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Negative Regulation of LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by AP-1 in Macrophage Cell Line J774A.1
Biochemical and Biophysical Research Communications 289, 1031–1038 (2001) doi:10.1006/bbrc.2001.6123, available online at http://www.idealibrary.com on
Negative Regulation of LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by AP-1 in Macrophage Cell Line J774A.1 Takako Kizaki,* ,1 Kenji Suzuki,* Yoshiaki Hitomi,* Kazuya Iwabuchi,† Kazunori Onoe´,† Shuko Haga,‡ Hitoshi Ishida,§ Tomomi Ookawara, ¶ Keiichiro Suzuki, ¶ and Hideki Ohno* *Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, Mitaka, Japan; †Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; ‡Institute of Health and Sport Sciences, University of Tsukuba, Tsukuba, Japan; §Department of Internal Medicine III, Kyorin University, School of Medicine, Mitaka, Japan; and ¶ Department of Biochemistry, Hyogo University of Medicine, Nishinomiya, Japan
Received November 22, 2001
The level of NOS II mRNA was markedly increased during 24 h lipopolysaccharide (LPS) stimulation, but showed no further increase thereafter. On the other hand, the level of NOS II mRNA in J774A.1 cells transfected with an expression vector containing the rat csk cDNA (J.Csk) was significantly increased during 3 h LPS stimulation, but rather decreased thereafter. Although no significant difference was observed in the activation of NF-B by LPS among parental J774A.1, J774A.1 transfected with promoterless vector (J.pBK), and J.Csk cells, activity of c-Jun N-terminal kinase (JNK) and nuclear translocation of nuclear factor activator protein-1 (AP-1) were markedly upregulated in the J.Csk cells. Then luciferase reporter vectors containing NOS II promoter with mutations in two AP-1like sites (U site, ⴚ1126⬃ⴚ1120; L site, ⴚ524⬃ⴚ518) were transiently transfected in J774A.1 cells. The promoter activity following LPS stimulation for 24 h was significantly increased by mutation at the L site, but not by mutation at the U site, suggesting that NOS II expression is negatively regulated, at least in part, through the AP-1-like L site in response to LPS. © 2001 Elsevier Science
Key Words: macrophage; NOS II; negative regulation; AP-1.
Nitric oxide (NO) is a free radical produced from the catalytic oxidation of the guanidino group of L-arginine by nitric oxide synthase (NOS). Inducible NOS (NOS 1
To whom correspondence and reprint requests should be addressed at Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo 181-8611, Japan. Fax: ⫹81-422-44-4427. E-mail: [email protected].
II) is not generally present in resting cells but is induced by various stimuli, such as bacterial lipopolysaccharide (LPS), tumour necrosis factor (TNF), interluekin-1␥, and interferon-␥. NO has been shown to have a number of important biological functions, including tumor cell killing and host defense against intracellular pathogens. On the other hand, increased expression of NOS II is known to be associated with disorders as diverse as septic and hemorrhagic shock, rheumatoid arthritis, and chronic infections (1–5). Thus, NOS II gene expression must be tightly controlled not only by positive but also by negative regulation. Although considerable advances have been made in elucidating the intracellular signals essential for positive signaling in NOS II expression (6 –12), knowledge of the specific signal transduction components that negatively regulate NOS II expression is not fully understood. Protein tyrosine kinases of src-family (src-PTK) were expressed in various hematolymphoid cells in a lineage specific manner. The src-PTK are associated with surface receptor molecules specifically expressed on each cell lineage, and play essential roles in intracellular transduction of signals initiated at the level of surface molecules that lack catalytic domains. Thus, the srcPTK may be involved in activation, proliferation, differentiation, and transformation of the cells (13, 14). The predominant Src family kinases expressed in macrophages, Hck, Fgr, and Lyn, have been implicated in a number of studies as being the initial signal transducers for tyrosine phosphorylation events after LPS stimulation (15–18). The activity of src-PTK is negatively regulated by a key enzyme, C-terminal Src kinase (Csk) (20 –22). Csk inactivates src-PTK by phosphorylating tyrosine at the C-terminal regulatory site (Tyr-
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527) (19). Indeed, the targeted disruption of the csk gene caused constitutive activation of src-PTK (23, 24). On the contrary, overexpression of Csk in some cell lines repressed cellular signaling mediation of src-PTK (25, 26), suggesting that a balance between src-PTK and Csk plays distinct roles in positive/negative regulation of signal transduction cascade initiated through srk-PTK. We have previously demonstrated that overexpression of Csk downregulates the production of interleukin (IL)-1a, IL-6, TNF-␣, and NO following stimulation with LPS in a macrophage cell line, J774A.1 (27). Thus, in the present study, the mechanisms of negative regulation of NOS II expression in responses to LPS were examined by analyzing effects of overexpression of Csk on the signal transduction cascade in J774A.1 cells. MATERIALS AND METHODS Electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared as previously described (27). The NF-B oligonucleotide probe (5⬘-AGT TGA GGG GAC TTT CCC AGG-3⬘) was purchased from Promega (Madison, WI) and the AP-1 oligonucleotide probe (5⬘-CGC TTG ATG AGT CAG CCG GAA-3⬘) were purchased from Promega Co. (Tokyo, Japan). Each probe was labeled with [␥- 32P]ATP using T4 polynucleotide kinase (Bio Labs) and purified in Microspin columns (Pharmacia Biotech, Uppsala, Sweden). For EMSA 1 g nuclear proteins were preincubated with EMSA buffer [10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl 2, 0.5 mM DTT, 0.5 mM EDTA, 4% glycerol, 1 g poly(dI– dC)] at room temperature for 20 min and then incubated with radiolabeled oligonucleotide probe for 5 min. The mixture was applied to a 7% polyacrylamide gel that had previously been electrophoresed at 100 V for 30 min. Gel was run at 100 V in TGE (5 mM Tris–HCl, pH 8.3, 38 mM glycine, 0.1 mM EDTA) followed by transference to 3MM Whatman paper, drying under vacuum at 80°C for 30 min, and quantification of the band intensities was then estimated according to an autoradiograph or analysis with a BAS 2000 imaging system (Fuji Photo Film Co. Ltd., Kanagawa, Japan). To identify the protein components of the NF-B complex or AP-1 complex, 1 l of specific polyclonal antibodies to p50 or p65 for NF-B or to c-Jun, Jun B, or Jun D for AP-1 (Santa Cruz Biotechnology) were added to the nuclear extracts, and the mixture was then incubated at 4°C for 1 h before the radiolabeled oligonucleotide probe was added. Oligonucleotide probes containing either two AP-1-like motif in 1.6 kb NOS II promoter (5⬘-CGC TTG ATC AAT CAG CCG GAA-3⬘ and 5⬘-CGC TTG ATC ACT CAG CCG GAA-3⬘) were used as competitors. Western blotting analysis. Nuclear proteins (20 g) were boiled in loading buffer (Tris–HCl, pH 6.8, 2% SDS, 5% glycerol, and 10% 2-mercaptoethanol), separated in 10% SDS–PAGE, and transferred to polyvinylidene difluoride membrane (Applied Biosystems, Foster City, CA). Membranes were blocked with 3% nonfat dried milk in Tris-buffered saline (TBS) for 1 h. Primary antibodies against c-Jun (Santa Cruz Biotechnology, Inc.) was applied at appropriate dilutions. After washing three times in TBS (25 mM Tris–HCl, 500 mM NaCl) containing 0.1% Tween 20 (TBS-T), secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, DAKO Japan, Kyoto, Japan] were applied at appropriate dilutions. Membranes were washed in TBS-T three times and the immunoreactivity was visualized with an enhanced chemiluminescence reagents (ECL, Amersham–Pharmacia Biotech Ltd. Tokyo, Japan). Protein kinase assay for JNK. Cell were stimulated according to experimental protocols and lysed using lysis buffer containing 10 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM PMSF, 1 mM sodium
orthvanadate, 2 g/ml aprotinin, 50 mM NaF, 1% Nonidet P-40. Cell lysates were subjected to centrifugation at 12,000g for 10 min at 4°C. The soluble fraction was incubated with Abs against JNK at 4°C for 1 h. After the addition of 20 l protein A/G–Sephosrose 4B, the reaction mixtures were incubated at 4°C for 2 h and then subjected to microcentrifugation. The beads were washed three times with buffer A, then once with kinase buffer containing 10 mM Hepes, pH 7.4, 10 mM MgCl 2, 3 mM MnCl 2, and incubated with kinase buffer containing 1 g of GST– c-jun and 370 kBq [␥- 32P]ATP (NENTM Life Science Products, Inc., Wilmington, DE) at 30°C for 30 min. Thereafter, the beads were boiled for 5 min and applied to 9% SDS– polyacrylamide gel electrophoresis. The gels were dried, exposed for 30 min, and analyzed with BAS 2000 imaging system (Fuji Photo Film Co. Ltd., Kanagawa, Japan). Northern analysis. The expression NOS II mRNA was determined by Northern analysis. The RNA was extracted with TRIzol Reagent (Life Technologies). Total RNA (25 g) was electrophoresed under denaturing conditions through a 1% (w/v) agarose– formaldehyde gel and then transferred to Nyrone membranes. The blots were hybridized with 32P-labeled NOS II probes and analyzed with BAS 2000 imaging system. Generation of NOS II luciferase reporter constructs. The fulllength murine NOS II promoter fragment (bp ⫺1580 to ⫹159) were cloned into the pGL3-enhancer luciferase reporter gene vector (Promega, Madison, WI) (pGL3-NOS IIwild). Point mutations of the two AP-1-like sequences [upper site (Umut) and lower site (Lmut)] were introduced by site-directed mutagenesis via overlap extension using PCR and then subcloned into the pGL3-enhancer vector (pGL3-NOS IIUmut, pGL3-NOS IILmut, and pGL3-NOS IIU ⫹ Lmut). All plasmids were checked for accuracy by sequencing. Transient transfection and luciferase assay. J774 A.1 cells were transfected using LipofectAMINE Reagent (Life Technologies, Inc.) with constructs containing the luciferase reporter gene. At 80% confluency, after washing in serum-free medium, cells were incubated with 0.4 g pGL3-enhancer construct, 0.04 g pRL-SV, and 4 l of LipofectAMINE Reagent/well (12-well dishes) for 3 h at 37°C. After 24 h, the culture medium was replaced with fresh medium containing 1 g/ml LPS. Cell were harvested 24 h later, and luciferase activity was determined using a dual luciferase assay system kit (Promega). Briefly cells were washed twice in phosphate-buffered saline (PBS) and lysed by adding 100 l of a 1⫻ lysis buffer. After 15 min at room temperature, the lysate was removed from the plate and centrifuged at 12,000g. To 10 l of the supernatant, 100 l of a luciferase assay reagent was added, and luciferase activity was immediately measured using a BLR-301 luminescence reader (ALOKA). Firefly luciferase activity was normalized for Renilla luciferase activity. Cell lysates were analyzed for protein content using the BCA protein assay reagent (Pierce). Luminescence units (firefly luciferase activity/Renilla luciferase activity) were normalized for total protein content.
RESULTS AND DISCUSSION Time Course of NOS II mRNA Expression and NOS II Promoter Activity Following LPS Stimulation The time course study showed that the level of the NOS II mRNA in J774A.1 cells was markedly increased during 24 h of LPS stimulation, but was not further increased thereafter (Fig. 1), thereby suggesting that the transcriptional rate of NOS II mRNA changed during the LPS stimulation. To investigate whether the expression of NOS II mRNA was changed at the transcriptional level, J774A.1 cells were transiently transfected with a luciferase reporter gene con-
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II expression was accelerated in Csk cells under the LPS stimulation. Effects of Csk Overexpression on the Activation of NF-B Following LPS Stimulation
FIG. 1. NOS II mRNA expression in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) Time course of NOS II mRNA expression was analyzed by Northern blotting analysis. Two separate experiments were carried out, and representative result is shown. (B) Relative NOS II mRNA expression shown in A was analyzed by densitometry.
struct containing 1.6 kb murine NOS II promoter. As shown in Fig. 2, luciferase activity was significantly reduced in the cells stimulated with LPS for 24 h compared with that in cells stimulated with LPS for 6 h. The promoterless vector containing the luciferase gene was unaffected by LPS treatment, confirming that these effects were specific to the murine NOS II promoter. These findings suggest that the transcription of NOS II mRNA was rapidly induced by LPS stimulation but was downregulated by prolonged LPS stimulation. In our previous study, we demonstrated that production of NO following LPS stimulation for 48 h was markedly reduced in J774A.1 cells transfected with expression vector containing rat csk cDNA (Csk) compared with those in parental J774A.1 or J774A.1 cells transfected with vector alone (pBK) (27). Thus, time course studies of the level of NOS II mRNA in Csk and pBK cells were performed. As shown in Fig. 1, the level of the NOS II mRNA in pBK cells was markedly increased during 24 h of LPS stimulation as well as in J774A.1 cells. On the other hand, NOS II mRNA was rapidly increased in J.Csk cells as in J774A.1 and J.pBK cells following LPS stimulation for 3 h, but was rather decreased thereafter. It seemed, therefore, likely that the negative transduction cascade for NOS
Exposure of macrophages to bacterial LPS initiates a signal transduction cascade that leads to increased production of NO, secretion of proinflammatory cytokines, and acquisition of enhanced bactericidal/ tumoricidal activity. Therefore, regulation of this signaling pathway is critical for controlling the initial phases of the immune response to foreign organisms. The transcriptional regulation of NOS II has been extensively characterized. Among a number of signaling pathways which have been implicated in LPS responses in macrophages, the most rapidly induced response is activation of nuclear factor-B (NF-B) (28, 29). Two NF-B sites, one located in the region I between positions ⫺48 and ⫺209 and another located in the region II between positions ⫺913 and ⫺1029 in murine NOS II promoter, are necessary for NOS II expression by LPS stimulation. Thus, the time course of the activation of NF-B after treatment with LPS was evaluated by an electrophoretic mobility gel shift assay (EMSA). Since protein components of NF-B are encoded by a set of genes called “immediate early genes” whose transcription is rapidly induced, nuclear extracts were prepared from J774A.1, J.pBK, and J.Csk stimulated with LPS for 1, 2, 5, 10, and 15 min. No significant difference was observed in NF-B spe-
FIG. 2. NOS II promoter activity in J774A.1 cells following LPS stimulation. J774A.1 cells were transiently transfected with luciferase reporter vector alone (pGL3) or with the vector containing 1.6 kb NOS II promoter (pGL3-NOS promoter). Luciferase activities were analyzed after stimulation with LPS for 6, 12, and 24 h. *P ⬍ 0.05.
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FIG. 3. NF-B activities in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) J774A.1, J.pBK, or J.Csk cells were stimulated with LPS for 1, 2, 5, 10, or 15 min, and activation of NF-B was analyzed by EMSA. (B) J774A.1, J.pBK, or J.Csk cells were stimulated with LPS for 0.5, 1, 2, or 3 h, and activation of NF-B was analyzed by EMSA.
cific DNA-protein complex formation among the three cells (Fig. 3A). Then, more late responses of NF-B to LPS stimulation were analyzed. However, we could not find any distinct difference in the activation of NF-B among the three cells during 3 h LPS stimulation (Fig. 3B). In addition, after 24 h of LPS stimulation, the levels of activated NF-B was dwindled in all three cells (data not shown). Therefore, it was suggested that the activation of the major mediator, NF-B, in LPS signal transduction occurs normally in the J.Csk cells as well as in the J774 or J.pBK cells. Antibodies against p65 partially reduced the band of DNA–protein complexes and produced one supershift (data not shown). On the other hand, all DNA-protein complexes were supershifted by antibodies against p50 and formation of two supershifted bands were detected (data not shown), thus suggesting that the proteins binding to NF-B were p65/p50 heterodimer and p50/p50 homodimer. Effects of Csk Overexpression on the Activation of Jun N-Terminal Kinase Cascade by LPS Stimulation Mitogen-activated protein kinases (MAPK) are comprised of three principal family members: c-jun
N-terminal kinases (JNK)/stress-activated protein kinases, extracellular signal-regulated kinases, and p38 MAPK and have been shown to activate a number of transcription factors. Among them, activator protein-1 (AP-1) is ubiquitous transcription factors and pleiotropic regulators of the inducible expression of many genes that encode proteins involved in the modulation of inflammatory and host defense processes in eukaryotic cells (30). The activities of AP-1 components are modulated by phosphorylation. This form of posttranslational control is best understood for the phosphorylation of c-Jun by the JNK. Then, the time course study of JNK activity after stimulation of LPS in J774A.1, J.pBK, and J.Csk cells was performed using GST– cJun as a substrate. JNK activity was detected in J774A.1and J.pBK cells at 1 h and 3 h of LPS stimulation, respectively (Fig. 4A). The activity was the highest at 6 h of LPS treatment and then decreased with incubation in both cells. On the other hand, JNK activity was observed in J.Csk cells even before LPS stimulation and was further enhanced by LPS stimulation. Then the nuclear translocation of c-Jun was analyzed in the three cells after stimulation of LPS. The level of nuclear translocation of c-Jun in J.Csk was
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FIG. 4. Activation of JNK cascade in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) Time course of JNK activity in J774A.1, J.pBK, and J.Csk cells after stimulation of LPS was investigated using GST-c-Jun as a substrate. (B) Nuclear extract was prepared from the three cells stimulated with LPS for 0, 24, and 48 h and subjected to Western blot analysis with anti-c-Jun antibodies.
markedly higher than that in J774A.1 or J.pBK cells (Fig. 4B), consisting with the results shown in Fig. 4A. We further analyzed the activation of AP-1 in J774A.1, J.pBK, and J.Csk cells following stimulation with LPS by EMSA. In both J774A.1 and J.pBK cells, activation of AP-1 was observed after 12 h of LPS stimulation (Fig. 5A). Meanwhile, gel shift band was observed in J.Csk cells after 1 h of LPS stimulation and was further increased with stimulation. To identify proteins binding to AP-1 motif, antibodies against Jun related proteins (c-Jun, Jun B, or Jun D) were added to the mixture of nuclear extract and radiolabeled AP-1 probe. As shown in Fig. 5B, the intensity of shifted band observed in the mixture of nuclear extract and radiolabeled AP-1 probe became faint by addition of anti-c-Jun antibodies (Fig. 5B, lane 2), although supershifted band was not observed. On the other hand, supershifted bands were observed in the presence of either anti-JunB or anti-JunD antibodies (Fig. 5B, lanes 3 and 4). Likewise, in the supershift experiment using the mixture of the nuclear extract from J.Csk cells stimulated with LPS for 6 h and radiolabeled AP-1 probe, anti-c-Jun antibodies decreased the intensity of the shifted band, and either anti-Jun B or antiJun D antibody formed a supershift band (Fig. 5B, lanes 5– 8). Unrelated antibodies against p65 (a major component of NF-B) did not affect the gel-shift profile (data not shown), suggesting that c-Jun, JunB, and
JunD proteins participated in the binding to AP-1 motif. We have previously analyzed the effects of Csk overexpression on the tyrosine phosphorylation of intracellular proteins and showed significant induction of tyrosine phosphorylation of multiple cellular proteins in Csk cells, suggesting that the overexpression of Csk downregulates the Src associated signal transduction cascade but upregulates other signal transduction cascades (27). It seems likely, therefore, that overexpression of Csk accelerates the activation of the JNK pathway under LPS stimulation. Two transcriptional elements of AP-1 are located in human NOS II promoter region on the positive strand (31). Marks-Konczalik et al. (32) demonstrated that mutation of these AP-1 sites reduced promoter activities, suggesting that two AP-1 sites on the positive strand play an important role for induction of human NOS II transcription. On the other hand, in 1.6 kb murine NOS II promoter (33), there is no perfectly matched AP-1 site [TGA(C/G)T(C/A)A], but two AP-1like sites on the positive strand and one AP-1-like site on the negative strand are found in the promoter region. In these sites, one base is changed at rather flexible fourth or sixth position. To examine whether AP-1-like sites on the negative strand participates in the negative regulation of NOS II promoter activity, we focused one site [lower site (L), ⫺524⬃⫺518] on the negative strand and another similar site [upper site (U), ⫺1126⬃⫺1120] on the negative strand in which
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FIG. 5. Activation of AP-1 in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) J774A.1, J.pBK, or J.Csk cells were stimulated with LPS for various duration, and activation of AP-1 was analyzed by EMSA. Representative results from three separate experiment are shown. (B) The components of AP-1 were investigated. The nuclear extracts from J774A.1 cells stimulated with LPS for 24 h (lanes 1– 4) or from J.Csk cells stimulated with LPS for 6 h (lanes 5– 8) were incubated with 1 l of specific polyclonal antibodies to c-Jun, JunB, or JunD for 1 h at 4°C before the incubation with radiolabeled AP-1 oligonucleotide probe. (C) The nuclear extracts from J774A.1 cells stimulated with LPS for 24 h were incubated with radiolabeled AP-1 oligonucleotide probe in the absence or presence of either oligonucleotides containing AP-1-like U-site sequence or L-site sequence in the murine NOS II promoter.
two bases are changed at fourth and sixth position (Fig. 6). Then two oligomers containing either L or U sequence were synthesized and the competition assay was performed in EMSA. DNA-protein complexes were specifically reduced by an oligonucleotide with L-site motif but not by an oligonucleotide with U-site motif (Fig. 5C). These findings indicate that activated AP-1 components in the nuclear extract from J774A.1 cells stimulated with LPS interact with the AP-1-like L-site motif in the murine NOS II promoter.
Effect of Mutation in AP-1-like Binding Sequences of NOS II Promoter on NOS II Gene Expression by LPS Stimulation To investigate whether the two AP-1-like sites described above participate in the regulation of NOS II mRNA transcription, luciferase reporter gene constructs containing 1.6 kb murine NOS II promoter bearing a three-base mutation in either or both AP-1like binding motifs (U and L sites) were generated (Fig.
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FIG. 6. Two AP-1-like sites in 1.6 kb of the murine NOS II promoter. Sequences of the two AP-1-like sites and mutated sequences are illustrated. Asterisks indicate bases different from those of the AP-1 consensus motif. Mutated bases in AP-1-like sites are shown by bold letters.
6). J774A.1 cells were transiently transfected with wild-type and those three mutated NOS II promoters. Because down-regulation of the transcription of NOS II mRNA was observed in J774A.1 cells stimulated with LPS for 24 h, luciferase assay was performed after LPS stimulation for 24 h. As shown in Fig. 7, luciferase activity in cells transfected with the luciferase reporter vector containing NOS II promoter with mutations in the upper AP-1-like site (Umut) was not different from that in cells transfected with wild type NOS II promoter. Meanwhile, mutations in AP-1-like site (Lmut) or in both sites (U ⫹ Lmut) significantly increased luciferase activity in cells stimulated with LPS. These findings together with the results shown in Fig. 5 suggest that large amounts of AP-1 activated by JNK in an early stage of LPS stimulation bound to AP-1-like negative regulatory L site, resulting in the accelerated negative regulation of NOS II expression. Negative regulation of signal transduction cascades is necessary for homeostasis in a variety of systems. In hematopoietic and immune system cells, failure to regulate signal transduction during cell activation can result in hyperresponsive states that lead to significant pathological sequelae, such as autoimmunity and excessive inflammation (34 –36). Because the hyperresponsive states that result from failed regulation mimic several human diseases (37–39), understanding the molecular basis of negative signaling is increasing importance. The findings reported here suggest that
FIG. 7. Effects of mutations in the murine NOS II gene AP-1-like sites on responses to LPS stimulation. J774A.1 cells were transiently transfected with the vector containing 1.6 kb NOS II promoter (WT), or with the vector with mutations in the U site (Umut), in the L site (Lmut), or in the both U and L sites (U ⫹ Lmut). Luciferase activities were analyzed after stimulation with LPS for 24 h. Representative results from three separate experiment, each performed in triplicate or quadruplicate, are shown.
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NOS II gene expression is negatively regulated, at least in part, through AP-1-like site at position ⫺524⬃⫺518. Although the murine NOS II promoter comprises various elements such as NF-B, NF-IL6, TNF-RE, ISRE NF1, and Oct, NF-B, and AP-1 are well-defined transcription factors activated by LPS. It has been demonstrated that NF-B is required for NOS II transcription (40). Therefore, further studies on the negative regulation of NOS II expression by AP-1 and other transcription factors are needed. ACKNOWLEDGMENTS This study was supported in part by grants from the Promotion and Mutual Aid Corporation for Private Schools of Japan and from the Japanese Ministry of Education, Science, Sports, and Culture.
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Negative Regulation of LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by AP-1 in Macrophage Cell Line J774A.1 Takako Kizaki,* ,1 Kenji Suzuki,* Yoshiaki Hitomi,* Kazuya Iwabuchi,† Kazunori Onoe´,† Shuko Haga,‡ Hitoshi Ishida,§ Tomomi Ookawara, ¶ Keiichiro Suzuki, ¶ and Hideki Ohno* *Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, Mitaka, Japan; †Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; ‡Institute of Health and Sport Sciences, University of Tsukuba, Tsukuba, Japan; §Department of Internal Medicine III, Kyorin University, School of Medicine, Mitaka, Japan; and ¶ Department of Biochemistry, Hyogo University of Medicine, Nishinomiya, Japan
Received November 22, 2001
The level of NOS II mRNA was markedly increased during 24 h lipopolysaccharide (LPS) stimulation, but showed no further increase thereafter. On the other hand, the level of NOS II mRNA in J774A.1 cells transfected with an expression vector containing the rat csk cDNA (J.Csk) was significantly increased during 3 h LPS stimulation, but rather decreased thereafter. Although no significant difference was observed in the activation of NF-B by LPS among parental J774A.1, J774A.1 transfected with promoterless vector (J.pBK), and J.Csk cells, activity of c-Jun N-terminal kinase (JNK) and nuclear translocation of nuclear factor activator protein-1 (AP-1) were markedly upregulated in the J.Csk cells. Then luciferase reporter vectors containing NOS II promoter with mutations in two AP-1like sites (U site, ⴚ1126⬃ⴚ1120; L site, ⴚ524⬃ⴚ518) were transiently transfected in J774A.1 cells. The promoter activity following LPS stimulation for 24 h was significantly increased by mutation at the L site, but not by mutation at the U site, suggesting that NOS II expression is negatively regulated, at least in part, through the AP-1-like L site in response to LPS. © 2001 Elsevier Science
Key Words: macrophage; NOS II; negative regulation; AP-1.
Nitric oxide (NO) is a free radical produced from the catalytic oxidation of the guanidino group of L-arginine by nitric oxide synthase (NOS). Inducible NOS (NOS 1
To whom correspondence and reprint requests should be addressed at Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo 181-8611, Japan. Fax: ⫹81-422-44-4427. E-mail: [email protected].
II) is not generally present in resting cells but is induced by various stimuli, such as bacterial lipopolysaccharide (LPS), tumour necrosis factor (TNF), interluekin-1␥, and interferon-␥. NO has been shown to have a number of important biological functions, including tumor cell killing and host defense against intracellular pathogens. On the other hand, increased expression of NOS II is known to be associated with disorders as diverse as septic and hemorrhagic shock, rheumatoid arthritis, and chronic infections (1–5). Thus, NOS II gene expression must be tightly controlled not only by positive but also by negative regulation. Although considerable advances have been made in elucidating the intracellular signals essential for positive signaling in NOS II expression (6 –12), knowledge of the specific signal transduction components that negatively regulate NOS II expression is not fully understood. Protein tyrosine kinases of src-family (src-PTK) were expressed in various hematolymphoid cells in a lineage specific manner. The src-PTK are associated with surface receptor molecules specifically expressed on each cell lineage, and play essential roles in intracellular transduction of signals initiated at the level of surface molecules that lack catalytic domains. Thus, the srcPTK may be involved in activation, proliferation, differentiation, and transformation of the cells (13, 14). The predominant Src family kinases expressed in macrophages, Hck, Fgr, and Lyn, have been implicated in a number of studies as being the initial signal transducers for tyrosine phosphorylation events after LPS stimulation (15–18). The activity of src-PTK is negatively regulated by a key enzyme, C-terminal Src kinase (Csk) (20 –22). Csk inactivates src-PTK by phosphorylating tyrosine at the C-terminal regulatory site (Tyr-
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527) (19). Indeed, the targeted disruption of the csk gene caused constitutive activation of src-PTK (23, 24). On the contrary, overexpression of Csk in some cell lines repressed cellular signaling mediation of src-PTK (25, 26), suggesting that a balance between src-PTK and Csk plays distinct roles in positive/negative regulation of signal transduction cascade initiated through srk-PTK. We have previously demonstrated that overexpression of Csk downregulates the production of interleukin (IL)-1a, IL-6, TNF-␣, and NO following stimulation with LPS in a macrophage cell line, J774A.1 (27). Thus, in the present study, the mechanisms of negative regulation of NOS II expression in responses to LPS were examined by analyzing effects of overexpression of Csk on the signal transduction cascade in J774A.1 cells. MATERIALS AND METHODS Electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared as previously described (27). The NF-B oligonucleotide probe (5⬘-AGT TGA GGG GAC TTT CCC AGG-3⬘) was purchased from Promega (Madison, WI) and the AP-1 oligonucleotide probe (5⬘-CGC TTG ATG AGT CAG CCG GAA-3⬘) were purchased from Promega Co. (Tokyo, Japan). Each probe was labeled with [␥- 32P]ATP using T4 polynucleotide kinase (Bio Labs) and purified in Microspin columns (Pharmacia Biotech, Uppsala, Sweden). For EMSA 1 g nuclear proteins were preincubated with EMSA buffer [10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl 2, 0.5 mM DTT, 0.5 mM EDTA, 4% glycerol, 1 g poly(dI– dC)] at room temperature for 20 min and then incubated with radiolabeled oligonucleotide probe for 5 min. The mixture was applied to a 7% polyacrylamide gel that had previously been electrophoresed at 100 V for 30 min. Gel was run at 100 V in TGE (5 mM Tris–HCl, pH 8.3, 38 mM glycine, 0.1 mM EDTA) followed by transference to 3MM Whatman paper, drying under vacuum at 80°C for 30 min, and quantification of the band intensities was then estimated according to an autoradiograph or analysis with a BAS 2000 imaging system (Fuji Photo Film Co. Ltd., Kanagawa, Japan). To identify the protein components of the NF-B complex or AP-1 complex, 1 l of specific polyclonal antibodies to p50 or p65 for NF-B or to c-Jun, Jun B, or Jun D for AP-1 (Santa Cruz Biotechnology) were added to the nuclear extracts, and the mixture was then incubated at 4°C for 1 h before the radiolabeled oligonucleotide probe was added. Oligonucleotide probes containing either two AP-1-like motif in 1.6 kb NOS II promoter (5⬘-CGC TTG ATC AAT CAG CCG GAA-3⬘ and 5⬘-CGC TTG ATC ACT CAG CCG GAA-3⬘) were used as competitors. Western blotting analysis. Nuclear proteins (20 g) were boiled in loading buffer (Tris–HCl, pH 6.8, 2% SDS, 5% glycerol, and 10% 2-mercaptoethanol), separated in 10% SDS–PAGE, and transferred to polyvinylidene difluoride membrane (Applied Biosystems, Foster City, CA). Membranes were blocked with 3% nonfat dried milk in Tris-buffered saline (TBS) for 1 h. Primary antibodies against c-Jun (Santa Cruz Biotechnology, Inc.) was applied at appropriate dilutions. After washing three times in TBS (25 mM Tris–HCl, 500 mM NaCl) containing 0.1% Tween 20 (TBS-T), secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, DAKO Japan, Kyoto, Japan] were applied at appropriate dilutions. Membranes were washed in TBS-T three times and the immunoreactivity was visualized with an enhanced chemiluminescence reagents (ECL, Amersham–Pharmacia Biotech Ltd. Tokyo, Japan). Protein kinase assay for JNK. Cell were stimulated according to experimental protocols and lysed using lysis buffer containing 10 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM PMSF, 1 mM sodium
orthvanadate, 2 g/ml aprotinin, 50 mM NaF, 1% Nonidet P-40. Cell lysates were subjected to centrifugation at 12,000g for 10 min at 4°C. The soluble fraction was incubated with Abs against JNK at 4°C for 1 h. After the addition of 20 l protein A/G–Sephosrose 4B, the reaction mixtures were incubated at 4°C for 2 h and then subjected to microcentrifugation. The beads were washed three times with buffer A, then once with kinase buffer containing 10 mM Hepes, pH 7.4, 10 mM MgCl 2, 3 mM MnCl 2, and incubated with kinase buffer containing 1 g of GST– c-jun and 370 kBq [␥- 32P]ATP (NENTM Life Science Products, Inc., Wilmington, DE) at 30°C for 30 min. Thereafter, the beads were boiled for 5 min and applied to 9% SDS– polyacrylamide gel electrophoresis. The gels were dried, exposed for 30 min, and analyzed with BAS 2000 imaging system (Fuji Photo Film Co. Ltd., Kanagawa, Japan). Northern analysis. The expression NOS II mRNA was determined by Northern analysis. The RNA was extracted with TRIzol Reagent (Life Technologies). Total RNA (25 g) was electrophoresed under denaturing conditions through a 1% (w/v) agarose– formaldehyde gel and then transferred to Nyrone membranes. The blots were hybridized with 32P-labeled NOS II probes and analyzed with BAS 2000 imaging system. Generation of NOS II luciferase reporter constructs. The fulllength murine NOS II promoter fragment (bp ⫺1580 to ⫹159) were cloned into the pGL3-enhancer luciferase reporter gene vector (Promega, Madison, WI) (pGL3-NOS IIwild). Point mutations of the two AP-1-like sequences [upper site (Umut) and lower site (Lmut)] were introduced by site-directed mutagenesis via overlap extension using PCR and then subcloned into the pGL3-enhancer vector (pGL3-NOS IIUmut, pGL3-NOS IILmut, and pGL3-NOS IIU ⫹ Lmut). All plasmids were checked for accuracy by sequencing. Transient transfection and luciferase assay. J774 A.1 cells were transfected using LipofectAMINE Reagent (Life Technologies, Inc.) with constructs containing the luciferase reporter gene. At 80% confluency, after washing in serum-free medium, cells were incubated with 0.4 g pGL3-enhancer construct, 0.04 g pRL-SV, and 4 l of LipofectAMINE Reagent/well (12-well dishes) for 3 h at 37°C. After 24 h, the culture medium was replaced with fresh medium containing 1 g/ml LPS. Cell were harvested 24 h later, and luciferase activity was determined using a dual luciferase assay system kit (Promega). Briefly cells were washed twice in phosphate-buffered saline (PBS) and lysed by adding 100 l of a 1⫻ lysis buffer. After 15 min at room temperature, the lysate was removed from the plate and centrifuged at 12,000g. To 10 l of the supernatant, 100 l of a luciferase assay reagent was added, and luciferase activity was immediately measured using a BLR-301 luminescence reader (ALOKA). Firefly luciferase activity was normalized for Renilla luciferase activity. Cell lysates were analyzed for protein content using the BCA protein assay reagent (Pierce). Luminescence units (firefly luciferase activity/Renilla luciferase activity) were normalized for total protein content.
RESULTS AND DISCUSSION Time Course of NOS II mRNA Expression and NOS II Promoter Activity Following LPS Stimulation The time course study showed that the level of the NOS II mRNA in J774A.1 cells was markedly increased during 24 h of LPS stimulation, but was not further increased thereafter (Fig. 1), thereby suggesting that the transcriptional rate of NOS II mRNA changed during the LPS stimulation. To investigate whether the expression of NOS II mRNA was changed at the transcriptional level, J774A.1 cells were transiently transfected with a luciferase reporter gene con-
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II expression was accelerated in Csk cells under the LPS stimulation. Effects of Csk Overexpression on the Activation of NF-B Following LPS Stimulation
FIG. 1. NOS II mRNA expression in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) Time course of NOS II mRNA expression was analyzed by Northern blotting analysis. Two separate experiments were carried out, and representative result is shown. (B) Relative NOS II mRNA expression shown in A was analyzed by densitometry.
struct containing 1.6 kb murine NOS II promoter. As shown in Fig. 2, luciferase activity was significantly reduced in the cells stimulated with LPS for 24 h compared with that in cells stimulated with LPS for 6 h. The promoterless vector containing the luciferase gene was unaffected by LPS treatment, confirming that these effects were specific to the murine NOS II promoter. These findings suggest that the transcription of NOS II mRNA was rapidly induced by LPS stimulation but was downregulated by prolonged LPS stimulation. In our previous study, we demonstrated that production of NO following LPS stimulation for 48 h was markedly reduced in J774A.1 cells transfected with expression vector containing rat csk cDNA (Csk) compared with those in parental J774A.1 or J774A.1 cells transfected with vector alone (pBK) (27). Thus, time course studies of the level of NOS II mRNA in Csk and pBK cells were performed. As shown in Fig. 1, the level of the NOS II mRNA in pBK cells was markedly increased during 24 h of LPS stimulation as well as in J774A.1 cells. On the other hand, NOS II mRNA was rapidly increased in J.Csk cells as in J774A.1 and J.pBK cells following LPS stimulation for 3 h, but was rather decreased thereafter. It seemed, therefore, likely that the negative transduction cascade for NOS
Exposure of macrophages to bacterial LPS initiates a signal transduction cascade that leads to increased production of NO, secretion of proinflammatory cytokines, and acquisition of enhanced bactericidal/ tumoricidal activity. Therefore, regulation of this signaling pathway is critical for controlling the initial phases of the immune response to foreign organisms. The transcriptional regulation of NOS II has been extensively characterized. Among a number of signaling pathways which have been implicated in LPS responses in macrophages, the most rapidly induced response is activation of nuclear factor-B (NF-B) (28, 29). Two NF-B sites, one located in the region I between positions ⫺48 and ⫺209 and another located in the region II between positions ⫺913 and ⫺1029 in murine NOS II promoter, are necessary for NOS II expression by LPS stimulation. Thus, the time course of the activation of NF-B after treatment with LPS was evaluated by an electrophoretic mobility gel shift assay (EMSA). Since protein components of NF-B are encoded by a set of genes called “immediate early genes” whose transcription is rapidly induced, nuclear extracts were prepared from J774A.1, J.pBK, and J.Csk stimulated with LPS for 1, 2, 5, 10, and 15 min. No significant difference was observed in NF-B spe-
FIG. 2. NOS II promoter activity in J774A.1 cells following LPS stimulation. J774A.1 cells were transiently transfected with luciferase reporter vector alone (pGL3) or with the vector containing 1.6 kb NOS II promoter (pGL3-NOS promoter). Luciferase activities were analyzed after stimulation with LPS for 6, 12, and 24 h. *P ⬍ 0.05.
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FIG. 3. NF-B activities in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) J774A.1, J.pBK, or J.Csk cells were stimulated with LPS for 1, 2, 5, 10, or 15 min, and activation of NF-B was analyzed by EMSA. (B) J774A.1, J.pBK, or J.Csk cells were stimulated with LPS for 0.5, 1, 2, or 3 h, and activation of NF-B was analyzed by EMSA.
cific DNA-protein complex formation among the three cells (Fig. 3A). Then, more late responses of NF-B to LPS stimulation were analyzed. However, we could not find any distinct difference in the activation of NF-B among the three cells during 3 h LPS stimulation (Fig. 3B). In addition, after 24 h of LPS stimulation, the levels of activated NF-B was dwindled in all three cells (data not shown). Therefore, it was suggested that the activation of the major mediator, NF-B, in LPS signal transduction occurs normally in the J.Csk cells as well as in the J774 or J.pBK cells. Antibodies against p65 partially reduced the band of DNA–protein complexes and produced one supershift (data not shown). On the other hand, all DNA-protein complexes were supershifted by antibodies against p50 and formation of two supershifted bands were detected (data not shown), thus suggesting that the proteins binding to NF-B were p65/p50 heterodimer and p50/p50 homodimer. Effects of Csk Overexpression on the Activation of Jun N-Terminal Kinase Cascade by LPS Stimulation Mitogen-activated protein kinases (MAPK) are comprised of three principal family members: c-jun
N-terminal kinases (JNK)/stress-activated protein kinases, extracellular signal-regulated kinases, and p38 MAPK and have been shown to activate a number of transcription factors. Among them, activator protein-1 (AP-1) is ubiquitous transcription factors and pleiotropic regulators of the inducible expression of many genes that encode proteins involved in the modulation of inflammatory and host defense processes in eukaryotic cells (30). The activities of AP-1 components are modulated by phosphorylation. This form of posttranslational control is best understood for the phosphorylation of c-Jun by the JNK. Then, the time course study of JNK activity after stimulation of LPS in J774A.1, J.pBK, and J.Csk cells was performed using GST– cJun as a substrate. JNK activity was detected in J774A.1and J.pBK cells at 1 h and 3 h of LPS stimulation, respectively (Fig. 4A). The activity was the highest at 6 h of LPS treatment and then decreased with incubation in both cells. On the other hand, JNK activity was observed in J.Csk cells even before LPS stimulation and was further enhanced by LPS stimulation. Then the nuclear translocation of c-Jun was analyzed in the three cells after stimulation of LPS. The level of nuclear translocation of c-Jun in J.Csk was
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FIG. 4. Activation of JNK cascade in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) Time course of JNK activity in J774A.1, J.pBK, and J.Csk cells after stimulation of LPS was investigated using GST-c-Jun as a substrate. (B) Nuclear extract was prepared from the three cells stimulated with LPS for 0, 24, and 48 h and subjected to Western blot analysis with anti-c-Jun antibodies.
markedly higher than that in J774A.1 or J.pBK cells (Fig. 4B), consisting with the results shown in Fig. 4A. We further analyzed the activation of AP-1 in J774A.1, J.pBK, and J.Csk cells following stimulation with LPS by EMSA. In both J774A.1 and J.pBK cells, activation of AP-1 was observed after 12 h of LPS stimulation (Fig. 5A). Meanwhile, gel shift band was observed in J.Csk cells after 1 h of LPS stimulation and was further increased with stimulation. To identify proteins binding to AP-1 motif, antibodies against Jun related proteins (c-Jun, Jun B, or Jun D) were added to the mixture of nuclear extract and radiolabeled AP-1 probe. As shown in Fig. 5B, the intensity of shifted band observed in the mixture of nuclear extract and radiolabeled AP-1 probe became faint by addition of anti-c-Jun antibodies (Fig. 5B, lane 2), although supershifted band was not observed. On the other hand, supershifted bands were observed in the presence of either anti-JunB or anti-JunD antibodies (Fig. 5B, lanes 3 and 4). Likewise, in the supershift experiment using the mixture of the nuclear extract from J.Csk cells stimulated with LPS for 6 h and radiolabeled AP-1 probe, anti-c-Jun antibodies decreased the intensity of the shifted band, and either anti-Jun B or antiJun D antibody formed a supershift band (Fig. 5B, lanes 5– 8). Unrelated antibodies against p65 (a major component of NF-B) did not affect the gel-shift profile (data not shown), suggesting that c-Jun, JunB, and
JunD proteins participated in the binding to AP-1 motif. We have previously analyzed the effects of Csk overexpression on the tyrosine phosphorylation of intracellular proteins and showed significant induction of tyrosine phosphorylation of multiple cellular proteins in Csk cells, suggesting that the overexpression of Csk downregulates the Src associated signal transduction cascade but upregulates other signal transduction cascades (27). It seems likely, therefore, that overexpression of Csk accelerates the activation of the JNK pathway under LPS stimulation. Two transcriptional elements of AP-1 are located in human NOS II promoter region on the positive strand (31). Marks-Konczalik et al. (32) demonstrated that mutation of these AP-1 sites reduced promoter activities, suggesting that two AP-1 sites on the positive strand play an important role for induction of human NOS II transcription. On the other hand, in 1.6 kb murine NOS II promoter (33), there is no perfectly matched AP-1 site [TGA(C/G)T(C/A)A], but two AP-1like sites on the positive strand and one AP-1-like site on the negative strand are found in the promoter region. In these sites, one base is changed at rather flexible fourth or sixth position. To examine whether AP-1-like sites on the negative strand participates in the negative regulation of NOS II promoter activity, we focused one site [lower site (L), ⫺524⬃⫺518] on the negative strand and another similar site [upper site (U), ⫺1126⬃⫺1120] on the negative strand in which
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FIG. 5. Activation of AP-1 in J774A.1, J.pBK, or J.Csk cells following LPS stimulation. (A) J774A.1, J.pBK, or J.Csk cells were stimulated with LPS for various duration, and activation of AP-1 was analyzed by EMSA. Representative results from three separate experiment are shown. (B) The components of AP-1 were investigated. The nuclear extracts from J774A.1 cells stimulated with LPS for 24 h (lanes 1– 4) or from J.Csk cells stimulated with LPS for 6 h (lanes 5– 8) were incubated with 1 l of specific polyclonal antibodies to c-Jun, JunB, or JunD for 1 h at 4°C before the incubation with radiolabeled AP-1 oligonucleotide probe. (C) The nuclear extracts from J774A.1 cells stimulated with LPS for 24 h were incubated with radiolabeled AP-1 oligonucleotide probe in the absence or presence of either oligonucleotides containing AP-1-like U-site sequence or L-site sequence in the murine NOS II promoter.
two bases are changed at fourth and sixth position (Fig. 6). Then two oligomers containing either L or U sequence were synthesized and the competition assay was performed in EMSA. DNA-protein complexes were specifically reduced by an oligonucleotide with L-site motif but not by an oligonucleotide with U-site motif (Fig. 5C). These findings indicate that activated AP-1 components in the nuclear extract from J774A.1 cells stimulated with LPS interact with the AP-1-like L-site motif in the murine NOS II promoter.
Effect of Mutation in AP-1-like Binding Sequences of NOS II Promoter on NOS II Gene Expression by LPS Stimulation To investigate whether the two AP-1-like sites described above participate in the regulation of NOS II mRNA transcription, luciferase reporter gene constructs containing 1.6 kb murine NOS II promoter bearing a three-base mutation in either or both AP-1like binding motifs (U and L sites) were generated (Fig.
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FIG. 6. Two AP-1-like sites in 1.6 kb of the murine NOS II promoter. Sequences of the two AP-1-like sites and mutated sequences are illustrated. Asterisks indicate bases different from those of the AP-1 consensus motif. Mutated bases in AP-1-like sites are shown by bold letters.
6). J774A.1 cells were transiently transfected with wild-type and those three mutated NOS II promoters. Because down-regulation of the transcription of NOS II mRNA was observed in J774A.1 cells stimulated with LPS for 24 h, luciferase assay was performed after LPS stimulation for 24 h. As shown in Fig. 7, luciferase activity in cells transfected with the luciferase reporter vector containing NOS II promoter with mutations in the upper AP-1-like site (Umut) was not different from that in cells transfected with wild type NOS II promoter. Meanwhile, mutations in AP-1-like site (Lmut) or in both sites (U ⫹ Lmut) significantly increased luciferase activity in cells stimulated with LPS. These findings together with the results shown in Fig. 5 suggest that large amounts of AP-1 activated by JNK in an early stage of LPS stimulation bound to AP-1-like negative regulatory L site, resulting in the accelerated negative regulation of NOS II expression. Negative regulation of signal transduction cascades is necessary for homeostasis in a variety of systems. In hematopoietic and immune system cells, failure to regulate signal transduction during cell activation can result in hyperresponsive states that lead to significant pathological sequelae, such as autoimmunity and excessive inflammation (34 –36). Because the hyperresponsive states that result from failed regulation mimic several human diseases (37–39), understanding the molecular basis of negative signaling is increasing importance. The findings reported here suggest that
FIG. 7. Effects of mutations in the murine NOS II gene AP-1-like sites on responses to LPS stimulation. J774A.1 cells were transiently transfected with the vector containing 1.6 kb NOS II promoter (WT), or with the vector with mutations in the U site (Umut), in the L site (Lmut), or in the both U and L sites (U ⫹ Lmut). Luciferase activities were analyzed after stimulation with LPS for 24 h. Representative results from three separate experiment, each performed in triplicate or quadruplicate, are shown.
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NOS II gene expression is negatively regulated, at least in part, through AP-1-like site at position ⫺524⬃⫺518. Although the murine NOS II promoter comprises various elements such as NF-B, NF-IL6, TNF-RE, ISRE NF1, and Oct, NF-B, and AP-1 are well-defined transcription factors activated by LPS. It has been demonstrated that NF-B is required for NOS II transcription (40). Therefore, further studies on the negative regulation of NOS II expression by AP-1 and other transcription factors are needed. ACKNOWLEDGMENTS This study was supported in part by grants from the Promotion and Mutual Aid Corporation for Private Schools of Japan and from the Japanese Ministry of Education, Science, Sports, and Culture.
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