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J Biol Chem, Vol. 274, Issue 44, 31559-31564, October 29, 1999

Role of the Cyclic AMP-Protein Kinase A Pathway in Lipopolysaccharide-induced Nitric Oxide Synthase Expression in RAW 264.7 Macrophages
INVOLVEMENT OF CYCLOOXYGENASE-2^*

Ching-Chow Chen, Kuo-Tung Chiu, Yi-Tau Sun, and Wei-Chyuan Chen

From the Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The signaling pathway for lipopolysaccharide (LPS)-induced nitric oxide (NO) release in RAW 264.7 macrophages involves the protein kinase C and p38 activation pathways (Chen, C. C., Wang, J. K., and Lin, S. B. (1998) J. Immunol. 161, 6206-6214; Chen, C. C., and Wang, J. K. (1999) Mol. Pharmacol. 55, 481-488). In this study, the role of the cAMP-dependent protein kinase A (PKA) pathway was investigated. The PKA inhibitors, KT-5720 and H8, reduced LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression. The direct PKA activator, Bt₂cAMP, caused concentration-dependent NO release and iNOS expression, as confirmed by immunofluorescence studies. The intracellular cAMP concentration did not increase until after 6 h of LPS treatment. Two cAMP-elevating agents, forskolin and cholera toxin, potentiated the LPS-induced NO release and iNOS expression. Stimulation of cells with LPS or Bt₂cAMP for periods of 10 min to 24 h caused nuclear factor-B (NF-B) activation in the nuclei, as shown by detection of NF-B-specific DNA-protein binding. The PKA inhibitor, H8, inhibited the NF-B activation induced by 6- or 12-h treatment with LPS but not that induced after 1, 3, or 24 h. The cyclooxygenase-2 (COX-2) inhibitors, NS-398 and indomethacin, attenuated LPS-induced NO release, iNOS expression, and NF-B DNA-protein complex formation. LPS induced COX-2 expression in a time-dependent manner, and prostaglandin E₂ production was induced in parallel. These results suggest that 6 h of treatment with LPS increases intracellular cAMP levels via COX-2 induction and prostaglandin E₂ production, resulting in PKA activation, NF-B activation, iNOS expression, and NO production.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric oxide (NO)¹ has been identified as an important signaling molecule involved in regulating a wide range of biological activities in the neural, vascular, and immune systems (1). NO and its metabolites mediate a number of host defense functions mediated by activated macrophages, including antimicrobial and tumoricidal activity, implicated in the pathogenesis of tissue damage associated with acute and chronic inflammation (2, 3). Macrophages generate NO from the guanidino moiety of L-arginine via a reaction catalyzed by the inducible form of nitric oxide synthase (iNOS) (4). iNOS has been identified in a wide variety of cell types including macrophages, mesangial cells, vascular smooth muscle cells, keratinocytes, chondrocytes, osteoclasts, and hepatocytes and can be induced by many immune stimuli (1, 5). Changes in NO formation in iNOS-expressing cells usually correlate with similar changes in iNOS mRNA levels, indicating that a major part of iNOS regulation occurs at the transcription level. The promoter region of the iNOS gene contains several binding sites for transcriptional factors, such as nuclear factor-B (NF-B) and activator protein-1, as well as for various members of the CCAAT/enhancer-binding protein, activating transcription factor/cAMP-response element-binding protein, and Stat families of transcriptional factors (6). Of these, the proteins of the NF-B family appear to be essential for the enhanced iNOS gene expression seen in macrophages exposed to the active component of endotoxin, lipopolysaccharide (LPS) (7). In unstimulated cells, NF-B is retained in the cytoplasm by binding to IB but is released by signal induction and translocates to the nucleus, activating the responsive gene (8). In macrophages, iNOS induction by LPS requires initiation of gene expression and de novo protein synthesis over a period of several hours (9).

The intracellular signaling pathways by which LPS causes iNOS expression in macrophages involve a series of events resulting in the transmission of the signal from the plasma membrane through the cytoplasm to the nucleus, where iNOS gene expression is up-regulated. Previous studies have shown that LPS first binds to LPS-binding protein and then to membrane CD14 and that it also activates phosphatidylinositol-phospholipase C and phosphatidylcholine-PLC by tyrosine phosphorylation, thus causing PKC activation (10). Tyrosine phosphorylation also causes p38 activation (11). These phosphorylation processes result in stimulation of NF-B DNA-protein binding and the initiation of iNOS expression and NO release (10, 11). An increase in intracellular cAMP levels is an important intracellular signaling mechanism involved in the regulation of gene expression. Certain in vitro studies have shown that an increase in cAMP levels causes iNOS induction (12-14), whereas in other studies, increased cAMP levels caused a reduction in iNOS (15, 16). In the present study, we explored the intracellular signaling pathway for the LPS-induced increase in cAMP levels and its involvement in LPS-stimulated NO production in RAW 264.7 macrophages. The results show that, after 6 h of treatment, LPS can increase cAMP levels by induction of cyclooxygenase-2 (COX-2) and formation of prostaglandin E₂ (PGE₂), resulting in the activation of PKA and NF-B, iNOS expression, and NO production. The PKA activation pathway explored in this study had a delayed onset (6 h), whereas the previously reported PKC and p38 activation pathways have rapid onsets (10 min) (10, 11).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Affinity-purified rabbit polyclonal anti-iNOS antibody was obtained from Transduction Laboratories (Lexington, KY). Dulbecco's modified Eagle's medium, fetal calf serum, penicillin, and streptomycin were purchased from Life Technologies, Inc. The NF-B probe was from Santa Cruz Biotechnology (Santa Cruz, CA). LPS (from Escherichia coli serotype 0127:B8), forskolin, Bt₂cAMP, cholera toxin (CTX), sulfanilamide, and N-(1-naphthyl)-ethylenediamine were from Sigma. KT-5720 and NS-398 were from Calbiochem. H8 was from Seikagaku (Tokyo, Japan). T4 polynucleotide kinase was from New England Biolabs (Beverly, MA). Rabbit polyclonal anti-Cox-2 antibody was from Cayman Chemicals (Ann Arbor, MI). Poly(dI·dC), a cAMP enzyme immunoassay kit, horseradish peroxidase-labeled donkey anti-rabbit second antibody, and the ECL detection reagent were from Amersham Pharmacia Biotech. Reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was from Cappel (Aurora, OH).

Cell Culture-- RAW 264.7 cells, a murine macrophage cell line, were obtained from the ATCC and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in 12-well plates (nitrite assay, iNOS and COX-2 expression, and PGE₂ production), on 24-mm glass coverslips in 35-mm dishes (immunofluorescence staining), in 6-well plates (cAMP assay), or in 10-cm dishes (NF-B gel shift assay).

Determination of NO Concentration-- NO production in culture supernatants was assessed by measuring nitrite, its stable degradation product, using Griess reagent as described previously (10). The Dulbecco's modified Eagle's medium was changed to phenol red-free medium before the cells were stimulated for 24 h with 1 µg/ml LPS or 100 µM Bt₂cAMP. After stimulation, the supernatants were centrifuged and mixed with an equal volume of Griess reagent and then incubated for 10 min at room temperature before measuring the absorbance at 550 nm in a microplate reader. NaNO₂ was used as a standard. In pretreatment experiments, the cells were incubated for 30 min with KT-5720 or H8 (PKA inhibitors), with actinomycin C or cycloheximide (transcriptional or translational inhibitors, respectively), or with NS-398 or indomethacin (COX-2 inhibitors) before the addition of LPS or Bt₂cAMP.

Immunofluorescence Staining-- RAW cells grown on coverslips were treated for 24 h with LPS or Bt₂cAMP in growth medium and then rapidly washed with phosphate-buffered saline and fixed at room temperature for 10 min with 2% paraformaldehyde. After washing with phosphate-buffered saline, the cells were blocked for 15 min with 1% bovine serum albumin in TTBS (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) containing 0.1% Triton X-100, incubated with anti-iNOS antibody (1:100) for 1 h, washed extensively, and stained for 30 min with anti-rabbit IgG-fluorescein (1:2,000). After additional washes, the coverslips were mounted on glass slides using mounting medium (2% N-propyl gallate in 60% glycerol, 0.1 M phosphate-buffered saline, pH 8.0). Optical sections of the immunostained cells were observed and photographed using a Zeiss Axiovert inverted microscope equipped with the photo MicroGraph digitized integration system (MGDS).

Preparation of Cell Extracts and Western Blot Analysis of iNOS and COX-2-- Following treatment with LPS or Bt₂cAMP, with or without pretreatment with various inhibitors, the cells were harvested and collected. Cell lysates were prepared and subjected to SDS-polyacrylamide gel electrophoresis using 7.5% (iNOS) or 10% (COX-2) running gels as described previously (10). The proteins were transferred to nitrocellulose, and the membrane was incubated successively with 0.1% milk in TTBS at room temperature for 1 h, with rabbit antibody specific for iNOS or COX-2 for 1 h, and with horseradish peroxidase-labeled anti-rabbit antibody for 30 min. After each incubation, the membrane was washed extensively with TTBS. The immunoreactive band was detected using ECL detection reagent and developed with Hyperfilm-ECL.

Determination of Intracellular cAMP Concentrations-- After cells were treated with LPS for 1, 3, 6, 12, or 24 h or with CTX for 24 h or with forskolin for 10 min, the reaction was terminated by aspiration of the growth medium and addition of 0.1 N HCl. The cells were scraped into Eppendorff tubes and the suspensions were centrifuged; the supernatants were then neutralized with 10 N NaOH and assayed for cAMP levels using an enzyme immunoassay kit from Amersham Pharmacia Biotech.

Preparation of Nuclear Extracts and the Electrophoretic Mobility Shift Assay (EMSA)-- Control cells or H8, NS-398, or indomethacin-pretreated cells were treated with LPS or Bt₂cAMP for various amounts of time, and then nuclear extracts were prepared as described previously (10). A double-stranded oligonucleotide probe containing NF-B binding sequences was purchased (5'-AGTTGAGGGGACTTTCCCAGGGC-3', Santa Cruz Biotechnology) and end-labeled with [-³²P]ATP using T4 polynucleotide kinase. The nuclear extracts (6-10 µg) were incubated at 30 °C for 20 min with 1 ng of ³²P-labeled NF-B probe (40,000-60,000 cpm) in 10 µl of binding buffer containing 1 µg of poly(dI·dC), 15 mM HEPES, pH 7.6, 80 mM NaCl, 1 mM EGTA, 1 mM dithiothreitol, and 10% glycerol as described previously (10). DNA-nuclear protein complexes were separated from the DNA probe by electrophoresis on a native 6% polyacrylamide gel, and the gel was vacuum-dried and subjected to autoradiography using an intensifying screen at 80 °C. The quantitative data were obtained using a computing densitometer and ImageQuant software (Molecular Dynamics).

PGE₂ Production-- After treatment of cells with LPS for 1, 3, 6, 12, or 24 h, PGE₂ levels in the culture media were measured using an enzyme immunoassay kit from Amersham Pharmacia Biotech.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibitory Effect of PKA Inhibitors on LPS-induced NO Production and iNOS Expression and Effect of Bt₂cAMP-- To determine whether PKA was involved in the LPS-induced NO production, the PKA inhibitors, KT-5720 and H8, were used. When cells were pretreated for 30 min with 1 or 3 µM KT-5720 or with 30 or 50 µM H8, LPS-induced NO production (Fig. 1A) and iNOS expression (Fig. 1, B and C) were inhibited in a dose-dependent manner. The respective levels of inhibition were 31 or 62% for 1 or 3 µM KT-5720 and 35 or 65% for 30 or 50 µM H8.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of PKA inhibitors on LPS-induced nitrite release and iNOS expression in RAW 264.7 macrophages. A, cells were pretreated with 1 or 3 µM KT-5720 or 30 or 50 µM H8 for 30 min before incubation with 1 µg/ml of LPS for 24 h; the medium was then removed and analyzed for nitrite release. The results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05 compared with LPS alone. In iNOS expression studies (B and C), the cells used in the nitrite assay were subjected to electrophoresis and Western blotting using iNOS-specific antibody as described under "Experimental Procedures."

Because both LPS-induced NO production and iNOS expression were inhibited by KT-5720 or H8, indicating the involvement of the PKA pathway in the LPS effect, the direct PKA activator, Bt₂cAMP, was used. Exposure of RAW cells to Bt₂cAMP for 24 h resulted in both nitrite production (Fig. 2A) and iNOS expression (Fig. 2B) in a dose-dependent manner, with maximum nitrite release (37.2 ± 0.2 nmol/10⁶cells/24 h; n = 3) being obtained using 100 µM Bt₂cAMP (Fig. 2A). In the following NO release experiment, the cells were treated with 100 µM Bt₂cAMP for 24 h. Under these conditions, either the transcriptional inhibitor, actinomycin D, or the translational inhibitor, cycloheximide, inhibited the Bt₂cAMP-induced nitrite production and iNOS expression (data not shown). Bt₂cAMP-induced iNOS expression was also demonstrated by immunofluorescence staining; as shown in Fig. 3, iNOS expression was not seen in the basal state (Fig. 3B) but was induced in the cytoplasm after treatment with either LPS (Fig. 3D) or Bt₂cAMP (Fig. 3F).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent Bt2cAMP-induced stimulation of nitrite release and iNOS expression in RAW 264.7 macrophages. Cells were incubated at 37 °C with various concentrations of Bt2cAMP for 24 h (A); the medium was then removed and analyzed for nitrite. The results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. B, cell lysates from the nitrite assay were subjected to electrophoresis and Western blotting using iNOS-specific antibody as described under "Experimental Procedures."

View larger version (119K): [in this window] [in a new window]   Fig. 3.   iNOS is located in the cytoplasm. Immunofluorescent staining of RAW 264.7 macrophages with affinity-purified iNOS antibody (1:100) is shown. Cells were fixed and stained as described under "Experimental Procedures." A and B, control; C and D, after 24-h treatment with LPS; E and F, Bt2cAMP. The same field from a representative culture was viewed by indirect immunofluorescence for iNOS staining (B, D, and F) and by phase-contrast microscopy (A, C, and E). The bar represents 200 µm.

Because the PKA pathway had been shown to be involved in LPS-induced NO production and because Bt₂cAMP stimulated NO production, intracellular cAMP levels were measured following LPS treatment. When cells were treated with 1 µg/ml LPS for various times, cAMP levels increased slightly after 3 h (121% of basal), reached a maximum at 6 h (243% of basal), and then declined (161% of basal after 12 h) (Fig. 4A). Following treatment of cells with 1 µg/ml CTX for 24 h or with 100 µM forskolin for 10 min, cAMP levels increased to 292 and 202% of basal, respectively (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Changes in the intracellular cAMP concentration in RAW 264.7 macrophages following LPS treatment. RAW 264.7 macrophages were treated with 1 µg/ml LPS for the indicated time intervals (A) or with 1,000 ng/ml CTX for 24 h or 100 µM forskolin for 10 min (B). The intracellular cAMP concentration was measured as described under "Experimental Procedures." The results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. The cAMP concentration in the basal state was 1.88 ± 0.2 pmol/mg protein (n = 3). *, p < 0.05 compared with the basal level.

Effect of Cyclic AMP-elevating Agents on LPS-induced NO Production and iNOS Expression-- Forskolin or CTX themselves had no effect on nitrite production but enhanced the LPS-stimulated increase in nitrite production and iNOS expression (Fig. 5, A-C). Ten or 30 µM forskolin, which had no effect on cAMP levels in RAW cells (data not shown), also had no effect on LPS-induced NO production and iNOS expression, whereas 100 µM forskolin, which increased cAMP levels 2-fold (Fig. 4B), also increased LPS-induced NO production and iNOS expression (Fig. 5, A and B). CTX potentiated the LPS effect over the range of 10-1,000 ng/ml (Fig. 5, A and C). A similar parallel enhancement of the LPS-stimulated increase in NO production and iNOS expression was seen using Bt₂cAMP (Fig. 6).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Effect of forskolin or CTX on LPS-induced nitrite release and iNOS expression. A, cells were incubated for 24 h with the indicated concentrations of forskolin or CTX plus 1 µg/ml LPS; the medium was then removed and analyzed for nitrite. The results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. For iNOS expression studies (B and C), cell lysates from the nitrite assay were subjected to electrophoresis and Western blotting using iNOS-specific antibody as described under "Experimental Procedures." *, p < 0.05 compared with LPS alone.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Effect of Bt2cAMP on LPS-induced nitrite release and iNOS expression. A, the cells were incubated for 24 h with the indicated concentrations of Bt2cAMP plus 1 µg/ml LPS; the medium was then removed and analyzed for nitrite. The results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. For iNOS expression studies (B), cell lysates from the nitrite assay were subjected to electrophoresis and Western blotting using iNOS-specific antibody as described under "Experimental Procedures." *, p < 0.05 compared with LPS alone.

Kinetics of NF-- B-specific DNA-Protein Complex Formation in Nuclei Stimulated with LPS or Bt₂cAMP and the Inhibitory Effect of H8The time course of NF-B activation after treatment with 1 µg/ml LPS or 100 µM Bt₂cAMP was studied. Nuclear extracts prepared from RAW cells were assayed for activated NF-B in an EMSA. As shown in Fig. 7A, NF-B-specific DNA-protein complex formation increased after treatment with LPS for 1, 3, 6, 12, or 24 h. When cells were exposed to 100 µM Bt₂cAMP for 10 min, increased formation of the NF-B-specific DNA-protein complex was also seen (Fig. 7B), whereas after treatment with Bt₂cAMP for 3 or 24 h, the intensity of these complexes decreased but was still stronger than in resting cells (Fig. 7B). The bands in the upper and lower complex were previously identified as the p65/p50 heterodimer and p50/p50 homodimer, respectively (10). After pretreatment of the cells for 30 min with 50 µM H8, the activation of NF-B-specific DNA-protein complex formation induced following 1, 3, or 24 h of LPS treatment was not affected, whereas that induced following 6 or 12 h of LPS treatment was inhibited, the extent of inhibition being 46% and 26%, respectively (Fig. 8A). The activation of NF-B-specific DNA complex formation seen after 6 h of LPS treatment was inhibited by H8 in a dose-dependent manner (30, 50, and 75 µM) (Fig. 8B).

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Kinetics of NF-B-specific DNA-protein complex formation in nuclear extracts of RAW 264.7 macrophages stimulated with LPS or Bt2cAMP. Cells were treated with 1 µg/ml LPS for 1, 3, 6, 12, or 24 h (A) or with 100 µM Bt2cAMP for 10 min or 1, 3, or 24 h (B); then nuclear extracts were prepared and NF-B DNA-protein binding activity in the extracts was determined by EMSA as described under "Experimental Procedures."

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Effect of H8 on LPS-induced NF-B DNA-protein complex formation in nuclear extracts of RAW 264.7 macrophages. Cells were pretreated with 50 µM H8 for 30 min before incubation with 1 µg/ml LPS for various time intervals (A) or pretreated with 30, 50, or 75 µM H8 for 30 min before incubation with 1 µg/ml LPS for 6 h (B). NF-B DNA-protein binding activity in nuclear extracts was determined by EMSA as described under "Experimental Procedures." Densitometric analyses (A) are expressed as the mean ± S.E. of seven independent experiments (B). *, p < 0.05 compared with LPS alone.

Inhibitory Effect of COX-2 Inhibitors on LPS-induced NO Production, iNOS Expression, and NF-- B DNA-Protein Complex Formation and Induction of COX-2 by LPSThe fact that the cAMP-PKA pathway had been shown to be involved in LPS-induced NO production and iNOS expression, that LPS caused an increase in cAMP levels after 6 h of treatment, and that H8 inhibited LPS-induced NF-B-specific DNA-protein complex formation following 6 h of treatment indicated that the cAMP formation was a delayed response. To determine whether the increased cAMP levels were because of PG formation produced as a result of COX-2 expression, the COX-2 inhibitors, NS-398 and indomethacin, were used. As shown in Fig. 9A, LPS-induced NO production and iNOS expression were inhibited by 10 µM NS-398 or indomethacin. The inhibition of NO production was 35 and 38%, respectively. NF-B DNA-protein complex formation induced after 6 h of treatment with LPS was also inhibited by 10 µM NS-398 or indomethacin (Fig. 9B). LPS-elicited COX-2 expression was also examined. Exposure of RAW cells to 1 µg/ml LPS resulted in a time-dependent COX-2 expression; no expression was seen after 1 h of treatment, but expression was observed at 3 h and continued to increase to 24 h (Fig. 10A). Fig. 10B shows the time-dependent production of PGE₂ in response to 1 µg/ml LPS. The basal release of PGE₂ was 0.838 pg/µg of total protein, whereas after treatment with LPS for 1, 3, 6, 12, or 24 h, this rose to 1.82, 9.23, 37.8, 71.2, and 112.3 pg/µg protein, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Effect of NS-398 or indomethacin on LPS-induced nitrite release, iNOS expression (A), and NF-B DNA-protein complex formation (B). A, the cells were pretreated with 10 µM NS-398 (NS) or indomethacin (Ind) for 30 min before incubation with 1 µg/ml LPS for 24 h; the medium was then removed and analyzed for nitrite. The results are expressed as the mean ± S.E. of three independent experiments performed in duplicate. *, p < 0.05 compared with LPS alone. In the iNOS expression studies, the cells from the nitrite assay were subjected to electrophoresis and Western blotting using iNOS-specific antibody as described under "Experimental Procedures." B, cells were pretreated with 10 µM NS-398 or indomethacin for 30 min before incubation with 1 µg/ml LPS for 6 h. NF-B DNA-protein binding activity in nuclear extracts was determined as described under "Experimental Procedures."

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Time-dependent LPS-induced stimulation of COX-2 expression and PGE2 production. Cells were incubated at 37 °C with 1 µg/ml LPS for various time intervals; the medium was then removed and analyzed for PGE2 production by enzyme-linked immunosorbent assay (B), while the cells were lysed and subjected to Western blotting using anti-COX-2 antibody as described under "Experimental Procedures" (A). B, the results are expressed as the mean ± S.E. of one typical experiment performed in duplicate; similar results were obtained in three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The effects of cAMP on iNOS expression have been of increasing interest since the first report that cAMP-elevating agents induced iNOS in cultured vascular smooth muscle cells and that this induction was synergistic with that elicited by inflammatory cytokines (17). Similar effects have also been seen in renal mesangial cells (13, 18) and brown adipocytes (19). Although cAMP alone does not induce iNOS in unstimulated cardiac myocytes, it augments iNOS induction in interleukin-1-stimulated cells (20). In 3T3 fibroblasts, different signaling pathways, including elevation of cAMP, lead to the induction of iNOS by NF-B mediation (21). In contrast, elevation of cellular cAMP levels has been shown to down-regulate iNOS in LPS- or cytokine-activated astrocytes, hepatocytes, or Kuffer cells (15, 16, 22). In RAW 264.7 macrophages, NF-B/Rel is positively regulated by the cAMP cascade, thus helping to initiate iNOS gene expression in response to LPS stimulation, and inhibition of adenylate cyclase attenuates LPS-induced activation of iNOS gene expression (12), indicating that, in inducing iNOS expression in these cells, LPS acts by increasing cAMP levels. In the present study, the PKA inhibitors, KT-5720 and H8, inhibited LPS-induced NO release and iNOS expression in a dose-dependent manner, indicating that the LPS effect is indeed related to the cAMP-PKA activation pathway. The cAMP analogue, Bt₂cAMP, also increased NO release and iNOS expression; immunofluorescence staining also demonstrated iNOS expression in the cytoplasm. LPS caused a time-dependent increase in cAMP levels that was maximal with 6-h treatment and then declined. In J774 macrophages, the increase in cAMP levels occurs after 6 h of treatment with LPS (23), and PKA is involved in the LPS-induced activation of junB and NF-B (24). As previously reported (10, 11), activation of NF-B-specific DNA-protein complex formation was seen after 10-min to 24-h treatment with LPS, and a similar time course of activation of this complex was seen using Bt₂cAMP (Fig. 7B). In contrast with the inhibition seen using PKC or p38 inhibitors (10, 11), when cells were pretreated for 30 min with H8, the NF-B-specific DNA-protein complex formation seen after 1 h of LPS treatment was unaffected (Fig. 8A). However, the complex formation seen after 6 h of LPS treatment was inhibited by H8, thus correlating with the maximal cAMP level seen after 6 h of treatment (Fig. 4A). Thus, in contrast with the PKC and p38 activation pathways, which are rapid (10 min) (10, 11), the cAMP-PKA activation pathway is a delayed event in LPS-induced NF-B activation. cAMP may modulate NF-B activation and iNOS transcription via cAMP-dependent PKA-mediated phosphorylation of the cAMP response element-binding protein (25). In RAW cells, the rapid activation of NF-B by PKC and p38 pathways, together with the delayed activation of NF-B by the cAMP-PKA pathway, contributes to the LPS-induced iNOS expression and NO release.

Because the cAMP-PKA activation pathway is a much delayed event (6 h) in LPS-induced NF-B activation, the mechanism involved in LPS-induced increase in cAMP levels was further explored. Both NS-398 and indomethacin had an inhibitory effect on LPS-induced NO release, iNOS expression, and NF-B activation, indicating the involvement of COX-2 expression in LPS-stimulated NO release. When the effect of various periods of LPS treatment was studied, no COX-2 expression was seen in unstimulated cells or after 1 h of treatment, but COX-2 expression was seen after 3 h of treatment and continued to increase to 24 h. COX is a key enzyme in prostanoid synthesis, as it catalyzes the conversion of arachidonic acid to PGH2, which is then metabolized by one or more terminal synthases to a variety of active prostanoids (26). It possesses both fatty acid cyclooxygenase activity and PG hydroperoxidase activity (converting PGG2 to PGH2). COX-2 is a COX isoform that is induced in a number of cells by proinflammatory stimuli and is thought to contribute to the generation of prostanoids at sites of inflammation (27, 28); it is considered to be responsible for high production of PGs (29). PGE₂ production following LPS treatment was also measured, and the increases after 1, 3, 6, 12, or 24 h of treatment were, respectively, 2-, 11-, 45-, 85-, and 134-fold of basal levels, paralleling the increase in COX-2 expression. PGE₂ acts via receptor-mediated generation of cAMP and activation of PKA (30). As seen in a study on the effects of interleukin-1 on human bronchial smooth muscle cells (31), in the present study, induction of PGE₂ synthesis precedes the increase in cAMP, and PGE₂ acts as an autocrine factor for adenylate cyclase activation. LPS-induced tumor cell killing in EC4 cells is also because of increased levels of cAMP, and this effect is inhibited by indomethacin (32). In peritoneal macrophages, LPS is reported to act via PGE₂ to increase cAMP levels (33).

In summary, in RAW 264.7 cells, LPS increases iNOS expression via a prostanoid- and cAMP-dependent pathway, and this is followed by PKA activation of NF-B. The increase in PGE₂ is because of COX-2 expression. This effect has a more delayed onset (6 h) compared with those involving the PKC and p38 activation pathways (10 min). A schematic representation of the signaling pathway for the LPS-induced NO release in RAW cells is shown in Fig. 11.

View larger version (31K): [in this window] [in a new window]   Fig. 11.   Schematic representation of the signaling pathway for LPS-induced NO release in RAW 264.7 macrophages. LPS binds to LPS-binding protein (LBP); the complex then binds to membrane CD14 (mCD14) and activates phosphatidylinositol-phospholipase C (PI-PLC) and phosphatidylcholine-PLC (PC-PLC) via tyrosine phosphorylation to induce PKC activation. Tyrosine phosphorylation also induces p38 activation. These two pathways have rapid onset (10 min). LPS also induces COX-2 expression, PGE2 formation, cAMP production, and PKA activation. This pathway has a more delayed onset (6 h). These three pathways result in stimulation of NF-B-specific DNA-protein binding, initiating iNOS expression and NO release. DAG, diacylglycerol; TK, tyrosine kinase; PIP2, phosphatidylinositol bisphosphate; IP3, inositol triphosphate; MAPK, mitogen-activated protein kinase.

	FOOTNOTES

^* This work was supported by a research grant from the National Science Council of Taiwan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd., 1st Section, Taipei 10018, Taiwan. Tel.: 886-2-23970800 (ext. 8321); Fax: 886-2-23947833; E-mail: ccchen@ha.mc.ntu.edu.tw.

	ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; PKC, protein kinase C; Bt₂cAMP, dibutyryl cyclic AMP; PKA, protein kinase A; NF-B, nuclear factor B; COX, cyclooxygenase; PG, prostaglandin; EMSA, electrophoretic mobility shift assay; CTX, cholera toxin; TTBS, Tris-buffered saline/Tween 20.

	REFERENCES

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