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Originally published In Press as doi:10.1074/jbc.M408444200 on October 13, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54023-54031, December 24, 2004
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The Function of Mitogen-activated Protein Kinase Phosphatase-1 in Peptidoglycan-stimulated Macrophages*

Edward G. Shepherd, Qun Zhao, Stephen E. Welty, Thomas N. Hansen, Charles V. Smith, and Yusen Liu{ddagger}

From the Center for Developmental Pharmacology and Toxicology, Children's Research Institute, Children's Hospital, the Department of Pediatrics, Ohio State University, Columbus, Ohio 43205

Received for publication, July 26, 2004 , and in revised form, October 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitogen-activated protein (MAP) kinases play a pivotal role in the macrophages in the production of proinflammatory cytokines triggered by lipopolysaccharides. However, their function in the responses of macrophages to Gram-positive bacteria is poorly understood. Even less is known about the attenuation of MAP kinase signaling in macrophages exposed to Gram-positive bacteria. In the present study, we have investigated the regulation of MAP kinases and the role of MAP kinase phosphatase (MKP)-1 in the production of pro-inflammatory cytokines using murine RAW264.7 and primary peritoneal macrophages after peptidoglycan stimulation. Treatment of macrophages with peptidoglycan resulted in a transient activation of JNK, p38, and extracellular signal-regulated kinase. Most interestingly, MKP-1 expression was potently induced by peptidoglycan, and this induction was concurrent with MAP kinase dephosphorylation. Triptolide, a diterpenoid triepoxide, potently blocked the induction of MKP-1 by peptidoglycan and prolonged the activation of JNK and p38. Overexpression of MKP-1 substantially attenuated the production of tumor necrosis factor (TNF)-{alpha} induced by peptidoglycan, whereas knockdown of MKP-1 by small interfering RNA substantially increased the production of both TNF-{alpha} and interleukin-1{beta}. Finally, we found that in primary murine peritoneal macrophages, MKP-1 induction following peptidoglycan stimulation also coincided with inactivation of JNK and p38. Blockade of MKP-1 induction resulted in a sustained activation of both JNK and p38 in primary macrophages. Our results reveal that MKP-1 critically regulates the expression of TNF-{alpha} and interleukin-1{beta} in RAW264.7 cells and further suggest a central role for this phosphatase in controlling the inflammatory responses of primary macrophages to Gram-positive bacterial infection.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sepsis represents a major challenge to the health care system, affecting about 751,000 people, causing ~215,000 deaths, and costing nearly $17 billion annually in the United States (1). According to a recent report, the incidence of sepsis is rising at an astonishing annual rate of ~8.7%, despite substantial prevention efforts and advancements in treatment (1, 2). Moreover, Gram-positive bacteria have become the predominant organisms in sepsis cases since 1987 and have accounted for more than 52% of all cases of sepsis in 2000 (1). Staphylococcus aureus is a leading cause of nosocomial pneumonia and wound infections and represents one of the bacteria most commonly isolated from patients with sepsis (1, 2). For decades, group B Streptococcus has been the single most frequent cause of sepsis in newborns, and it remains a primary cause of neonatal morbidity and mortality (3, 4). Streptococcus pneumoniae is the leading agent causing invasive bacterial infections in children and is among the most common bacteria causing meningitis in children and young adults (5). Recently, multidrug-resistant species of Gram-positive bacteria have emerged, further exacerbating the threat to public health (6).

Sepsis is associated with important hemodynamic alterations, including decreased central blood volume, systolic alterations of ventricular function, and severe peripheral vasodilatation leading to profound alterations in blood distribution (7). Although the mechanisms leading to hemodynamic disturbances and organ failure in patients with severe sepsis are not yet fully understood, pro-inflammatory cytokines, such as TNF-{alpha},1 IL-1{beta}, and IL-6, appear to play an important role in mediating the pathophysiological process (8–10). As critical constituents of the antimicrobial arsenal produced primarily by macrophages, these inflammatory cytokines induce local inflammation and recruit neutrophils to the infection site, thereby containing the invading pathogens (11, 12). Moreover, pro-inflammatory cytokines are vital for the initiation of the acute-phase responses in the liver and contribute to the induction of adaptive immunity mediated by lymphocytes (13). Although these cytokines play a critical role in host defense against pathogenic infection, their overproduction can cause septic shock, multiple organ dysfunction syndrome, and even death (14). The critical roles of TNF-{alpha} in immune defense and in the pathogenesis of sepsis are highlighted by findings illustrating that mice lacking a TNF-{alpha} receptor gene are resistant to septic shock but unable to control local bacterial infection (15, 16). In addition to acute inflammatory disorders, excessive release of pro-inflammatory cytokines is also implicated in a variety of chronic inflammatory diseases including Crohn's disease, rheumatoid arthritis, psoriasis, asthma, and systemic lupus erythematosus (17). Thus, both the induction and termination of pro-inflammatory cytokine production are crucial for maintaining an appropriate immune defense while avoiding potentially devastating pathological consequences.

Cytokine biosynthesis in macrophages relies on a series of signal transduction cascades initiated by microbial components through Toll-like receptors (TLRs) (18–20). These pathways are most thoroughly studied with regard to lipopolysaccharides (LPS), a cell wall component of Gram-negative bacteria (18). Recognition of LPS by TLR-4 triggers a cascade of signaling events that lead to activation of transcription factor NF-{kappa}B and the MAP kinase pathways, including extracellular signal-regulated kinase (ERK), JNK, and p38 subfamilies (18, 21). The MAP kinase family plays a crucial role in mediating the induction of pro-inflammatory cytokines, including TNF-{alpha}, IL-1{beta}, and IL-6, through multiple mechanisms involving both transcriptional and post-transcriptional regulatory events (18, 21, 22). Peptidoglycan (PepG) and lipoteichoic acid are two major cell wall components of Gram-positive bacteria (23, 24). Unlike LPS that is recognized by TLR-4, both PepG and lipoteichoic acid are recognized by TLR-2 (20, 25, 26). Both PepG and lipoteichoic acid have been reported to activate MAP kinases and induce production of inflammatory cytokines (2, 27–29), yet the roles of MAP kinases in this process are still not well defined. Also poorly understood are the mechanisms responsible for terminating the MAP kinase cascades during the macrophage responses to Gram-positive bacteria. Previously, we have demonstrated that MKP-1 acts as a feedback control mediator and serves to restrain the production of pro-inflammatory cytokines in LPS-stimulated macrophages (30). In the present report, we have studied the role of MKP-1 during the macrophage responses to Gram-positive bacteria by using PepG and murine RAW264.7 or primary macrophages as a model system. We found that MKP-1 was highly induced by PepG, and its induction coincided with the inactivation of both JNK and p38. MKP-1 overexpression inhibited the production of TNF-{alpha} induced by PepG, whereas down-regulation of MKP-1 by siRNA increased the production of both TNF-{alpha} and IL-1{beta}. By using thioglycollate-elicited murine peritoneal macrophages, we demonstrated that MKP-1 induction occurred concomitantly with inactivation of p38 and JNK, whereas suppressing MKP-1 induction with triptolide delayed the inactivation of these MAP kinases. Our results suggest that MKP-1 plays an important role in controlling the inflammatory responses of macrophages to Gram-positive bacterial infection.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mice—Pathogen-free female C57BL6 and C3H/HeN mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). The TLR-4-deficient female C3H/HeJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). These mice were maintained at 24 °C in an atmosphere with relative humidity between 30 and 70% on a 12-h day-night rhythm on Harlan Tecklad irradiated diet (Harlan Sprague-Dawley). All animals received humane care in accordance with the National Institutes of Health guidelines and were sacrificed by CO2 inhalation.

Reagents—PepG, isolated from S. aureus (Sigma), was dissolved in PBS through sonication and added to the culture medium to the indicated concentrations. Triptolide (Calbiochem) was dissolved in Me2SO and added to the culture medium to the indicated concentrations. The MEK inhibitor U0126 (Promega, Madison, WI), the JNK inhibitor SP600125 (Biomol, Plymouth Meeting, PA), and the p38 inhibitor SB203580 (Calbiochem) were dissolved in Me2SO and added to the medium 30 min before the addition of PepG. Polymyxin B (Sigma) was dissolved in serum-free medium.

Plasmids—The mammalian expression vector pSR{alpha}-FLAG and pSR{alpha}-FLAG-MKP-1 are as described previously (30, 31). The mammalian expression vector pDsRed2 was purchased from Clontech. The JIP-1 expression vector was kindly provided by Dr. Roger Davis (32). To construct a vector expressing a hairpin small interfering RNA (siRNA) against mouse MKP-1, a pair of complementary oligonucleotides (5'-TCGAGGTCTTCTTTCTCCAAGGAGTTCAAGAGACTCCTTGGAGAAAGAAGACTTTTT-3' and 5'-CTAGAAAAAGTCTTCTTTCTCCAAGGAGTCTCTTGAACTCCTTGGAGAAAGAAGACC-3') was designed, synthesized by Integrated DNA Technologies (Coralville, IA), and annealed. The resultant double-stranded oligonucleotides were cloned between the SalI and XbaI sites of pSuppressorNeo (Imgenex, San Diego, CA) using standard molecular biology techniques (33). The authenticity of the plasmid construct was confirmed by sequencing.

Isolation of Peritoneal Macrophages—Thioglycollate-elicited peritoneal macrophages were isolated from C3H/HeN, C3H/HeJ, or C57BL6 mice by peritoneal lavage as described previously (34). Briefly, mice were injected intraperitoneally (2 ml/mouse) with 3% Brewer Thioglycollate Medium (BD Diagnostic, Sparks, MD). Four days later, cells were harvested by lavage with cold RPMI 1640 medium (Invitrogen) containing 5% FBS (HyClone Laboratories, Logan, UT). Peritoneal cells were recovered by centrifugation, resuspended in RPMI 1640 medium containing 5% FBS, and plated into tissue culture plates. Cells were allowed to adhere for 2 h, washed free of nonadherent cells, and maintained in RPMI 1640 medium containing 5% FBS.

Cell Culture and Transfection—RAW264.7 cells were purchased from American Tissue Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2. RAW264.7 cells were transfected with pcDNA3 (Invitrogen) together with either the pSR{alpha}-FLAG-MKP-1, or the empty vector pSR{alpha}, or with the MKP-1 siRNA expression plasmid, using FuGENE 6 transfection reagent (Roche Diagnostics) according to the manufacturer's specifications. After transfection, cells were selected in medium containing 500 µg/ml of G418 (Roche Diagnostics) for 2 weeks, and individual G418-resistant clones were isolated. These clones were propagated and screened for MKP-1 expression by Western blot analyses either using a monoclonal antibody against the FLAG epitope (Sigma) or using a rabbit polyclonal antibody against MKP-1 (Santa Cruz Biotechnology, Santa Cruz, CA). Stable RAW264.7 clones expressing either FLAG-tagged MKP-1 or an MKP-1 siRNA or carrying the empty vector were maintained in medium containing 120 µg/ml G418.

To examine the effect of JIP-1 overexpression on PepG-stimulated TNF-{alpha} production, RAW264.7 cells were transiently transfected with pDsRed2 (encoding a red fluorescent protein) together with either pcDNA3 or the JIP-1 expression vector at a ratio of 1:10 using Lipofectamine 2000 (Invitrogen), according to the manufacturer's recommendation. Sixteen hours later, cells were trypsinized, and DsRed-positive cells were isolated through fluorescence-activated cell sorting. These DsRed-positive cells were plated into 6-well plates and stimulated with 10 µg/ml PepG on the next day.

Western Blotting and ELISA—To isolate total lysate protein, cells were harvested in a lysis buffer containing 10 mM HEPES (pH 7.4), 50 mM {beta}-glycerophosphate, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 10 mM NaF, 1 mM Na3VO4, 20 nM microcystin-LR, 2 µM leupeptin, 2 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Western blot analysis was essentially conducted as described previously by using ECL reagent (Amersham Biosciences) (30, 35). MKP-1 levels were assessed by Western blotting using a rabbit polyclonal antibody (Santa Cruz Biotechnology). Phosphorylated ERK, JNK, and p38 were detected using rabbit polyclonal phospho-specific antibodies from Cell Signaling Technology (Beverly, MA). Total JNK1 levels were determined by Western blot analysis using a polyclonal antibody against JNK1 (Santa Cruz Biotechnology). Total p38 was detected using a monoclonal antibody (Transduction Laboratories). The levels of active MAP kinase-activated protein kinase-2 (hereafter referred to as MK2) were determined by Western blotting using a rabbit polyclonal antibody (Cell Signaling). FLAG-tagged MKP-1 was detected using a monoclonal antibody (clone M2) against FLAG (Sigma). {beta}-Actin was detected using a monoclonal antibody purchased from Sigma. IL-1{beta} in the cell lysates was detected by Western blot analysis using a rabbit polyclonal antibody against mouse IL-1{beta} (Chemicon, Temecula, CA). TNF-{alpha} concentrations in the culture medium were measured using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations.

Immune Complex Kinase Assays—JNK1 and MK2 activities were measured by immune complex kinase assays as described previously (36–38). Briefly, endogenous MK2 was immunoprecipitated from 500 µg of RAW264.7 lysate protein using 3 µg of rabbit polyclonal antiserum (kindly provided by Dr. Jacques Landry, l'Université Laval, Quebec, Canada) and protein A-Sepharose (Amersham Biosciences). Endogenous JNK1 was immunoprecipitated from 500 µg of cell lysates using 1 µg of a rabbit polyclonal antibody against JNK1 (Santa Cruz Biotechnology) and protein A-Sepharose (36). After extensive washing, the kinase activity in the MK2 immune complexes was assayed using [{gamma}-32P]ATP and recombinant heat shock protein 25 (HSP25) (Stressgen Biotechnologies, Victoria, Canada) as a substrate (37, 38). JNK1 activity was assayed using recombinant GST-c-Jun-(1–143) as a substrate (39).

Northern Blot Analysis—Total RNA was isolated with STAT-60 (Tel-Test, Friendswood, TX). Northern blot analysis was performed using a mouse MKP-1 cDNA as a probe, as described previously (30, 40). The membrane was stripped and reprobed with an oligonucleotide corresponding to 18 S rRNA to normalize for differences in sample loading (30, 40).

Statistics—The results from the experiments assessing the effects of MAP kinase or MKP-1 modulation on TNF-{alpha} production were analyzed by one-, two-, or three-way analysis of variance with the least significant difference post hoc test, using an SPSS statistical software program (Aspire Software International, Leesburg, VA). The TNF-{alpha} values were transformed to a logarithmic scale to normalize the variances. All comparisons are interpreted as being statistically significant at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of RAW264.7 Macrophages with PepG Causes a Transient Activation of MAP Kinases—To investigate the effect of PepG on MAP kinases in macrophages, subconfluent RAW264.7 cells were stimulated with 10 µg/ml PepG, and cell lysates were harvested after specified periods. The activities of ERK and p38 were assessed by Western blot analyses using phospho-specific antibodies that recognize either active ERK or active p38 (Fig. 1A). Both ERK and p38 were activated rapidly in response to PepG, with peak activities attained at about 30 min after the addition of PepG. p38 activity then gradually decreased, returning to close to basal levels by 2 h. In contrast, ERK activity induced by PepG was sustained, although slight decreases in phospho-ERK levels were observed after 2 h. Western blot analysis of these blots with housekeeping protein {beta}-actin confirmed equivalent protein loading among all the samples. The kinetics of JNK1 activation was examined by immune complex kinase assays, using a recombinant GST-c-Jun protein as a substrate. As observed with p38, JNK1 was rapidly activated in response to PepG treatment, as indicated by the incorporation of 32P into GST-c-Jun (Fig. 1B, lower panel). Although significant activation of JNK1 was detected as early as 15 min after PepG treatment, maximal activity of JNK1 was observed between 30 and 45 min. As with p38, JNK1 activity was rapidly attenuated after the initial peak (Fig. 1B). Western blot analysis of the cell lysates indicated that the total JNK1 protein levels did not change with PepG treatment (Fig. 1B, upper panel).



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  		FIG. 1.
PepG transiently activates MAP kinases in macrophages. A, activation kinetics of ERK and p38. Western blot analysis was also performed using an antibody against {beta}-actin to control for sample loading. B, kinetics of JNK1 activation. Total JNK1 protein was detected by Western blot analysis using an antibody against total JNK1 (upper panel). JNK1 activity was assessed by immune complex kinase assays using [{gamma}-32P]ATP and recombinant GST-c-Jun as a substrate (lower panel). The activity was quantitated using a PhosphorImager and presented as "fold relative to control."

 
Inactivation of JNK and p38 in PepG-stimulated RAW264.7 Macrophages Correlates with Accumulation of MKP-1 Protein—MKP-1 is a dual specificity protein phosphatase, widely expressed in a variety of cell types (41–43). Recent studies have indicated that MKP-1 protein preferentially inactivates p38 and JNK (44). To investigate whether MKP-1 plays a role in the dephosphorylation of JNK and p38 after PepG stimulation, the kinetics of MKP-1 protein induction after PepG stimulation was examined by Western blot analysis (Fig. 2A). MKP-1 protein levels were practically undetectable in control cells. Upon PepG treatment, MKP-1 protein levels gradually increased, becoming detectable by 45 min and reaching peak levels between 1 and 2 h. The reciprocal relationship between MKP-1 induction and inactivation of both JNK and p38 supported the notion that MKP-1 may be involved in the dephosphorylation of these kinases, thereby contributing to the termination of the signaling pathways driving cytokine synthesis.



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  		FIG. 2.
PepG stimulation potently induces MKP-1 expression. RAW264.7 cells were stimulated with PepG (10 µg/ml). A, increase of MKP-1 protein levels following PepG stimulation. To control for loading, the membrane was stripped and blotted with a {beta}-actin antibody. B, induction of MKP-1 mRNA by PepG stimulation. The same membrane was stripped and re-hybridized with a probe corresponding to 18 S rRNA (lower panel). The MKP-1 mRNA signals were normalized to 18 S rRNA and expressed as fold relative to control.

 
To understand the mechanisms of MKP-1 induction by PepG, MKP-1 mRNA levels were examined by Northern blot analysis (Fig. 2B). Mirroring the pattern of MKP-1 protein expression, basal MKP-1 mRNA levels were essentially undetectable in control cells. Upon PepG stimulation, the MKP-1 mRNA levels rapidly increased, reaching maximum peak by 1 h. These levels substantially decreased by 2 h, but remained elevated compared with base line for at least 6 h.

It has been shown that in response to LPS stimulation, MK2 is phosphorylated by p38 and plays an important role in mediating the production of pro-inflammatory cytokines (45–48). As MK2 is a downstream target of p38, we examined whether MKP-1 induction was correlated with down-regulation of MK2 activity. The effect of PepG treatment on MK2 activity was examined by Western blot analysis using an antibody that specifically recognizes MK2 phosphorylated at residue Thr-334 (Fig. 3A, upper panel). Upon PepG stimulation, MK2 was rapidly phosphorylated at Thr-334 with kinetics similar to that of p38 activation. Comparable loading of the sample proteins in the Western blot was verified by immunoblotting with an antibody against {beta}-actin (Fig. 3A, lower panel). Consistent with the substantial increase in MK2 phosphorylation, the kinase activity of MK2 was markedly increased upon PepG treatment, as indicated by the immune complex kinase assays, using recombinant HSP25 and [{gamma}-32P]ATP as substrates (Fig. 3B). Like p38, both the phosphorylation and the kinase activity of MK2 were diminished by 2 h after PepG treatment (Fig. 3). These results are consistent with the notion that MKP-1 may play an important role in controlling the activity of MK2.



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  		FIG. 3.
PepG induces a transient MK2 activation. RAW264.7 cells were stimulated with PepG (10 µg/ml). A, kinetics of MK2 phosphorylation. Western blot analysis on the samples was also carried out using a {beta}-actin antibody to control for loading. B, kinetics of MK2 activation. MK2 activity was assessed by immune complex kinase assays using [{gamma}-32P]ATP and recombinant HSP25 as a substrate. The activity was quantitated using a PhosphorImager and presented as fold relative to control.

 
Previously, it has been reported that another commercially available bacterial cell wall component isolated from S. aureus, lipoteichoic acid, contains endotoxin that contributes to activation of mouse macrophages (49). To rule out the possibility that potential LPS contamination in the PepG preparation could obscure the results of our observation, we took two approaches to eliminate the effect of potential LPS contamination on MAP kinase activation. First, we incubated PepG with polymyxin B, a relatively specific endotoxin inhibitor, for 30 min before the mixture was added into RAW264.7 culture. As indicated in Fig. 4A, the presence of polymyxin B did not exhibit an appreciable effect on the activation of any MAP kinase subfamily. To rule out completely the effects of residual LPS to the cellular responses, we compared MAP kinase activation and MKP-1 induction between macrophages isolated from C3H/HeN and C3H/HeJ mice. It has been shown that C3H/HeJ mice are deficient in TLR-4 receptor functions and that macrophages isolated from such mice are unresponsive to LPS stimulation (50, 51). In contrast, C3H/HeN mice harbor a wild type Tlr-4 gene, and macrophages derived from these mice respond vigorously to LPS (50, 51). In response to PepG stimulation, all three MAP kinase subfamilies, including ERK, JNK and p38, responded vigorously in thioglycollate-elicited peritoneal macrophages isolated from both C3H/HeJ and C3H/HeN mice. No appreciable differences in the activation of these kinases were observed between the two mice strains. Likewise, MKP-1 was also highly induced by PepG in the primary macrophages derived from both strains of mice (Fig. 4B). Comparable protein loading among all samples was indicated by the similar intensities of the {beta}-actin bands detected by Western blotting. Taken together, these results indicated that there was no significant LPS contamination in the PepG used in our experiments.



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  		FIG. 4.
Neither elimination of LPS by polymyxin B nor deficiency in TLR-4 activity exhibits significant effects on MAP kinase activation. A, effect of polymyxin B (PMB) on MAP kinase activation in response to PepG. PepG was first incubated with either polymyxin B or an equal volume of serum-free medium for 30 min, and the mixtures were then added into the cell culture. Thirty minutes later, cells were harvested and analyzed by Western blot analysis using antibodies against activated MAP kinases. The blot was stripped and blotted with a {beta}-actin antibody to control for loading. Two separate experiments were shown. Final concentrations of both PepG and polymyxin B were 10 µg/ml. B, the effect of Tlr-4 status on PepG-triggered MAP kinase activation and MKP-1 induction. Peritoneal macrophages isolated from thioglycollated-elicited C3H/HeN or C3H/HeJ mice were stimulated with PepG (10 µg/ml), and lysates were analyzed by Western blot analysis using antibodies against activated MAP kinases, or MKP-1, or {beta}-actin to control for loading.

 
To dissect the relationship between MKP-1 induction and inactivation of JNK and p38, we utilized a pharmacological inhibitor to block MKP-1 transcription and examined the effects of MKP-1 blockade on MAP kinase dephosphorylation. We have found previously that triptolide, a novel diterpenoid triepoxide (52), blocks MKP-1 induction by LPS (30). Pretreatment of cells with triptolide also blocked MKP-1 induction by PepG in a dose-dependent manner (Fig. 5A). In the absence of triptolide, both JNK and p38 reached their peak activities at about 30 min following PepG stimulation, and their activities then decreased close to basal levels by 2 h. In contrast, pretreatment of cells with 0.5 µM of triptolide blocked PepG-induced MKP-1 protein accumulation and markedly delayed the dephosphorylation of both JNK and p38. More potent attenuation of both the MKP-1 induction and the dephosphorylation of JNK and p38 was observed with higher concentration of triptolide (Fig. 5A). Equal protein loading among the samples was verified by immunoblotting with an antibody against {beta}-actin.



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  		FIG. 5.
Triptolide blocks MKP-1 induction and prolongs MAP kinase activities. A, dose-dependent blockade of PepG-stimulated MKP-1 induction by triptolide, and the effects of this MKP-1 blockade on JNK and p38 activities. RAW264.7 cells were pretreated with different doses of triptolide prior to stimulation with 10 µg/ml PepG. Cells were then harvested and analyzed by Western blotting. Cells stimulated with PepG (10 µg/ml) for 30 min served as a positive control. Comparable protein loading was confirmed by blotting with a {beta}-actin antibody. B, effect of MKP-1 blockade on the kinetics of JNK and p38 activation. RAW264.7 cells were pretreated with either Me2SO or 1 µM of triptolide for 30 min before stimulation with PepG (10 µg/ml). MKP-1 protein levels and the activities of JNK and p38 were detected by Western blot analysis. Immunoblotting of samples with an antibody against total p38 verified comparable loading among the samples.

 
The effects of MKP-1 blockade on the kinetics of MAP kinase activation after PepG stimulation were also investigated (Fig. 5B). In the absence of triptolide, MKP-1 protein levels were substantially increased by 1 h after PepG stimulation, which was associated with an almost complete dephosphorylation of p38 and a considerable decrease in JNK phosphorylation. Dephosphorylation of JNK was more prominent after 2 h. In the presence of triptolide, the accumulation of MKP-1 protein was almost completely abolished. The fact that triptolide did not block or delay the activation of any of the MAP kinases indicates that triptolide did not act to block MKP-1 induction by interrupting the initial cellular responses to PepG. Consistent with the hypothesis that MKP-1 participates in the attenuation of JNK and p38 activities, triptolide markedly delayed the dephosphorylation of both p38 and JNK, which normally occurs shortly after PepG stimulation. In addition to p38 and JNK, triptolide also modestly protected ERKs from dephosphorylation (data not shown). Western blot analysis with an antibody against total p38 confirmed comparable protein loading among all the samples. These results provided strong support for a critical role of MKP-1 in the feedback control of p38 and JNK during the macrophage response to PepG.

Overexpression of MKP-1 Attenuates TNF-{alpha} Production by PepG-stimulated Macrophages—It has been well established that TNF-{alpha} biosynthesis in macrophages in response to LPS stimulation is regulated through a complex mechanism mediated by MAP kinases, including ERK, JNK, and p38 (53–55). To investigate the roles of MAP kinases in TNF-{alpha} production by PepG-stimulated macrophages, RAW264.7 cells were pretreated with either the MEK1/2 inhibitor U0126, the JNK inhibitor SP600125, or the p38 inhibitor SB203580 30 min prior to stimulation with PepG. TNF-{alpha} secreted to the medium by the PepG-treated RAW264.7 cells was measured by ELISA using commercial kits (Fig. 6A). PepG potently induced the production of TNF-{alpha}. TNF-{alpha} concentration in the medium harvested from PepG-stimulated cells was greater than 16 ng/ml, whereas TNF-{alpha} in medium harvested from cells stimulated with vehicle (PBS) was essentially undetectable. Pretreatment of the cells with U0126 resulted in a modest decrease in PepG-stimulated TNF-{alpha} production, reducing the TNF-{alpha} secretion by 35%. Likewise, the p38 inhibitor SB203580 also modestly attenuated the production of TNF-{alpha}, decreasing TNF-{alpha} secretion by 45%. Remarkably, the JNK inhibitor SP600125 potently inhibited PepG-triggered TNF-{alpha} secretion, resulting in a 75% decrease in TNF-{alpha} production. Recently, it has been reported that SP600125 can significantly inhibit a variety of protein kinases in vitro (56), thus raising concerns on the actual contribution of the JNK pathway to PepG-induced TNF-{alpha} production. Concordantly, we found that SP600125, at a concentration of 20 µM, modestly inhibited p38 activation while having little effect on the ERK activity in PepG-stimulated RAW264.7 macrophages (data not shown). To clarify the contribution of JNK in TNF-{alpha} production stimulated by PepG, a JIP-1-based approach was used to block JNK, because it has been demonstrated that overexpression of JIP-1 specifically inhibits JNK while exhibiting little effect on either ERK or p38 (32). RAW264.7 cells were transiently transfected with a plasmid encoding for the red fluorescent protein (pDsRed2) together with either an empty vector (pcDNA3) or the JIP-1 expression construct. The DsRed-positive cells were isolated through fluorescence-activated cell sorting and allowed for recovery overnight. These cells were then stimulated with PepG for 4 and 6 h, and TNF-{alpha} production was determined by ELISA. Compared with cells transfected with an empty vector, cells expressing JIP-1 produced significantly less TNF-{alpha} (Fig. 6B). Taken together, these results indicate that MAP kinases are critical mediators in the signal transduction pathways leading to TNF-{alpha} production in PepG-stimulated macrophages.



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  		FIG. 6.
Overexpression of MKP-1 attenuates PepG-induced TNF-{alpha} production. A, effects of pharmacological inhibitors on TNF-{alpha} biosynthesis. RAW267.4 cells were plated into 6-well plates (2 x 105 cells per well). The next day, cells were first treated with Me2SO or U0126 (10 µM), SB203580 (10 µM), or SP600125 (20 µM) for 30 min and then stimulated with either PepG (10 µg/ml) or PBS for 6 h. Data represent the mean ± S.E. from three independent experiments. * indicates significant difference compared with Me SO, p < 0.05. B, inhibition of TNF-{alpha} production by JIP-1 overexpression. RAW264.7 cells were transiently transfected with pDsRed2 together with either pcDNA3 or the JIP-1 expression vector at a ratio of 1:10 using LipofectAMINE 2000. DsRed-positive cells were purified through fluorescence-activated cell sorting, plated into 6-well plates (104 cells per well), and stimulated with 10 µg/ml PepG the next day. Data represent the mean ± S.E. from three independent experiments. *, significantly different from cells transfected with pcDNA3, p < 0.05. C, establishment of stable RAW264.7 clones that overexpress FLAG-tagged MKP-1, as detected by Western blotting using a FLAG antibody. Clone D2 carries the empty vector; clones PC29 and PC39 express FLAG-MKP-1. D, effect of FLAG-MKP-1 expression on TNF-{alpha} biosynthesis. Cells of clones D2, PC29, and PC39 were plated into 6-well plates (5 x 104 cells per well) and stimulated with PepG (10 µg/ml). Data represent the mean ± S.E. from three independent experiments. * indicates significant difference between the two groups, p < 0.05.

 
To investigate if increased MKP-1 expression affects the production of pro-inflammatory cytokines after PepG treatment, RAW264.7 cells were transfected with either a mammalian expression construct expressing FLAG-tagged MKP-1 or an empty vector (pSR{alpha}) together with pcDNA3, which carries a neomycin (G418) resistance gene. The transfected cells were selected in medium containing G418, and individual clones were isolated. These clones were propagated and screened for FLAG-MKP-1 expression by Western blot analyses using a monoclonal antibody against the FLAG epitope. Among these clones, two independent clones, designated hereby as PC29 and PC39, were found to express relatively higher levels of FLAG-MKP-1 (Fig. 6C), although their {beta}-actin contents were similar to that in cells carrying an empty vector pSR{alpha} (clone D2). These clones were stimulated with PepG, and the TNF-{alpha} secretion by these clones was determined by ELISA. Compared with clone D2, which carried the empty vector pSR{alpha}, both PC29 and PC39 produced substantially lower amounts of TNF-{alpha} (Fig. 6D). Clone PC29 expressed a moderate amount of FLAG-MKP-1 protein and produced about half of the amount of TNF-{alpha} secreted by clone D2. PC39 cells, which expressed slightly more FLAG-MKP-1 protein than PC29 cells (Fig. 6C), secreted about 70% less TNF-{alpha} compared with clone D2 (Fig. 6D).

Knockdown of MKP-1 Expression by Small Interfering RNA Augments TNF-{alpha} Production by PepG-stimulated Macrophages—To investigate whether MKP-1 acts as a major determinant or a minor contributing factor in attenuating the biosynthesis of pro-inflammatory cytokines, a plasmid-based small interfering RNA approach was used to suppress MKP-1 expression. A U6 promoter-based plasmid vector targeting a 19-nucleotide sequence, +352–370, in the open read frame of mouse MKP-1 mRNA was created to produce a hairpin siRNA, and the plasmid was transfected into RAW264.7 cells. After selecting in G418, individual clones were isolated and screened for MKP-1 induction after LPS stimulation. Clone GH1L was found to express substantially less MKP-1 protein relative to the control clone (D2) after both LPS stimulation (data not shown) and PepG treatment (Fig. 7A). Compared with clone D2, GH1L displayed a greater than 70% decrease in MKP-1 protein levels by 60 min after PepG stimulation and a greater than 85% reduction in MKP-1 levels after 2 h. The differences between the MKP-1 levels were not due to differences in sample loading, as {beta}-actin levels did not differ between these two clones (Fig. 7A, top panel). To assess the effect of MKP-1 knockdown on the kinetics of p38 activation, cells from both D2 and GH1L clones were stimulated with PepG over a 6-h period, and p38 activation was examined by Western blotting (Fig. 7B). PepG stimulation resulted in a robust transient p38 activation in D2 cells, and the activity of p38 descended substantially after 60 min. In contrast, in GH1L cells the inactivation of p38 after the initial peak was significantly delayed. In fact, p38 activity at 60 min was comparable with that seen at 30 min after PepG stimulation, although it also returned to close to basal levels by 2 h. Most interestingly, p38 activity displayed a subtle, nevertheless reproducible, increase at later time points in GH1L cells, but not in D2 cells, supporting a role of MKP-1 in restraining the inflammatory responses.



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  		FIG. 7.
PepG-induced MKP-1 expression is attenuated by an MKP-1 siRNA. RAW264.7 cells were transfected with a U6-driven MKP-1 siRNA expression cassette and selected in G418 for 3 weeks. Individual clones were isolated and tested for MKP-1 induction. A, comparison of MKP-1 induction by PepG between a control clone (D2) and a clone expressing an MKP-1 siRNA (GH1L). Cells were stimulated with PepG (10 µg/ml), and analyzed by Western blotting using MKP-1 and {beta}-actin antibodies. The intensities of MKP-1 bands were determined by densitometry using ImageMaster 1D Elite Program and expressed in arbitrary units. B, effect of MKP-1 knockdown on p38 activity. Cells from clone D2 or clone GH1L were stimulated with 10 µg/ml PepG, and p38 activity was examined. The membrane was stripped and blotted with {beta}-actin to control for loading.

 
To determine the effect of MKP-1 down-regulation on PepG-stimulated TNF-{alpha} production, cells originating from both GH1L and D2 clones were stimulated with either 1 or 5 µg/ml PepG for 4 and 6 h. The medium was harvested, and TNF-{alpha} was determined by ELISA (Fig. 8A). Upon PepG stimulation, cells expressing the MKP-1 siRNA (GH1L) produced significantly higher amounts of TNF-{alpha} than the cells carrying an empty vector (D2). The differences in TNF-{alpha} production between the two clones were significant at both 4 and 6 h with both concentrations of PepG. Compared with the control clone, cells expressing the MKP-1 siRNA produced approximately 150% more TNF-{alpha} at the 4-h time point and about 100% more at the 6-h time point.



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  		FIG. 8.
Knockdown of MKP-1 expression results in increased production of TNF-{alpha} and IL-1{beta}. A, effect of MKP-1 knockdown on TNF-{alpha} production. Cells carrying control vector (D2) or expressing MKP-1 siRNA (GH1L) were plated into 6-well plates (5x104 cells per well). The next day, cells were either stimulated with 1 or 5 µg/ml PepG or left unstimulated. Data represent the mean ± S.E. from three independent experiments. * indicates significant difference between the two groups, p < 0.05. B, effect of MKP-1 knockdown on IL-{beta} biosynthesis. Cells were treated as in Fig. 7B. The intensities of IL-1{beta} bands were determined by densitometry using ImageMaster 1D Elite Program and expressed in arbitrary (Arb.) units below the panel. The blot was blotted with {beta}-actin antibody to control for sample loading.

 
Like TNF-{alpha}, IL-1{beta} is also a critical inflammatory mediator produced by macrophages. p38 has been shown to play a crucial role in mediating the transcription of the IL-1{beta} gene in LPS-stimulated macrophages (57, 58). To examine the effect of MKP-1 down-regulation on PepG-stimulated IL-1{beta} biosynthesis, cells from both the D2 and the GH1L clones were stimulated with PepG for different periods and harvested. IL-1{beta} levels in the cell lysates were determined by Western blot analysis (Fig. 8B). IL-1{beta} was detected at 4 h after PepG treatment and continued to increase over the time period examined. Compared with the cells carrying a control vector (D2 cells), cells expressing the MKP-1 siRNA (GH1L cells) produced substantially higher amounts of IL-1{beta}. Taken together, these results firmly established that MKP-1 plays a crucial role in terminating the signaling pathways driving the production of pro-inflammatory cytokines in PepG-stimulated macrophages.

MKP-1 Induction Is Associated with Inactivation of p38 and JNK in PepG-Stimulated Primary Macrophages—To explore the role of MKP-1 in the inactivation of MAP kinases during Gram-positive bacterial infection in primary macrophages, peritoneal macrophages were isolated from thioglycollate-elicited C57BL6 mice. The kinetics of MAP kinase activation and the induction of MKP-1 following PepG stimulation in these primary macrophages were examined by Western blot analysis (Fig. 9A). All three MAP kinase were rapidly activated in response to PepG, reaching their peak levels in about 30 min. Both JNK and p38 then underwent rapid dephosphorylation, and their activities diminished to close to basal levels after 2 h, whereas ERK activity was only modestly decreased. Consistent with the notion that MKP-1 is responsible for the inactivation, inactivation of JNK and p38 coincided with MKP-1 induction. MKP-1 protein was undetectable in control cells. Upon PepG stimulation, MKP-1 protein levels were rapidly elevated, becoming visible within 30 min and peaking at about 60 min. Immunoblotting of the same membrane with {beta}-actin verified comparable sample loading (Fig. 9A, bottom panel).



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  		FIG. 9.
MKP-1 induction in response to PepG treatment in primary peritoneal macrophages is associated with inactivation of JNK and p38. A, kinetics of MAP kinase activation and induction of MKP-1 by PepG. Primary macrophages were stimulated with PepG (10 µg/ml) and analyzed by Western blotting. Comparable loading was confirmed by blotting with an antibody against {beta}-actin. B, the effects of MKP-1 blockade by triptolide on MAP kinase activation. Peritoneal macrophages were pretreated with different doses of triptolide for 30 min before stimulation with PepG (10 µg/ml) for 2 h. Unstimulated macrophages and those stimulated with PepG for 30 min were used as negative and positive controls. Comparable loading was confirmed by blotting with {beta}-actin antibody.

 
To study the role of MKP-1 in MAP kinase regulation during PepG stimulation, the primary macrophages were pre-treated with increasing amounts of triptolide to block MKP-1 expression. The effects of triptolide on MAP kinase activities were examined by Western blotting (Fig. 9B). In the absence of triptolide, MKP-1 was highly induced, which coincided with inactivation of JNK and p38 and a significant decrease of ERK phosphorylation. Triptolide blocked MKP-1 induction by PepG and delayed the dephosphorylation of all three MAP kinases in a dose-dependent manner (Fig. 9B). Thus, MKP-1 also appears to play an important role in the attenuation of MAP kinases in primary macrophages.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The signal transduction pathways mediating the initiation of the biosynthesis of TNF-{alpha} and other pro-inflammatory cytokines are studied extensively (18, 20, 59, 60). However, the mechanisms responsible for terminating the production of these cytokines are poorly understood. In this study, we have focused on the role that MKP-1 plays in macrophages during the cellular responses to PepG stimulation. Through the use of a relatively specific endotoxin inhibitor, polymyxin B, we demonstrated that the PepG preparation used in our experiments did not contain a significant LPS contamination (Fig. 4A). This conclusion was further supported through the experiments utilizing primary macrophages isolated from mice deficient in the LPS receptor, TLR-4 (Fig. 4B). Our studies indicated that MKP-1 plays a crucial role in mediating the termination of the JNK and p38 MAP kinase pathways during the responses to PepG, and contributes to "switching off" the signals directing the biosynthesis of pro-inflammatory cytokines. We demonstrated the following. 1) MKP-1 was potently induced in response to PepG, and its protein accumulation correlated with dephosphorylation of both JNK and p38 (Figs. 1 and 2). 2) Blocking MKP-1 induction by triptolide led to a prolonged activation of both JNK and p38 after PepG simulation (Fig. 5). 3) Increasing MKP-1 expression by introducing an MKP-1 expression cassette resulted in attenuated TNF-{alpha} production (Fig. 6). 4) Selective down-regulation of MKP-1 expression through siRNA significantly augmented PepG-stimulated production of both TNF-{alpha} and IL-1{beta} (Figs. 7 and 8). Furthermore, we demonstrated that MKP-1 induction is associated with inactivation of MK2 (Fig. 3), a serine/threonine kinase critical for the production of inflammatory cytokines during LPS stimulation (47). Taken together, our studies illustrate an important mechanism by which macrophages terminate the signaling cascades mediating cytokine production during the responses to Gram-positive bacterial infection.

Previously, we have shown that MKP-1 induction correlates with inactivation of both p38 and JNK and that overexpression of MKP-1 inhibits the production of TNF-{alpha} in LPS-stimulated RAW264.7 macrophages. These observations suggested that MKP-1 could play a role in restraining pro-inflammatory cytokine biosynthesis via feedback control of JNK and p38 (30). However, previous studies did not address the extent to which MKP-1 mediated these effects. In mammalian cells, there are at least 10 MKP family members (43, 61). In RAW264.7 macrophages, at least three MKP family members have been shown to exhibit increased expression in response to LPS, including MKP-1, MKP-M, and PAC-1 (62). Most interestingly, those three MKPs exhibit distinct substrate preferences for different MAP kinases; MKP-1 preferentially inactivates p38 and JNK (44), whereas MKP-M displays a strong selectivity toward JNK (62), and PAC-1 preferentially dephosphorylates ERK and p38 (63). In addition to these phosphatases, MKP-2 is also expressed in RAW264.7 macrophages, although its expression was not affected by inflammatory stimuli (data not shown). It is possible that MKP family members act together to inactivate the MAP kinase signaling cascades and terminate the production of TNF-{alpha}. In the present study, we designed an MKP-1 siRNA to specifically block the expression of endogenous MKP-1 (Fig. 7). We demonstrated that MKP-1 siRNA substantially attenuated the PepG-induced increases in MKP-1 protein, decreasing MKP-1 protein levels by ~75%. As a result, p38 inactivation was delayed relative to cells that did not express MKP-1 siRNA. In control cells, p38 activity was substantially decreased by 60 min following PepG stimulation. However, in the cells expressing MKP-1 siRNA, p38 activity was still at its peak levels at 60 min (Fig. 7B). Moreover, MKP-1 knockdown not only delayed the initial dephosphorylation of p38 but also resulted in an appreciable increase in p38 activity at the later stage (4 – 6 h). Consequently, TNF-{alpha} production triggered by PepG in these MKP-1 down-regulated cells increased by more than 100%, and IL-1{beta} biosynthesis was also substantially augmented (Fig. 8). These results strongly suggest that MKP-1 acts as a critical regulator in both TNF-{alpha} and IL-1{beta} biosyntheses in PepG-stimulated macrophages. Nevertheless, our studies do not exclude the possibility that in addition to MKP-1, other MKPs may also contribute to the termination of the signals driving the biosynthesis of pro-inflammatory cytokines in PepG-stimulated macrophages.

Although MAP kinases play an important role in TNF-{alpha} production in both LPS- and PepG-stimulated macrophages, the contribution of each MAP kinase subfamily to the process appears to differ considerably. It has been reported that the ERK pathway does not play a significant role in mediating TNF-{alpha} production in RAW264.7 macrophages stimulated with LPS (64, 65), whereas both JNK and p38 are required for TNF-{alpha} biosynthesis in these cells (66–68). We found that, in addition to JNK and p38, ERK also played a significant role in TNF-{alpha} production by PepG-stimulated RAW264.7 cells, because TNF-{alpha} biosynthesis in PepG-stimulated RAW264.7 macrophages was modestly inhibited by the MEK1/2 inhibitor U0126 (Fig. 5). The differential involvement of these MAP kinase subfamilies could partially explain the differences in biological responses elicited by the two bacterial components.

Perhaps the most exciting finding in this study is that MKP-1 induction is tightly associated with down-regulation of JNK and p38 in PepG-treated primary macrophages. We found that MKP-1 was potently induced upon PepG stimulation in primary peritoneal macrophages isolated from all three strains of mice, including C3H/HeN, C3H/HeJ, and C57BL6 (Figs. 4 and 9). Accumulation of MKP-1 protein after PepG treatment is associated with inactivation of JNK and p38. Further supporting the notion that MKP-1 may be responsible for terminating p38 and JNK, we found that blocking MKP-1 induction by triptolide markedly delayed the inactivation of these stress-activated protein kinases. The fact that triptolide also displayed an appreciable protective effect on phosphorylated/active ERK also suggests that MKP-1 could play a role in down-regulating the ERK pathway (Fig. 9). In view of the function of MKP-1 in the termination of MAP kinases during pro-inflammatory cytokine biosynthesis, our findings raise the possibility that up-regulation of MKP-1 expression could represent a novel approach to the treatment of sepsis and many chronic inflammatory diseases.


   FOOTNOTES
 
* This work was supported by NIAID Grant AI57798 (to Y. L.) and NICHD Grant HD43003 (to T. N. H.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Center for Developmental Pharmacology and Toxicology, Children's Research Institute, Children's Hospital, Dept. of Pediatrics, Ohio State University, 700 Children's Dr., Columbus, OH 43205. Tel.: 614-722-3703; Fax: 614-722-3455; E-mail: liuy{at}pediatrics.ohio-state.edu.

1 The abbreviations used are: TNF-{alpha}, tumor necrosis factor-{alpha}; TLR, Toll-like receptor; PepG, peptidoglycan; LPS, lipopolysaccharides; MAP, mitogen-activated protein; MK2, MAP kinase-activated protein kinase-2 or MAPKAPK-2; siRNA, small interfering RNA; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; JIP, JNK-interacting protein; MKP, MAP kinase phosphatase; IL, interleukin; GST, glutathione S-transferase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Jacques Landry, Jacques Huot, Roger Davis, and James Woodgett for kindly providing us with valuable reagents. We thank Mary Manson and Cynthia McAllister for excellent technical assistance. We are grateful to Dr. Myriam Gorospe for editing.



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