Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages

Shu Okugawa, Yasuo Ota, Takatoshi Kitazawa, Kuniko Nakayama, Shintaro Yanagimoto, Kunihisa Tsukada, Miki Kawada, and Satoshi Kimura

Department of Infectious Disease, Graduate School of Medicine, University of Tokyo, Tokyo 113-6855, Japan

Submitted 16 January 2003 ; accepted in final form 8 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms by which lipopolysaccharide (LPS) is recognized, and how such recognition leads to innate immune responses, are poorly understood. Stimulation with LPS induces the activation of a variety of proteins, including mitogen-activated protein kinases (MAPKs) and NF-{kappa}B. Activation of protein tyrosine kinases (PTKs) is also necessary for a number of biological responses to LPS. We used a murine macrophage-like cell line, RAW264.7, to demonstrate that Janus kinase (JAK)2 is tyrosine phosphorylated immediately after LPS stimulation. Anti-Toll-like receptor (TLR)4 neutralization antibody inhibits the phosphorylation of JAK2 and the c-Jun NH2-terminal protein kinase (JNK). Both the JAK inhibitor AG490 and the kinase-deficient JAK2 protein reduce the phosphorylation of JNK and phosphatidylinositol 3-kinase (PI3K) via LPS stimulation. Pharmacological inhibition of the kinase activity of PI3K with LY-294002 decreases the phosphorylation of JNK. Finally, we show that JAK2 is involved in the production of IL-1{beta} and IL-6. PI3K and JNK are also important for the production of IL-1{beta}. These results suggest that LPS induces tyrosine phosphorylation of JAK2 via TLR4 and that JAK2 regulates phosphorylation of JNK mainly through activation of PI3K. Phosphorylation of JAK2 via LPS stimulation is important for the production of IL-1{beta} via the PI3K/JNK cascade. Thus JAK2 plays a pivotal role in LPS-induced signaling in macrophages.

cytokine; toll-like receptor-4; c-Jun NH2-terminal kinase


THE IMMUNE SYSTEM IN MAMMALS consists of innate and acquired immunity. Acquired immunity is a sophisticated system that is mediated by antigen-specific T- and B cells. The mechanisms for recognition of nonself in acquired immunity have been investigated intensively. However, the mechanisms by which microbial products such as lipopolysaccharides (LPS) are recognized, and how this recognition ultimately leads to innate immune responses, are poorly understood (50).

Toll-like receptors (TLRs) are named for specific receptors in innate immunity that respond to microbial products. The TLR family comprises more than nine members, and the number is increasing (50). TLRs have been shown to be involved in the expression of genes of inflammatory cytokines and costimulatory molecules. The cytoplasmic part of TLRs is highly similar to that of the IL-1 receptor family and is currently referred to as the Toll/IL-1 receptor (TIR) domain. The signaling pathway via the TLR family is highly homologous to that of the IL-1 receptor family. TLR interacts with adaptor protein MyD88 in its TIR domain (8, 32). Once stimulated, MyD88 recruits IL-1 receptor-associated kinase (IRAK) to the receptor (35, 54). IRAK is activated and associates with tumor necrosis factor (TNF)-associated factor 6 (TRAF6), leading to the activation of several distinct signaling pathways, including mitogen-activated protein kinases (MAPKs) and NF-{kappa}B (34). This signal transduction pathway triggers production of proinflammatory cytokines, such as IL-1, IL-6, and TNF-{alpha} (35).

LPS is a major component of the outer membrane of gram-negative bacteria and is composed of polysaccharides that extend outward from the bacterial cell surface. LPS provokes a wide variety of immunologic responses, and TLR4 is a critical signal transducer for LPS. Two mouse strains, C3H/HeJ and C57BL/10ScCr, possess a mutation or deletion in their TLR4 gene and consequently are defective in their responses to LPS (41, 43). Furthermore, a study of TLR4-deficient mice confirmed that TLR4 is critically important for LPS signaling (20). It is now generally accepted that LPS from gram-negative bacteria stimulates inflammatory responses via TLR4 (4, 50).

The c-Jun NH2-terminal protein kinase (JNK) pathway, one of the MAPK pathways, is activated in response to a variety of physiological and stress-related stimuli (12, 25). Treatment with LPS also has resulted in JNK activation in macrophages (17, 21, 49) and leads to rapid activation of JNK in a murine macrophage-like cell line, RAW264.7, and a human monocyte cell line, THP-1. The JNK pathway is also required for LPS-induced translation of TNF-{alpha} and IL-1 mRNA (17, 21, 49).

In addition to activating MAPKs and NF-{kappa}B, LPS stimulation induces activation of a variety of proteins involved in signal transduction (18, 31). LPS stimulation activates the phosphatidylinositol 3-kinase (PI3K) pathway, although the molecular mechanisms by which LPS activates PI3K remain to be elucidated. The PI3K pathway has been reported to activate MAPKs and NF-{kappa}B-dependent gene expression (7, 21, 27, 28, 44, 47). On the other hand, the PI3K pathway has been demonstrated to negatively regulate LPS-induced signaling (16, 40).

Both in vitro and in vivo studies with tyrosine kinase inhibitors have demonstrated that activation of tyrosine kinases is necessary for a number of the biological responses to LPS, including activation of JNK (13, 15, 17, 37). The protein tyrosine kinases (PTKs) in the Src family are candidates for involvement in activating multiple signal-transducing molecules after LPS stimulation. The Src family kinases, Hck and Lyn, have both been implicated in the biological responses to LPS by macrophages (5, 14).

The Janus kinase (JAK) family is an important PTK, especially in hematopoietic cells (39, 45). The four mammalian members of the JAK family of PTKs, JAK1, JAK2, JAK3, and TYK2, are, with the exception of JAK3, ubiquitously expressed. JAK2 is involved in signaling by single-chain hormone receptors, the common {beta}-chain family, and certain members of the class II receptor cytokine family. JAK2 is also tyrosine phosphorylated after stimulation via several other receptors. Stromal cell-derived factor (SDF)-1{alpha} stimulation via chemokine receptor CXCR4 led to the tyrosine phosphorylation of JAK2 in a human progenitor cell line and in human T cell lines (53, 57). JAK2 is involved in the expression of type II nitric oxide synthase (iNOS) in skin-derived dendritic cells treated with LPS (10).

JAK2 is directly implicated in the activation of PI3K in several signal transduction cascades. Growth hormone and prolactin stimulate PI3K activation via JAK2 (55). JAK2 is also involved in the stimulation of PI3K in signaling pathways via granulocyte-macrophage colony-stimulating factor (GM-CSF) and chemokine receptor CXCR4 (3, 57).

Furthermore, JAK2 is involved in the LPS-induced expression of iNOS in skin-derived dendritic cells (11). In contrast, JAK2 regulates the production of iNOS negatively in vascular smooth muscle cells (30). Little is known about the molecular mechanisms by which JAK2 transduces the LPS-induced signals to the downstream molecules to produce the proinflammatory cytokines.

We report here that LPS induces tyrosine phosphorylation of JAK2 via TLR4. We also demonstrate that JAK2 regulates the phosphorylation of JNK, primarily through PI3K. Tyrosine phosphorylation of JAK2 is involved in the production of IL-1{beta} via JNK. Thus we have established that JAK2 plays an important role in LPS-induced signaling in macrophages.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. RAW264.7, a murine macrophage-like cell line, was obtained from American Type Culture Collection (Manassas, VA) and was maintained in Eagle's modified minimum essential medium supplemented with 2 mM glutamine (Sigma, St. Louis, MO), 100 U/ml penicillin, 100 µg/ml streptomycin (ICN, Aurora, OH), and 10% fetal bovine serum (Sigma).

Reagents and antibodies. LY-294002 (a specific inhibitor for PI3K), AG490 (a JAK inhibitor), and SP-600125 (a JNK inhibitor) were all purchased from Calbiochem (San Diego, CA). Polyclonal antibodies for the JAK family kinases, including anti-JAK1, anti-JAK2, anti-JAK3, and anti-TYK2, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-JNK1 antibody was also purchased from Santa Cruz Biotechnology. Phosphospecific antibody for JNK was purchased from Cell Signaling (Beverly, MA). Anti-phosphotyrosine (4G10) antibody, anti-PI3K (p85) antibody, and anti-JAK2 antibody were obtained from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were obtained from Dako.

Repurification of LPS. LPS from Escherichia coli O11:B4 was purchased from Sigma and repurified by the phenol extraction method of Hirschfeld et al. (19). Briefly, 5 mg of LPS was resuspended in 1 ml of endotoxin-free water containing 0.2% triethylamine (TEA) at room temperature. Deoxycholate (DOC) was added to the aliquot to a final concentration of 0.5%, followed by the addition of 1 ml of water-saturated phenol. The samples were vortexed intermittently for 5 min, and the phases were allowed to separate at room temperature for 5 min. The samples were placed on ice for 5 min, followed by centrifugation at 4°C for 2 min at 10,000 g. The top aqueous layer was transferred to a new tube, and the phenol phase was reextracted with 1 ml of 0.2% TEA-0.5% DOC. The aqueous phases were pooled and reextracted with 1 ml of water-saturated phenol. The pooled aqueous phases were adjusted to 75% ethanol and 30 mM sodium acetate and were precipitated at -20°C for 1 h. The precipitates were centrifuged at 4°C for 10 min at 10,000 g, washed in 1 ml of cold 100% ethanol, and air dried. The precipitates were resuspended in the original volume (1 ml) of 0.2% TEA.

Transfection of JAK2 plasmids. Wild-type JAK2 cDNA and kinase-deficient JAK2 (JAK2-KD) cDNA were kindly provided by Dr. T. Kadowaki (University of Tokyo). Construction of the plasmids containing wild-type JAK2 or the JAK2-KD under the control of the SR{alpha} promoter has been described previously (55).

RAW264.7 cells were transfected with 15 µg of JAK2, JAK2-KD, or control plasmid (vector) by using 60 µg of Lipofectamine (Invitrogen, Carlsbad, CA) in a 100-mm dish according to the manufacturer's protocol. The cells were used for experiments 48 h after transfection.

LPS stimulation and inhibition of kinase activity. RAW264.7 cells were stimulated with 1 µg/ml repurified LPS at 37°C for the indicated time. LPS stimulation was stopped by the addition of ice-cold phosphate-buffered saline. The cells were then lysed for immunoprecipitation and immunoblotting.

The activation of JAK2, PI3K, and JNK was inhibited by pretreatment with AG490, LY-294002, or SP-600125, respectively. The cells were treated in the presence or absence of each concentration of the inhibitors for 1 h before they were stimulated with LPS. When the supernatants were collected, cell viabilities were demonstrated to be >95% for each experiment. AG490, LY-294002, and SP-600125 were each dissolved in dimethyl sulfoxide. To create positive controls for tyrosine-phosphorylated proteins, we incubated cells for 20 min at 37°C in the presence of 0.1 mM orthovanadate treated with 0.1 mM hydrogen peroxide.

Immunoprecipitation and immunoblotting. Cells were lysed in ice-cold NP-40 lysis buffer containing 1% Nonidet P-40, 25 mM Tris · HCl (pH 7.5), 150 mM sodium chloride, 1 mM EDTA, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride. For immunoprecipitation studies, cell lysates were mixed with the indicated antibodies for 1 h. Cell lysates were then mixed with protein G-coupled Sepharose beads and rotated for 1 h at 4°C. After the beads were washed three times with ice-cold NP-40 lysis buffer, the precipitated proteins were boiled for 5 min and eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. For the precipitation of total cell lysates, cells were lysed directly by the addition of SDS-PAGE sample buffer containing 2-mercaptoethanol.

Immunoprecipitated proteins and total cell lysates were separated by SDS-PAGE under reducing conditions and were electrically transferred to a polyvinylidene difluoride membrane. The membrane was blocked for 1 h at room temperature with 1% bovine serum albumin in Tris-buffered saline (TBS) buffer. The membrane was then incubated with the indicated antibody, and the reactive bands were visualized with an HRP-coupled secondary antibody via an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech) according to the manufacturer's procedures. Intensity of the signals was measured with a digital imaging system (Aisin Cosmos)

Anti-TLR4 antibody neutralization assay. RAW264.7 cells were incubated with 20 µg/ml of the inhibitory rat monoclonal antibody for murine TLR4, MTS510, or a control rat IgG antibody for 1 h at 37°C to bind the antibodies to TLR4 at the cell surface. MTS510 antibody recognizes the TLR4-MD2 complex and inhibits the binding of LPS to TLR4 (2). The cells were lysed 1 min after the addition of LPS to create immunoprecipitations for JAK2. The immunoprecipitated proteins were Western blotted for anti-phosphotyrosine or anti-JAK2. To analyze the effects of TLR4 on JNK activation, we lysed cells 30 min after LPS addition. Total cell lysates were then Western blotted for phospho-JNK or JNK1 antibody.

Cytokine production. RAW264.7 cells were seeded at a density of 1 x 105/ml in 24-well plates. To assess the effect of JAK2, PI3K, or JNK on IL-1{beta} production, we preincubated cells at 37°C for 1 h with or without AG490, LY-294002, or SP-600125 at each concentration. The supernatants were then collected 24 h after LPS stimulation. The concentration of IL-1{beta} was measured by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Biosource International, Camarillo, CA) with a microplate reader (Bio-Rad, Hercules, CA).

The cells transfected with JAK2, JAK2-KD, or the control plasmid (vector) were also seeded at the same density 24 h after transfection, and the supernatants were collected 24 h after LPS stimulation. The concentration of IL-1{beta} and IL-6 was then measured by the method described above.

Statistical analysis. Comparison of the two data sets was performed by unpaired Student's t-tests with SPSS for Windows (SPSS, Chicago, IL). Differences were considered significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LPS stimulation induces tyrosine phosphorylation of JAK2. To determine whether members of the JAK family, including JAK1, JAK2, JAK3, and TYK2, are tyrosine phosphorylated after LPS stimulation, RAW264.7 cells were stimulated with 1 µg/ml of LPS for the indicated times. Cell lysates were immunoprecipitated with antibody specific for JAK1, JAK2, JAK3, or TYK2, and the immunoprecipitates were analyzed by Western blotting with anti-phosphotyrosine antibody.

LPS stimulation induced rapid tyrosine phosphorylation of JAK2 (Fig. 1A), and JAK2 phosphorylation was detected 1 min after stimulation with LPS. Tyrosine phosphorylation of JAK2 was also detected in cells serum starved for 1 h before stimulation with 1 µg/ml LPS (data not shown). On the other hand, LPS stimulation resulted in no detectable tyrosine phosphorylation of JAK1, JAK3, or TYK2 (Fig. 1B); however, we did detect tyrosine phosphorylation of JAK1, JAK3, and TYK2 in cells treated with the tyrosine phosphatase inhibitor pervanadate (Fig. 1C). These results indicate that LPS induces tyrosine phosphorylation of only JAK2 in RAW264.7 cells.



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Fig. 1. Lipopolysaccharide (LPS) induces tyrosine phosphorylation of Janus kinase (JAK)2. A: RAW264.7 cells were stimulated at 37°C with 1 µg/ml repurified LPS for the indicated times. Cell lysates were immunoprecipitated (IP) with antibody to JAK2. The immunoprecipitated proteins for JAK2 were separated by electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with antibody (Ab) to phosphotyrosine (p-Tyr) (4G10) (top). The membrane was then stripped of Ab and reprobed with anti-JAK2 Ab (bottom). B: cell lysates stimulated with LPS were immunoprecipitated with other JAK family kinases, including JAK1, JAK3, and TYK2, and immunoprecipitated proteins were Western blotted for 4G10 (top) or Abs specific for JAK1, JAK3, and TYK2 (bottom). C: total cell lysates treated with 0.1 mM pervanadate were used as positive controls for tyrosine phosphorylation of JAK family kinases.

 

JAK2 regulates activation of JNK. LPS was shown previously to induce activation of JNK, and JNK plays a pivotal role in LPS-mediated signal transduction. Specifically, JNK is thought to regulate the expression of various stress-induced proteins and the production of inflammatory cytokines (17, 18, 26). To determine whether we could induce JNK phosphorylation with LPS, we analyzed whole cell lysates by Western blotting with a phosphospecific antibody for JNK. Figure 2A demonstrates that maximal JNK phosphorylation occurred 30 min after the addition of LPS.



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Fig. 2. JAK2 regulates phosphorylation of c-Jun NH2-terminal protein kinase (JNK) in LPS-induced signaling. A: RAW264.7 cells were stimulated with 1 µg/ml of LPS for the indicated times. Total cell lysates were blotted with a phosphospecific Ab for JNK (p-JNK). The membrane was stripped of Ab and reprobed with anti-JNK1 Ab. B: RAW264.7 cells were pretreated for 1 h with JAK inhibitor AG490 (0, 10, 30, or 100 µM), followed by stimulation with 1 µg/ml LPS for 30 min. Phosphorylation of JNK was detected by immunoblotting for JNK with phosphospecific Ab. AG490 treatment before LPS stimulation had little effect on the basal level of kinase phosphorylation. The membrane was stripped of Ab and reprobed with anti-JNK1 Ab. C: RAW264.7 cells transfected with wild-type JAK2 (WT), kinasedeficient JAK2 (KD), or vector control were stimulated with 1 µg/ml LPS for 30 min at 48 h after transfection. Phosphorylation of JNK was detected with phosphospecific JNK Ab (top). The membrane was reprobed with anti-JNK1 Ab to verify the amounts of the loaded proteins (bottom).

 

We next examined the effect of AG490, a JAK inhibitor, on the activation of JNK. Treatment with AG490 inhibited LPS-induced activation of JNK in a dose-dependent manner (Fig. 2B), and the addition of 100 µM AG490 completely abrogated JNK phosphorylation.

Several experiments suggested that AG490 acts as a JAK-specific inhibitor (9, 33). However, the possibility that AG490 nonspecifically inhibits the activities of PTKs other than JAK2 could not be completely excluded. Therefore, we transfected JAK2, JAK2-KD, or the control into RAW264.7 cells and compared the intensities of JNK phosphorylation after LPS stimulation. JAK2-KD was constructed by deleting the kinase domain of the JAK2 COOH terminus. JNK phosphorylation was augmented in the cells transfected with wild-type JAK2 compared with that in the control cells (Fig. 2C); however, LPS-induced phosphorylation of JNK was significantly inhibited in cells transfected with JAK2-KD. These results indicate that JAK2 is required for LPS-induced JNK activation.

Treatment with neutralizing antibody for TLR4 inhibits activation of JNK and JAK2 in LPS-induced signaling. TLR4 recognizes LPS through the cooperation of CD14 and MD2 (1, 46, 56). Because CD14 and MD2 possess no cytoplasmic domains, they are thought to be enhancers of TLR4 signaling. To confirm that both JNK and JAK2 are involved in signal transduction via TLR4, we examined the effect of an anti-TLR4 neutralizing antibody (MTS510) that recognizes the murine TLR4-MD2 complex and inhibits the binding of LPS to the receptors (2). The cells were pretreated at 37°C for 1 h with MTS510 or a control antibody. Phosphorylation of JNK was clearly observed in cells pretreated with a control antibody (Fig. 3A) but was decreased in cells pretreated with MTS510. LPS-induced tyrosine phosphorylation of JAK2 also was inhibited only in cells treated with MTS510 (Fig. 3B). These data demonstrate that LPS-induced phosphorylation of JNK and JAK2 is mediated by TLR4.



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Fig. 3. LPS induces phosphorylation of both JNK and JAK2 via Toll-like receptor (TLR)4. A: RAW264.7 cells were pretreated with 20 µg/ml anti-TLR4 antibody MTS510 or a control rat Ab to mouse IgG for 1 h, followed by stimulation with 1 µg/ml LPS for 30 min. The Ab recognizes the TLR4-MD2 complex and inhibits the binding of LPS to the receptors. Phosphorylation of JNK was detected with the phosphospecific JNK Ab (top). The membrane was then reprobed with anti-JNK1 Ab to verify the amount of loaded protein (bottom). B: RAW264.7 cells were pretreated with 20 µg/ml anti-TLR4 Ab MTS510 or a control rat Ab to mouse IgG for 1 h and stimulated with 1 µg/ml of LPS for 1 min. Cell lysates were immunoprecipitated with anti-JAK2 Ab, and immunoprecipitated proteins were probed with anti-phosphotyrosine (top) or anti-JAK2 (bottom).

 

PI3K is activated after LPS stimulation and inhibits phosphorylation of JNK. Because PI3K is involved in LPS-induced signaling, tyrosine phosphorylation of PI3K was examined via immunoprecipitation with anti-PI3K (p85) antibody and then blotting of the immunoprecipitated proteins with anti-phosphotyrosine antibody. This experiment revealed that stimulation with LPS induces the tyrosine phosphorylation of PI3K. Tyrosine phosphorylation of PI3K reached a maximum 1 min after LPS stimulation and gradually declined thereafter (Fig. 4A).



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Fig. 4. Phosphatidylinositol 3-kinase (PI3K) is tyrosine phosphorylated and positively regulates phosphorylation of JNK in LPS-induced signaling. A: RAW264.7 cells were serum starved for 1 h and then stimulated at 37°C with 1 µg/ml repurified LPS for the indicated times. Total cell lysates of RAW264.7 were immunoprecipitated with PI3K (p85) Ab and probed with anti-phosphotyrosine Ab 4G10 (top) or anti-PI3K (bottom). B: cells were pretreated in the presence or absence of PI3K inhibitor LY-294002 (1–30 µM) for 1 h at 37°C and then stimulated with 1 µg/ml LPS for 30 min. Total cell lysates were blotted with phosphospecific JNK Ab (top) or anti-JNK1 as control (bottom).

 

The roles of PI3K in LPS-induced signaling remain to be elucidated. In LPS-mediated signaling, PI3K is reported to act upstream or downstream of JNK (42). To examine whether PI3K acts upstream or downstream from JNK activation, RAW264.7 cells were pretreated in the presence or absence of LY-294002, a PI3K-specific inhibitor, and were stimulated with LPS for 30 min. JNK phosphorylation decreased in a dose-dependent manner in cells treated with LY-294002 (Fig. 4B). On the other hand, treatment with the JNK inhibitor SP-600125 had no differential effect on the tyrosine phosphorylation of PI3K (data not shown). These results demonstrate that PI3K positively regulates the activation of JNK.

JAK2 enhances PI3K tyrosine phosphorylation and association of JAK2 and PI3K. We sought to determine the functional roles of JAK2 in LPS-induced activation of macrophages. The major function of the JAK family kinases is generally considered to be activation of the signal transducer and activator of transcription (STAT) transcription factors; however, this function is not the sole role of the JAK family. The JAK family also has been directly implicated in the activation of several tyrosine kinases, such as PI3K (3, 55). Therefore, we speculated that JAK2 is located upstream from PI3K activation in the LPS-induced signaling pathway of RAW264.7 cells.

First, we examined whether AG490 treatment inhibits tyrosine phosphorylation of PI3K. Cells were incubated for 1 h in the presence or absence of 30 µM AG490 and then stimulated with LPS for 1 min. The results demonstrated that tyrosine phosphorylation of PI3K decreased in cells treated with AG490 (Fig. 5A).



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Fig. 5. JAK2 enhances LPS-induced tyrosine phosphorylation of PI3K and the association of PI3K and JAK2. A: RAW264.7 cells were pretreated with or without 30 µg/ml AG490 for 1 h and stimulated with 1 µg/ml LPS for 1 min. Cell lysates were immunoprecipitated with anti-PI3K (p85) Ab and blotted with either anti-phosphotyrosine (top) or anti-p85 Ab as control (bottom). B: RAW264.7 cells transfected with kinase-deficient JAK2 or vector control were stimulated with 1 µg/ml LPS for 30 min at 48 h after transfection. Cell lysates were immunoprecipitated with anti-PI3K (p85) Ab and blotted with either anti-phosphotyrosine (top) or anti-p85 Ab as control (bottom). C: RAW264.7 cells were pretreated in the presence or absence of 30 µM AG490 for 1 h and then stimulated with 1 µg/ml LPS for 1 min. Total cell lysates were immunoprecipitated with anti-JAK2 and blotted with anti-p85 Ab (top) or anti-JAK2 Ab (bottom).

 

Next, we examined whether LPS-induced tyrosine phosphorylation of PI3K is decreased in cells transfected with JAK2-KD. As shown in Fig. 5B, tyrosine phosphorylation of PI3K was suppressed in cells transfected with JAK2-KD. These results confirm that JAK2 regulates PI3K tyrosine phosphorylation.

To determine whether JAK2 forms a complex with PI3K and affects the association of PI3K and JAK2, cells were pretreated in the presence or absence of AG490 and stimulated with LPS. Cell lysates were immunoprecipitated with anti-JAK2 antibody, and the immunoprecipitates were immunoblotted with anti-PI3K (p85) antibody. Figure 5C shows that LPS stimulation augments the association of JAK2 with PI3K; however, pretreatment with AG490 did not enhance the formation of a JAK2-PI3K complex after LPS stimulation. These results indicate that JAK2 acts upstream of PI3K and that JAK2 regulates the tyrosine phosphorylation of PI3K and enhances the association of JAK2 and PI3K in LPS-induced signaling pathways.

JAK2 plays a pivotal role in production of proinflammatory cytokines. MAPKs were reported previously (17, 21, 49) to play an important role in cytokine production. To examine whether JNK is involved in IL-1{beta} production, we pretreated cells with or without the JNK inhibitor SP-600125 for 1 h before LPS stimulation and collected the supernatants 24 h after the addition of LPS. To evaluate the functional roles of JNK, we measured IL-1{beta} concentrations with a commercially available ELISA kit for IL-1{beta}. Figure 6A shows that, compared with control cells, the production of IL-1{beta} was not inhibited in the cells treated with 1 µM SP-600125. However, the levels of IL-1{beta} significantly decreased in cells treated with 10 µM SP-600125. These results indicate that JNK is involved in IL-1{beta} production.



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Fig. 6. JAK2 regulates LPS-induced proinflammatory cytokine production. A: RAW264.7 cells were pretreated for 1 h with AG490, LY-294002, SP-600125, or dimethyl sulfoxide (control) at the indicated concentrations. Supernatants were collected in serum-free medium 24 h after LPS stimulation. Samples were analyzed for mouse IL-1{beta} with a commercial enzyme-linked immunosorbent assay ELISA kit (Biosource) according to the manufacturer's protocol. Inhibitors used in these experiments had no effect on basal level of IL-1{beta} production. Data represent means ± SD of 3 independent experiments; *P < 0.05 compared with control. B: RAW264.7 cells were pretreated for 1 h with AG490, LY-294002, SP-600125, AG490 + LY-294002, AG490 + LY-294002 + SP-600125, or dimethyl sulfoxide (control) at the indicated concentrations. The production of IL-1{beta} was measured as described in A. C and D: RAW264.7 cells were transfected with 15 µg of wild-type JAK2 (JAK2-WT), kinasedeficient JAK2 (JAK2-KD), or the control vector with 60 µg of Lipofectamine (Invitrogen) according to the manufacturer's protocol. Transfected cells were stimulated with LPS 48 h after transfection. The supernatants were collected in serum-free medium 24 h later. Samples were analyzed for mouse IL-1{beta} (C) or IL-6 (D) with the ELISA kit described in A according to the manufacturer's protocol. Data represent means ± SD of 3 independent experiments; *P < 0.05 compared with control.

 

We have shown (Fig. 4) that PI3K acts upstream of JNK activation in LPS-induced signaling. We evaluated whether PI3K is also involved in IL-1{beta} production: RAW264.7 cells were treated in the presence or absence of LY-294002 for 1 h before stimulation with LPS, and supernatants were collected 24 h later. The concentration of IL-1{beta} was measured with the same ELISA kit. Figure 6A demonstrates that treatment with LY-294002 inhibited the production of IL-1{beta} in a dose-dependent manner.

We next examined whether JAK2 is also involved in IL-1{beta} production, because we have already shown (Figs. 2 and 5) that JAK2 acts upstream of PI3K and JNK activation in LPS-induced signaling. Cells were treated in the presence or absence of 10 or 30 µM AG490 for 1 h before LPS stimulation, and supernatants were collected 24 h later. The concentration of IL-1{beta} was measured by the same method. Treatment with 10 µM AG490 weakly inhibited the production of IL-1{beta}. However, treatment with 30 µM AG490 significantly inhibited the production of IL-1{beta} (Fig. 6A).

We also examined whether treatment with a combination of two or three inhibitors has additive or synergistic effects on the production of IL-1{beta}. We pretreated cells with AG490 alone, LY-294002 alone, SP-600125 alone, AG490 plus LY-294002, AG490 plus LY-294002 plus SP-600125, or DMSO for 1 h before LPS stimulation and collected the supernatants 24 h after the addition of LPS. We measured IL-1{beta} concentrations with a commercially available ELISA kit for IL-1{beta} as described above. The amount of IL-1{beta} production was almost the same in the cells treated with AG490 alone as in the cells treated with the combination of AG490 plus LY-294002 and those treated with AG490 plus LY-294002 plus SP-600125 (Fig. 6B).

We then evaluated whether JAK2-KD inhibits IL-1{beta} production. Cells transfected with JAK2, JAK2-KD, or the control, were stimulated with LPS 48 h after transfection, and supernatants were collected 24 h later. IL-1{beta} concentration was measured as described above. Compared with cells transfected with the control vector, the production of IL-1{beta} was inhibited in cells transfected with JAK-KD (Fig. 6C). On the other hand, IL-1{beta} production was augmented in cells transfected with wild-type JAK2. These results demonstrate that JAK2 is also involved in IL-1{beta} production in the LPS-mediated signaling pathway.

Finally, we evaluated whether JAK2 is involved in the production of other proinflammtory cytokines. To establish whether JAK2 is also involved in the production of IL-6, cells transfected with JAK2, JAK2-KD, or the control were stimulated with LPS 48 h after transfection and supernatants were collected 24 h later. IL-6 concentration was measured as described above. Compared with cells transfected with the control vector, the production of IL-6 was inhibited in cells transfected with JAK2-KD (Fig. 6D). On the other hand, IL-6 production was augmented in cells transfected with wild-type JAK2. These results demonstrate that JAK2 is also involved in IL-6 production in the LPS-mediated signaling pathway (Fig. 6D).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
How the activation of signal-transducing molecules mediates the variety of biological responses to LPS via TLR4 remains unclear, although activation of tyrosine kinases is clearly necessary for a number of these biological responses to LPS (13, 15, 17, 37).

JAK2 is reported to be involved in the expression of iNOS in fetal skin-derived dendritic cells treated with LPS via NF-{kappa}B, as demonstrated with AG490 (10). In contrast, JAK2 has been shown to inhibit the LPS-induced expression of iNOS in vascular smooth muscle cells (30). The same study also showed that JAK2 appears to have a positive role in iNOS induction in RAW264.7 macrophages, as demonstrated with AG490 (30). However, the molecular mechanisms through which JAK2 is activated and transduces the signals to the downstream molecules remain to be determined. In this study we demonstrate the molecular mechanisms through which JAK2 is activated and transduces the signals to the production of proinflammatory cytokines. First, we have shown clearly that JAK2 is activated via TLR4. Second, we have identified the signaling cascade in which JAK2 is involved and have shown that JAK2 regulates phosphorylation of JNK primarily through activation of PI3K. Third, we have demonstrated that JAK2 is involved in the production of proinflammatory cytokines in macrophages.

We have shown that treatment with AG490 decreases JNK phosphorylation in a dose-dependent manner and that JAK2-KD inhibits JNK phosphorylation. These results demonstrate that the phosphorylation of JNK is an event occurring downstream of JAK2 activation. To determine whether both JNK and JAK2 are phosphorylated via TLR4, we examined phosphorylation of JNK and JAK2 in cells treated with anti-TLR4 neutralizing monoclonal antibody after LPS stimulation. This antibody recognizes the murine TLR4-MD2 complex and inhibits the binding of LPS to TLR4 (2). Our results demonstrating that phosphorylation of JNK and JAK2 is inhibited by treatment with anti-TLR4 neutralizing monoclonal antibody imply that phosphorylation of both JAK2 and JNK is mediated by TLR4. The intensity of the signal of the phosphorylated JNK or JAK2 was measured with a digital imaging system (Aisin Cosmos), which showed that phosphorylation of JNK was inhibited by ~75% because of the treatment with MTS510, as averaged over three experiments. LPS-induced tyrosine phosphorylation of JAK2 was inhibited by ~85% in cells treated with MTS510. This difference is probably due to the sensitivity of detecting the phosphorylated proteins, in particular the sensitivity of the antibodies. However, the possibility that phosphorylation of JNK is regulated in part by molecules other than JAK2 cannot be completely excluded. Repurification of LPS by phenol extraction was shown to eliminate the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice or those transfected with only TLR2 (29). Consequently, we repurified LPS by the phenol extraction method of Hirschfeld et al. (19) to exclude the possibility that cells were stimulated by contaminated endotoxin proteins via TLR2. These results indicate that LPS induces phosphorylation of JAK2 and JNK via TLR4.

LPS stimulation activates the PI3K pathway, although the molecular mechanisms by which PI3K is activated in LPS-induced signaling have not been elucidated. Activated PI3K allows the docking of protein kinase B/Akt that leads to the activation of downstream molecules. JAK2 is directly implicated in the activation of PI3K in several signal transduction cascades, and growth hormone or prolactin stimulates PI3K activation via JAK2 (55). The activation of PI3K is also mediated by JAK2 in GM-CSF-activated signaling pathways (3). We demonstrated in the present study that LPS-induced tyrosine phosphorylation of PI3K is inhibited by treatment with JAK inhibitor and transfection of JAK2-KD. These results indicate that JAK2 also activates PI3K in LPS-mediated signaling.

The role of PI3K in LPS-induced signaling remains controversial. The PI3K pathway has been shown to activate MAPKs and NF-{kappa}B-dependent gene expression. A recent study demonstrated that the PI3K/Akt pathway positively regulates NF-{kappa}B-dependent gene expression (21, 51). On the other hand, the PI3K pathway was shown to negatively regulate LPS-induced signaling. Inhibition of the PI3K/Akt pathway was recently reported to enhance LPS-induced activation of the MAPK pathways and downstream targets AP1 and Egr1 (16). In our experiments, treatment with LY-294002 inhibited JNK phosphorylation but tyrosine phosphorylation of PI3K was not inhibited in cells treated with the JNK inhibitor (data not shown). Our results indicate that PI3K is involved in the activation of JNK and support the idea that the PI3K pathway activates MAPKs. What is responsible for these differences remains to be determined. However, the differences may reflect the use of different cell types and different experimental conditions. Furthermore, JAK2 regulates phosphorylation of JNK, probably via PI3K, because JAK2 does regulate the tyrosine phosphorylation of PI3K.

JNK/stress-activated protein kinase (SAPK) is reported to be required for the LPS-induced translation of the mRNA of several cytokines (17, 21, 49). We speculate that both JAK2 and PI3K are involved in the production of cytokines such as IL-1{beta}, because activation of JNK is regulated by JAK2. We have demonstrated that inhibiting the kinase activity of JAK2 or PI3K suppresses IL-1{beta} production. SP-600125 also inhibited the release of IL-1{beta} (6). These results indicate that JAK2, PI3K, and JNK are all involved in the LPS-induced production of IL-1{beta}. Because the suppression level of IL-1{beta} production in the cells treated with a combination of two or three inhibitors was almost the same as that in the cells treated with AG490 alone, JAK2 regulates IL-1{beta} production primarily via PI3K and JNK. Although the molecules acting upstream of JAK2 in LPS-induced signaling remain unclear, the JAK2/PI3K/JNK cascade is clearly pivotal in the production of IL-1{beta} in LPS-induced signaling in RAW264.7 cells.

We could not detect the LPS-induced tyrosine phosphorylation of other JAK family kinases. Because we could detect tyrosine phosphorylation of these kinases in cells treated with pervanadate, our inability to detect their tyrosine phosphorylation after stimulation with LPS cannot be the result of our experimental conditions. AG490 is reported to be a JAK-specific inhibitor (33). AG490 has also been reported to have no effect on the activation of several other tyrosine kinases, including Lck, Lyn, Btk, Syk, and Src (33). However, AG490 has been reported to inhibit cyclin-dependent kinases (23) and to act as a partial blocker of c-Src tyrosine kinase activity (38). Because the possibility that AG490 inhibits the activity of other PTKs in a nonspecific manner cannot be completely excluded, we proved that JAK2-KD inhibits the phosphorylation of JNK or PI3K and the production of IL-1{beta}. We also demonstrated that JAK2 is involved in the production of another proinflammatory cytokine, IL-6.

We have shown that JAK2 regulates the production of IL-1{beta} positively in RAW264.7 cells. JAK2 was previously reported to be involved in the LPS-induced expression of iNOS in skin-derived dendritic cells and RAW 264.7 (10, 11, 30). In contrast, the JAK2 pathway suppresses LPS-stimulated iNOS induction in vascular smooth muscle cells (30). The cause of this difference remains to be determined. However, we speculate that the signaling molecules located downstream of JAK2 might determine whether JAK2 regulates the production of inflammatory cytokines or iNOS positively or negatively.

Stimulation with cytokines or LPS induces the suppressor of cytokine signaling (SOCS) proteins (48). Recently, SOCS-1 was shown to be rapidly induced by LPS and to negatively regulate LPS signaling (22, 36). Nakagawa et al. (36) demonstrated that cotransfection of COS7 cells with SOCS-1 and IRAK revealed the association of SOCS-1 with IRAK via the SH2 region of SOCS-1. However, Kinjyo et al. (22) could not detect the binding of SOCS-1 to TRAF6, IRAK, IKK, or TAK1. Because a role for SOCS proteins in the negative regulation of JAK kinases has been proven (24, 52), SOCS proteins may regulate the activation of JAK2 in LPS-induced signaling.

In the present study, we have demonstrated that LPS induces tyrosine phosphorylation of JAK2 via TLR4. Furthermore, JAK2 is required for the production of IL-1{beta} via the activation of the PI3K/JNK pathway. Thus LPS-induced activation of the JAK2 PTK pathway in macrophages is crucially important. An understanding of how LPS-induced signal transduction pathways are responsible for innate immunity may help in the development of improved treatments for the inflammatory responses that lead to sepsis and multiorgan failure.


    DISCLOSURES
 
This work was supported in part by a grant-in-aid for scientific research to Y. Ota from the Japanese Ministry of Education, Culture, Sports, Science and Technology.


    ACKNOWLEDGMENTS
 
We thank T. Kadowaki, University of Tokyo, for cDNAs of wild-type and dominant-negative JAK2. We are also grateful to L. E. Samelson (National Cancer Institute, National Institutes of Health) and K Okumura (Juntendo University) for critical reading of the manuscript. We also thank M. Kataoka for preparing the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Ota, Dept. of Infectious Disease, Graduate School of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (E-mail: yasuota-tky{at}umin.ac.jp).

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.


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