al., 1993; Novogrodsky et al., 1994; Hambleton et al., 1996). Moreover, tyrosine kinase 2 (Tyk2) is essential for LPS-induced endotoxin shock (Kamezaki et al., 2004). Tyk2 belongs to the JAK family, and the best-known substrate for these factors is the family of STAT proteins (Ihle, 1995, 2001; Rane and Reddy, 2000). The members of the JAK family, JAK1, JAK2, JAK3, and Tyk2, act as important protein tyrosine kinases (O'Shea et al., 2002; Schindler, 2002). JAK2 is reported to be involved in the LPS-induced expression of iNOS in skin-derived dendritic cells (Cruz et al., 2001); however, little is known about the molecular mechanisms by which JAK2 transduces the LPS-induced signals to downstream molecules to activate proinflammatory genes. Because higenamine (Kang et al., 1999a) and related isoquinolines (Kang et al., 1999b) effectively reduce iNOS gene expression in RAW 264.7 cells and smooth muscle cells by inhibition of NF-B, we speculate that the isoquinoline molecular backbone may be important for the inhibition of NF-B. In this process, phosphorylation of IB is vulnerable, hindering translocation of p65 from the cytosol to the nucleus (Kang et al., 2003). Indeed, it is possible to affect the LPS-activated protein tyrosine kinase pathway and its signaling cascade including MAPK or JAK/STATs by isoquinolines. Therefore, the purpose of the present study was to determine whether THI 53 (Fig. 1), a newly synthesized isoquinoline alkaloid, inhibits NO production as well as iNOS expression in RAW 264.7 murine macrophage cells and in mice stimulated by LPS. In addition, we aimed to determine the molecular mechanism by which THI 53 inhibits iNOS induction by LPS in RAW264.7 cells. The procedure for the total synthesis of THI 53 is now being prepared for publication in a relevant journal.

View larger version (15K): [in this window] [in a new window]   Fig. 1. The structure of the isoquinoline compound THI 53: 2-naphtylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline.

	Materials and Methods

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Materials. Dulbecco's modified Eagle's medium, fetal bovine serum, and antibiotics (penicillin/streptomycin) were purchased from Invitrogen (Carlsbad, CA). Anti-iNOS antibody was from Transduction Laboratories (Lexington, KY), and anti-p-Tyk2, anti-p-JNK, and anti-p-STAT-1 antibodies were from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-labeled goat anti-rabbit IgG was from Santa Cruz Biotechnology (Santa Cruz, CA). AG490, AG126, SB203580, and PD98059 were from Calbiochem (San Diego, CA). Enhanced chemiluminescence (ECL) Western blotting detection reagent was from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other chemicals, including LPS (Escherichia coli serotype 0128:B12), SP600125, and genistein, were from Sigma-Aldrich. (St. Louis, MO).

Cell Culture. RAW 264.7 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in RPMI 1640 medium supplemented with 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum.

Cell Viability. Cell viability was determined colorimetrically using the MTT assay. Cells at the exponential phase were seeded at 1 x 10⁴ cells/well in 24-well plates. After different treatments, 20 µl of 5 mg/ml MTT solution was added to each well (0.1 mg/well), and wells were incubated for 4 h. The supernatants were aspirated, the formazan crystals in each well were dissolved in 200 µl of dimethyl sulfoxide for 30 min at 37°C, and optical density at 570 nm was read on a Microplate Reader (Bio-Rad, Hercules, CA).

Cell Stimulation. RAW 264.7 cells were plated at a density of 1 x 10⁷ cells/100-mm dish. The cells were rinsed with fresh medium and stimulated with LPS (1 µg/ml) in the presence or absence of different concentrations of THI 53 (1–30 µM) simultaneously. THI 53 was dissolved in sterile distilled water and sterilized via a 0.2-µm filter.

Assay for Nitrite Production. Nitric oxide was measured as its stable oxidative metabolite, nitrite (NOx), as described by Kang et al. (1999a). At the end of the incubation, 100 µl of the culture medium was mixed with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). Light absorbance was measured at 550 nm, and the nitrite concentration was determined using a curve calibrated on sodium nitrite standards.

Western Blot Analysis. The cells were harvested and lysed with buffer containing 0.5% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl (pH 7.5), and protease inhibitors. The protein concentration of each sample was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL). To detect iNOS, 20 µg of the total protein was electrophoresed on a 10% polyacrylamide gel, and to detect phosphor-Tyk2, STAT-1, or JNK, 30 µg of the total protein was electrophoresed on a 12% polyacrylamide gel. The gels were transferred to polyvinylidene difluoride (PVDF) membranes by semidry electrophoretic transfer at 15 V for 60 to 75 min. The PVDF membranes were blocked overnight at 4°C in 5% bovine serum albumin (BSA). The cells were incubated with primary antibodies diluted 1:500 in Tris-buffered saline-Tween 20 containing 5% BSA for 2 h and then incubated with the secondary antibody at room temperature for 1 h. Anti-rabbit IgG was used as the secondary antibody (1:5000 dilution in Tris-buffered saline-Tween 20 containing 1% BSA). The signals were detected by ECL.

Quantitative Mouse IFN- Immunoassay. The quantity of IFN- secreted into the culture medium was analyzed using a commercially available mouse IFN- enzyme-linked immunosorbent assay kit (catalog number 42400-1; R&D Systems, Minneapolis, MN), according to the manufacturer's manual. Both the samples and standards were assayed in parallel.

Transfection. Transient transfections with NF-B-luciferase constructs were as described by Kim et al. (2006) using Lipofectin (Invitrogen). In brief, 5 x 10⁵ cells were plated on 60-mm plates the day before transfection and grown to 70% confluence. Cells were transfected with empty vector (pGL3 and/or pcDNA3) or with 1 µg of NF-B-luciferase plus 0.5 µg of pRL-TK-luciferase. Transfections were allowed to proceed for 4 h.

The transfected cells were washed with 4 ml of 1x phosphate-buffered saline (pH 7.4) and then stimulated with 1 µg/ml LPS. The cells were cultured in serum-free Dulbecco's modified Eagle's medium until harvested. Luciferase activity was normalized using a pRL-TK-luciferase activity (Renilla luciferase activity) for each sample.

Luciferase Assay. After experimental treatments, cells were washed twice with cold phosphate-buffered saline, lysed in a passive lysis buffer provided in the dual luciferase kit (Promega, Madison, WI), and assayed for luciferase activity using a TD-20/20 luminometer (Turner BioSystems, Inc., Sunnyvale, CA) according to the manufacturer's protocol. All transfections were done in triplicate. The data are presented as a ratio between firefly and Renilla luciferase activity.

Plasma Nitrite/Nitrate Measurement. Mice (ICR strain, 22–25 g, male) were divided into four groups: 1) LPS (10 mg/kg i.p., n = 4), 2) LPS plus THI 53 (20 mg/kg i.p., n = 4), 3) saline (i.p., n = 4), and 4) THI 53 (20 mg/kg i.p., n = 4). THI 53 was administered 30 min before LPS injection. Eight hours after LPS treatment, a whole blood sample was taken by cardiac puncture after anesthetizing the mice with pentobarbital. The plasma nitrate concentration was determined by reducing the nitrate enzymatically, using nitrate reductase from Aspergillus species. In brief, plasma samples were diluted 1:10 with distilled water and incubated with assay buffer: 50 mM KH₂PO₄, 0.6 mM NADPH, 5 mM FAD, and 10 U/ml nitrate reductase, pH 7.5, for 30 min at 37°C. Subsequently, culture medium was mixed with an equal volume of Griess reagent (mixture of 1 part of 1% sulfanilamide in 5% phosphoric acid and 1 part of 0.1% naphthylethylenediamine dihydrochloride in water) and incubated at room temperature for 10 min. The absorbance at 550 nm of the mixture was determined using a microplate reader. Mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996) and were treated ethically. The protocol was approved in advance by the Animal Research Committee of the Gyeongsang National University, Korea.

iNOS Protein Detection in LPS-Treated Mice. The lung tissues were homogenized in a buffer containing 50 mM Tris/Cl, pH 7.5, 1 mM EDTA, 1 mM leupeptin, 1 mM pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol and sonicated. The homogenates were then centrifuged at 7500g four times for 15 min, and the supernatants were subjected to SDS-polyacrylamide electrophoresis (7.5% gels). The separated proteins were transferred electrophoretically to PVDF membranes, and the membrane was incubated with anti-iNOS antibody complexes that were detected using ECL Western blotting detection reagents according to the manufacturer's instructions.

Statistical Evaluations. Data are expressed as the mean ± S.E.M. of results obtained from the number of animals used. Differences between data sets were assessed by one-way analysis of variance (ANOVA) followed by Newman-Keuls tests. P < 0.05 was accepted as statistically significant.

	Results

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Effects of THI 53 on LPS-Mediated NO Production and iNOS Protein Induction in RAW264.7 Cell Lines. Previously, nitrite increased time dependently, and the effect peaked 18 h after LPS treatment in cultured RAW 264.7 cells (Kang et al., 1999a). Thus, we treated the cells with LPS in the presence of THI 53 (5, 10, 20, and 30 µM) for 18 h. The accumulated nitrite was 3.8 ± 1.07 µM in control media, which increased to 19.5 ± 1.24 µM after LPS treatment for 18 h. Cotreatment with THI 53 decreased the nitrite concentration-dependently (Fig. 2A). Western blot analysis was performed to determine whether the reduced production of NO by THI 53 was caused by the inhibition of iNOS expression. THI 53 decreased LPS-mediated iNOS protein production in a concentration-dependent manner (Fig. 2B). More than 80% of cells were viable at treatments of up to 20 µM THI 53 in the presence of LPS.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Effects of THI 53 on NO production and iNOS protein level (A) and cell viability (B) in RAW 264.7 cells activated with LPS (1 µg/ml). A, RAW264.7 cells were stimulated with LPS for 18 h in the presence of different concentrations of THI 53 (5, 10, 20, and 30 µM). After treatment of LPS with and without THI 53 for 18 h, aliquots (100 µl) of the culture medium were mixed with an equal volume of Griess reagent. The absorbance at 570 nm was measured, and the nitrite concentration was determined using a curve calibrated to sodium nitrite standards. Treatment with THI 53 reduced LPS-induced iNOS protein levels in RAW 264.7 cells. B, cell viability was determined by MTT assay as described under Materials and Methods. Data represent the mean ± S.E.M. of triple determinations. One-way analysis of variance was used to compare group means, followed by Newman-Keuls tests (significance compared with the control: **, P < 0.01; significance compared with LPS: , P < 0.05; , P < 0.01).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effect of THI 53 on plasma NOx levels and iNOS induction in LPS-treated mice. Mice were pretreated with 20 mg/kg THI 53 for 30 min. Thereafter, mice were activated by LPS (10 mg/kg) for 8 h, and plasma NOx or iNOS protein levels in the lung tissues were measured after euthanasia. Data represent the mean ± S.E.M. of three experiments. One-way analysis of variance was used to compare the multiple group means followed by Newman-Keuls tests (significance compared with the control: **, P < 0.01; significance compared with LPS: , P < 0.01).

Differential Involvement of MAPK and JAK2 Pathways in iNOS Induction by LPS. As mentioned in the Introduction, the most extensively investigated intracellular signaling cascades involved in proinflammatory responses such as iNOS expression are the MAPK pathway and JAK/STAT pathway (including Tyk2). Therefore, we first confirmed the role of MAPK or JAK2 on LPS-mediated iNOS induction to clarify the action of THI 53 on the anti-inflammatory response. LPS-mediated iNOS protein induction was significantly inhibited by the specific tyrosine kinase inhibitor AG126 as well as by THI 53 (Fig. 4A). In addition, the JNK inhibitor SP600125 reduced the iNOS protein level induced by LPS, but the ERK inhibitor PD98059 or the p38 inhibitor SB203580 did not (Fig. 4B). Moreover, the JAK2 inhibitor AG490 prevented LPS-mediated iNOS protein induction in a dose-dependent manner.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Signaling pathway involved in LPS-induced iNOS expression in RAW264.7 cells. RAW 264.7 cells were stimulated with LPS (1 µg/ml) for 18 h after a 1-h pretreatment with various kinase inhibitors, and iNOS protein levels from cell lysate were detected by Western blotting. Top, AG126, a tyrosine kinase inhibitor given together with THI 53 reduced the iNOS level induced by LPS. Middle, SP600125, a specific JNK inhibitor, significantly inhibited LPS-mediated iNOS expression, but an ERK1/2 inhibitor PD98059 or the p38 inhibitor SB203580 did not. Bottom, AG490, a JAK2 inhibitor, also blocked LPS-induced iNOS expression. The results were confirmed by two independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Inhibition of THI 53 on LPS-mediated Tyk2 and STAT-1 phosphorylation. Cell lysates were extracted from cells treated with LPS at the indicated times, and Western blot analysis was performed using antibodies to phosphorylated Tyk2 and Tyk2 (A), to phosphorylated STAT-1 and STAT-1 (B), or to phosphorylated JNK and JNK (C). Pretreatment with THI 53 (20 µM) prevented activation of the LPS-induced Tyk2 or STAT-1 pathway; this did not prevent JNK phosphorylation by LPS but enhanced it. D, to investigate the involvement of tyrosine kinase or JNK in the STAT-1 pathway by LPS, cells were treated with a tyrosine kinase inhibitor [genistein (GT)] or a JNK inhibitor (SP600125). The experiments were repeated twice.

View larger version (19K): [in this window] [in a new window]   Fig. 6. The inhibitor of tyrosine phosphatase, Na3VO4, reversed the inhibition of NO production caused by THI 53 or by the tyrosine kinase inhibitor AG126 in dose-dependent manners. A, THI 53 (20 µM) or AG126 (20 µM), a tyrosine kinase inhibitor, reduced NO production in LPS-treated RAW 264.7 cells. Cells were pretreated with the tyrosine phosphatase inhibitor Na3VO4 (1 or 5 µM for 1 h) and were then stimulated with LPS. The tyrosine phosphatase inhibitor reversed the inhibition of LPS-induced NO production caused by THI 53 (B) or AG126 (C). Data represent the mean ± S.E.M. of triple determinations. One-way analysis of variance was used to compare the multiple group means followed by a Newman-Keuls test (significance compared with the control: **, P < 0.01; significance compared with LPS: , P < 0.05 or , P < 0.01; significance compared with THI 53 or AGS126: , P < 0.05 or , P < 0.01).

Phosphorylated STAT-1 is involved in iNOS expression. Because the JAK/STAT pathway is in the downstream of IFN- production and THI53 inhibited STAT-1 phosphorylation in our results, finally, we wanted to confirm whether THI 53 inhibits iNOS expression by LPS through the regulation of the IFN-/JAK-2-STAT-1 pathway. Thus, we investigated the effect of THI 53 on the induction of IFN- by LPS. As expected, LPS induced IFN- production in a time-dependent manner, started to increase prominently at 2 h, and showed a dramatic increase of IFN- production at 4 h of LPS treatment, which was efficiently inhibited by treatment of THI 53 (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effects of THI 53 on LPS-induced IFN- production in RAW 264.7 cells. An enzyme-linked immunosorbent assay was performed using the supernatant from the LPS-treated cells in a time-dependent manner. Before LPS treatment, cells were pretreated with THI 53 (20 µM) for 1 h. The data are presented as means ± S.E.M. of three independent experiments. One-way analysis of variance was used for comparisons of the multiple group means followed by Newman-Keuls test (significance compared with the control: **, P < 0.01; significance compared with LPS: , P < 0.05 or , P < 0.01).

View larger version (19K): [in this window] [in a new window]   Fig. 8. Effects of THI 53 on LPS-induced NF-B activation. A, cells were pretreated with THI 53 (5, 10, or 20 µM) for 1 h and then treated with LPS (1 µg/ml) for 1 h. After treatment, nuclear (NE) or cytoplasmic (CE) fractions were extracted, and the NF-B (p65) protein level was determined by Western blot analysis as described under Materials and Methods. The data are from two independent experiments. B, cells were transfected with empty vector or 1 µg of NF-B-luciferase plus 0.5 µg of pRL-TK-luciferase. Cells were allowed to recover for 24 h and then treated with 1 µg/ml LPS with/without THI 53 (5, 10, or 20 µMl). Cells were harvested 1 h after treatment. Luciferase activities are presented as fold activation relative to that of the untreated cells. Results are presented as the mean ± S.E.M. of three independent experiments. One-way analysis of variance was used to compare the multiple group means followed by a Newman-Keuls test (significance compared with the control: *, P < 0.05; significance compared with the LPS: , P < 0.05).

	Discussion

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We demonstrated here that THI 53 reduced NO production and iNOS protein induction concentration-dependently in LPS-treated RAW 264.7 cells. NO is massively generated from iNOS in endothelial and vascular smooth muscle cells several hours after a challenge with LPS and is known as an important pathological mediator of endotoxin shock (Kilbourn and Belloni, 1990). Therefore, selective inhibitors of iNOS with various chemical structures have been used as tools for the investigation of LPS-induced sepsis in animals. Some isoquinoline alkaloids inhibit NO production and iNOS induction in vascular smooth muscle cells and RAW 264.7 cells activated with LPS and/or IFN- (Kang et al., 1999a,b). We have confirmed that THI 52 inhibits both tumor necrosis factor- and iNOS mRNA expression in RAW 264.7 cells activated with LPS via regulation of NF-B activation (Kang et al., 2003). In the present study, we showed that THI 53, a newly synthesized isoquinoline alkaloid and a structural analog of THI 52, blocked NO production and iNOS protein induction in RAW264.7 macrophage cells and in lung tissues from mice after LPS stimulation. In addition, THI 53 inhibited LPS-mediated phosphorylations of Tyk2 and STAT-1, which are involved in iNOS expression. Furthermore, we demonstrated that LPS-phosphorylated JNK was also involved in iNOS induction; however, THI 53 did not inhibit the LPS-activated JNK pathway. Phosphorylated JNK peaked twice after LPS treatment, once at 1 h and later at 16 h, whereas phosphorylated STAT-1 peaked at 4 to 8 h. Therefore, we presume that LPS may activate Tyk2 and JNK, followed by activation of the JAK2/STAT-1 pathways; thereafter, JNK is phosphorylated again by activated STAT-1 (Fig. 9).

View larger version (19K): [in this window] [in a new window]   Fig. 9. Possible mechanisms by which THI 53 inhibits LPS-mediated inflammatory responses such as iNOS induction or NO production. LPS activates Tyk2 and JNK pathways, which are linked to the STAT-1 and NF-B pathways, resulting in iNOS induction. THI 53 can affect the IFN-/Tyk2/JAK2-STAT-1 and NF-B pathways but not the JNK pathway.

LPS is the major active agent in the pathogenesis of endotoxin-mediated shock. The binding of LPS to toll-like receptor 4 (TLR4) leads to the activation of monocytes and macrophages, which then release cytokines and NO. LPS activates both MyD88-dependent and MyD88-independent pathways, each of which leads to the activation of MAPKs and NF-B. In addition to the signaling cascade downstream of TLR4 (Akira, 2003), IFN- and IFN- are also involved in sensitivity to LPS. Deficiencies in the IFN- (Karaghiosoff et al., 2003) or IFN- receptors (Car et al., 1994) result in resistance to high-dose LPS challenge. Kamezaki et al. (2004) reported that the LPS-induced activations of NF-B and AP-1 were unaffected by the absence of Tyk2 and that IFN- and - signals activated by LPS challenge were severely affected in Tyk2-deficient mice. Tyk2 is a member of the JAK family and has been demonstrated to play a restricted role in IFN-/ signaling and to have an important role in interleukin-12 signaling (Karaghiosoff et al., 2000; Shimoda et al., 2000). Some IFN-/-induced biological activities, such as inhibition of the growth of bone marrow progenitor cells and NO production from macrophages after LPS stimulation, were abrogated in the absence of Tyk2. In addition, the role of STAT-1 in the induction by LPS of the iNOS gene in mouse macrophages has been reported (Gao et al., 1998). From these reports, we speculate that there may be two pathways related to LPS challenge: one via NF-B and the other via the link between Tyk2 and IFN. Our results also showed that THI 53 effectively inhibited LPS-induced iNOS expression, LPS-activated Tyk2, and STAT-1 phosphorylation, but its inhibitory effect on LPS-activated NF-B was relatively weak. In fact, Fig. 7 shows that LPS treatment induced IFN- production in a time-dependent manner, which is efficiently inhibited by treatment of THI 53. This may be because the LPS-mediated inflammatory response is mediated through not only the NF-B pathway but also the IFN-/Tyk2/JAK-2-STAT-1 pathway. Our result is supported by Jung et al. (2005), in which TLR4 agonists such as LPS induced NF-B activation and IFN- production in microglia, and neutralizing antibodies against IFN- attenuated TLR4-mediated microglial apoptosis. IFN- alone, however, did not induce a significant cell death.

Okugawa et al. (2003) reported that JAK2 regulated phosphorylation of JNK by LPS in RAW264.7 cells, and we showed that inhibition of JNK pathway with the specific JNK inhibitor SP600125 reduced phosphorylated STAT-1. Furthermore, we found that LPS activated the JNK and Tyk2-JAK2/STAT-1 pathways; consequently, phosphorylated STAT-1 reactivates JNK. The major function of the JAK family kinases is considered to be activation of STAT. Thus, from these reports, we can conclude that LPS-induced activation of JAK2 protein tyrosine kinase pathway in macrophages is central to mediation of inflammation through Tyk2/JAK2-STAT-1. In addition, we wish to highlight the role of JNK in LPS-induced inflammation by activation of STAT-1 phosphorylation or by affecting other transcription factors such as AP-1, even though JNK is not a point at which THI 53 acts to inhibit the LPS-mediated iNOS induction. We conclude that the ability of THI 53 to suppress NO production and iNOS expression by LPS is via regulation of the Tyk2 pathway linked to JAK2-STAT-1 and via NF-B activated by LPS. Thus, THI 53 is highly likely to be therapeutic in conditions in which up-regulation of iNOS is the main cause of health problems, such as septic shock.

	Acknowledgements

 
We thank Professor D. H. Lee, Sogang University, Seoul, Korea, for providing THI 53.

	Footnotes

 
This study was supported by the Korea Science and Engineering Foundation (R13-2005-005-01003-0).

H.J.K. and K.T. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.112052.

ABBREVIATIONS: iNOS, inducible nitric-oxide synthase; NO, nitric oxide; LPS, lipopolysaccharide; NF-B, nuclear factor-B; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; JNK, c-Jun N-terminal kinase; JAK, Janus protein tyrosine kinase; IB, inhibitor of NF-B; STAT, signal transducer and activator of transcription; Tyk2, tyrosine kinase 2; THI 53, 2-naphtylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; AG490, -cyano-(3,4-dihydroxy)-N-benzylcinnamide; AG126, -cyano-(3-hydroxy-4-nitro)cinnamonitrile; SB203580, [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole]; PD98029, ·; ECL, enhanced chemoluminescence; SP600125 (anthra[1,9-cd]pyrazole-6 (2H)-one); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; NOx, nitrite; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin; IFN, interferon; MAP, mitogen-activated protein; AP-1, activator protein-1; THI 52, 1-naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TLR4, Toll-like receptor 4; PD98029, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one.

¹ Current affiliation: Department of Pharmacology, College of Medicine, Yeungnam University, Daegu, Korea.

Address correspondence to: Dr. Ki Churl Chang, Department of Pharmacology, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, South Korea. E-mail: kcchang{at}gsnu.ac.kr

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