Title: The Function of Mitogen-activated Protein Kinase Phosphatase-1 in Peptidoglycan-stimulated Macrophages
Abstract: 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)-α induced by peptidoglycan, whereas knockdown of MKP-1 by small interfering RNA substantially increased the production of both TNF-α and interleukin-1β. 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-α and interleukin-1β 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. 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)-α induced by peptidoglycan, whereas knockdown of MKP-1 by small interfering RNA substantially increased the production of both TNF-α and interleukin-1β. 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-α and interleukin-1β 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. 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 (1Martin G.S. Mannino D.M. Eaton S. Moss M. N. Engl. J. Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4816) Google Scholar). 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 (1Martin G.S. Mannino D.M. Eaton S. Moss M. N. Engl. J. Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4816) Google Scholar, 2Wang J.E. Dahle M.K. Yndestad A. Bauer I. McDonald M.C. Aukrust P. Foster S.J. Bauer M. Aasen A.O. Thiemermann C. Crit. Care Med. 2004; 32: 546-552Crossref PubMed Scopus (56) Google Scholar). 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 (1Martin G.S. Mannino D.M. Eaton S. Moss M. N. Engl. J. Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4816) Google Scholar). 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 (1Martin G.S. Mannino D.M. Eaton S. Moss M. N. Engl. J. Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4816) Google Scholar, 2Wang J.E. Dahle M.K. Yndestad A. Bauer I. McDonald M.C. Aukrust P. Foster S.J. Bauer M. Aasen A.O. Thiemermann C. Crit. Care Med. 2004; 32: 546-552Crossref PubMed Scopus (56) Google Scholar). 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 (3Schrag S.J. Zywicki S. Farley M.M. Reingold A.L. Harrison L.H. Lefkowitz L.B. Hadler J.L. Danila R. Cieslak P.R. Schuchat A. N. Engl. J. Med. 2000; 342: 15-20Crossref PubMed Scopus (838) Google Scholar, 4Pearlman M. Obstet. Gynecol. 2003; 102: 414-415PubMed Google Scholar). 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 (5Hoffman J.A. Mason E.O. Schutze G.E. Tan T.Q. Barson W.J. Givner L.B. Wald E.R. Bradley J.S. Yogev R. Kaplan S.L. Pediatrics. 2003; 112: 1095-1102Crossref PubMed Scopus (114) Google Scholar). Recently, multidrug-resistant species of Gram-positive bacteria have emerged, further exacerbating the threat to public health (6Canton R. Coque T.M. Baquero F. Curr. Opin. Infect. Dis. 2003; 16: 315-325Crossref PubMed Scopus (117) Google Scholar). 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 (7Vieillard-Baron A. Prin S. Chergui K. Dubourg O. Jardin F. Am. J. Respir. Crit. Care Med. 2003; 168: 1270-1276Crossref PubMed Scopus (185) Google Scholar). 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-α, 1The abbreviations used are: TNF-α, tumor necrosis factor-α; 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. 1The abbreviations used are: TNF-α, tumor necrosis factor-α; 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. IL-1β, and IL-6, appear to play an important role in mediating the pathophysiological process (8Parrillo J.E. N. Engl. J. Med. 1993; 328: 1471-1477Crossref PubMed Scopus (1502) Google Scholar, 9Dinarello C.A. Chest. 1997; 112: S321-S329Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 10Beutler B. Krochin N. Milsark I.W. Luedke C. Cerami A. Science. 1986; 232: 977-980Crossref PubMed Scopus (1014) Google Scholar). 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 (11Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1997; 9: 4-9Crossref PubMed Scopus (1206) Google Scholar, 12Medzhitov R. Janeway Jr., C. N. Engl. J. Med. 2000; 343: 338-344Crossref PubMed Scopus (1727) Google Scholar). 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 (13Suffredini A.F. Fantuzzi G. Badolato R. Oppenheim J.J. O'Grady N.P. J. Clin. Immunol. 1999; 19: 203-214Crossref PubMed Scopus (322) Google Scholar). 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 (14Beutler B. J. Investig. Med. 1995; 43: 227-235PubMed Google Scholar). The critical roles of TNF-α in immune defense and in the pathogenesis of sepsis are highlighted by findings illustrating that mice lacking a TNF-α receptor gene are resistant to septic shock but unable to control local bacterial infection (15Rothe J. Lesslauer W. Lotscher H. Lang Y. Koebel P. Kontgen F. Althage A. Zinkernagel R. Steinmetz M. Bluethmann H. Nature. 1993; 364: 798-802Crossref PubMed Scopus (1149) Google Scholar, 16Pfeffer K. Matsuyama T. Kundig T.M. Wakeham A. Kishihara K. Shahinian A. Wiegmann K. Ohashi P.S. Kronke M. Mak T.W. Cell. 1993; 73: 457-467Abstract Full Text PDF PubMed Scopus (1536) Google Scholar). 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 (17O'Shea J.J. Ma A. Lipsky P. Nat. Rev. Immunol. 2002; 2: 37-45Crossref PubMed Scopus (525) Google Scholar). 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) (18Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar, 19Takeuchi O. Akira S. Int. Immunopharmacol. 2001; 1: 625-635Crossref PubMed Scopus (398) Google Scholar, 20Akira S. Curr. Opin. Immunol. 2003; 15: 5-11Crossref PubMed Scopus (471) Google Scholar). These pathways are most thoroughly studied with regard to lipopolysaccharides (LPS), a cell wall component of Gram-negative bacteria (18Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar). Recognition of LPS by TLR-4 triggers a cascade of signaling events that lead to activation of transcription factor NF-κB and the MAP kinase pathways, including extracellular signal-regulated kinase (ERK), JNK, and p38 subfamilies (18Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar, 21Han J. Thompson P. Beutler B. J. Exp. Med. 1990; 172: 391-394Crossref PubMed Scopus (502) Google Scholar). The MAP kinase family plays a crucial role in mediating the induction of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, through multiple mechanisms involving both transcriptional and post-transcriptional regulatory events (18Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar, 21Han J. Thompson P. Beutler B. J. Exp. Med. 1990; 172: 391-394Crossref PubMed Scopus (502) Google Scholar, 22Beutler B. Kruys V. J. Cardiovasc. Pharmacol. 1995; 25: 1-8Crossref PubMed Scopus (93) Google Scholar). Peptidoglycan (PepG) and lipoteichoic acid are two major cell wall components of Gram-positive bacteria (23Foster K.D. Conn C.A. Kluger M.J. Am. J. Physiol. 1992; 262: R211-R215PubMed Google Scholar, 24Kengatharan K.M. De Kimpe S. Robson C. Foster S.J. Thiemermann C. J. Exp. Med. 1998; 188: 305-315Crossref PubMed Scopus (206) Google Scholar). Unlike LPS that is recognized by TLR-4, both PepG and lipoteichoic acid are recognized by TLR-2 (20Akira S. Curr. Opin. Immunol. 2003; 15: 5-11Crossref PubMed Scopus (471) Google Scholar, 25Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, 26Michelsen K.S. Aicher A. Mohaupt M. Hartung T. Dimmeler S. Kirschning C.J. Schumann R.R. J. Biol. Chem. 2001; 276: 25680-25686Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Both PepG and lipoteichoic acid have been reported to activate MAP kinases and induce production of inflammatory cytokines (2Wang J.E. Dahle M.K. Yndestad A. Bauer I. McDonald M.C. Aukrust P. Foster S.J. Bauer M. Aasen A.O. Thiemermann C. Crit. Care Med. 2004; 32: 546-552Crossref PubMed Scopus (56) Google Scholar, 27Dziarski R. Jin Y.P. Gupta D. J. Infect. Dis. 1996; 174: 777-785Crossref PubMed Scopus (101) Google Scholar, 28Carl V.S. Brown-Steinke K. Nicklin M.J. Smith Jr., M.F. J. Biol. Chem. 2002; 277: 17448-17456Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29Wang J.E. Jorgensen P.F. Almlof M. Thiemermann C. Foster S.J. Aasen A.O. Solberg R. Infect. Immun. 2000; 68: 3965-3970Crossref PubMed Scopus (160) Google Scholar), 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 (30Chen P. Li J. Barnes J. Kokkonen G.C. Lee J.C. Liu Y. J. Immunol. 2002; 169: 6408-6416Crossref PubMed Scopus (252) Google Scholar). 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-α induced by PepG, whereas down-regulation of MKP-1 by siRNA increased the production of both TNF-α and IL-1β. 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. 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 pSRα-FLAG and pSRα-FLAG-MKP-1 are as described previously (30Chen P. Li J. Barnes J. Kokkonen G.C. Lee J.C. Liu Y. J. Immunol. 2002; 169: 6408-6416Crossref PubMed Scopus (252) Google Scholar, 31Hutter D. Yo Y. Chen W. Liu P. Holbrook N.J. Roth G.S. Liu Y. J. Gerontol. A Biol. Sci. Med. Sci. 2000; 55: B125-B134Crossref PubMed Scopus (39) Google Scholar). The mammalian expression vector pDsRed2 was purchased from Clontech. The JIP-1 expression vector was kindly provided by Dr. Roger Davis (32Dickens M. Rogers J.S. Cavanagh J. Raitano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (628) Google Scholar). 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 (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). 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 (34Handel-Fernandez M.E. Lopez D.M. Paulnock D.M. Macrophages. Oxford University Press, Oxford2000: 1-30Google Scholar). 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 pSRα-FLAG-MKP-1, or the empty vector pSRα, 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-α 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 β-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) (30Chen P. Li J. Barnes J. Kokkonen G.C. Lee J.C. Liu Y. J. Immunol. 2002; 169: 6408-6416Crossref PubMed Scopus (252) Google Scholar, 35Chen P. Hutter D. Yang X. Gorospe M. Davis R.J. Liu Y. J. Biol. Chem. 2001; 276: 29440-29449Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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). β-Actin was detected using a monoclonal antibody purchased from Sigma. IL-1β in the cell lysates was detected by Western blot analysis using a rabbit polyclonal antibody against mouse IL-1β (Chemicon, Temecula, CA). TNF-α 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 (36Liu Y. Gorospe M. Yang C. Holbrook N.J. J. Biol. Chem. 1995; 270: 8377-8380Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 37Hutter D. Chen P. Barnes J. Liu Y. Biochem. J. 2000; 352: 155-163Crossref PubMed Scopus (99) Google Scholar, 38Chen P. Hutter D. Liu P. Liu Y. Protein Expression Purif. 2002; 24: 481-488Crossref PubMed Scopus (21) Google Scholar). 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 (36Liu Y. Gorospe M. Yang C. Holbrook N.J. J. Biol. Chem. 1995; 270: 8377-8380Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). After extensive washing, the kinase activity in the MK2 immune complexes was assayed using [γ-32P]ATP and recombinant heat shock protein 25 (HSP25) (Stressgen Biotechnologies, Victoria, Canada) as a substrate (37Hutter D. Chen P. Barnes J. Liu Y. Biochem. J. 2000; 352: 155-163Crossref PubMed Scopus (99) Google Scholar, 38Chen P. Hutter D. Liu P. Liu Y. Protein Expression Purif. 2002; 24: 481-488Crossref PubMed Scopus (21) Google Scholar). JNK1 activity was assayed using recombinant GST-c-Jun-(1–143) as a substrate (39Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2413) Google Scholar). 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 (30Chen P. Li J. Barnes J. Kokkonen G.C. Lee J.C. Liu Y. J. Immunol. 2002; 169: 6408-6416Crossref PubMed Scopus (252) Google Scholar, 40Li J. Gorospe M. Hutter D. Barnes J. Keyse S.M. Liu Y. Mol. Cell. Biol. 2001; 21: 8213-8224Crossref PubMed Scopus (169) Google Scholar). The membrane was stripped and reprobed with an oligonucleotide corresponding to 18 S rRNA to normalize for differences in sample loading (30Chen P. Li J. Barnes J. Kokkonen G.C. Lee J.C. Liu Y. J. Immunol. 2002; 169: 6408-6416Crossref PubMed Scopus (252) Google Scholar, 40Li J. Gorospe M. Hutter D. Barnes J. Keyse S.M. Liu Y. Mol. Cell. Biol. 2001; 21: 8213-8224Crossref PubMed Scopus (169) Google Scholar). Statistics—The results from the experiments assessing the effects of MAP kinase or MKP-1 modulation on TNF-α 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-α values were transformed to a logarithmic scale to normalize the variances. All comparisons are interpreted as being statistically significant at p < 0.05. 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 β-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). 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 (41Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (569) Google Scholar, 42Sun J.B. Rask C. Olsson T. Holmgren J. Czerkinsky C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7196-7201Crossref PubMed Scopus (188) Google Scholar, 43Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (705) Google Scholar). Recent studies have indicated that MKP-1 protein preferentially inactivates p38 and JNK (44Franklin C.C. Kraft A.S. J. Biol. Chem. 1997; 272: 16917-16923Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). 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. 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 (45Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1500) Google Scholar, 46Ben Levy R. Leighton I.A. Doza Y.N. Attwood P. Morrice N. Marshall C.J. Cohen P. EMBO J. 1995; 14: 5920-5930Crossref PubMed Scopus (162) Google Scholar, 47Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (686) Google Scholar, 48Neininger A. Kontoyiannis D. Kotlyarov A. Winzen R. Eckert R. Volk H.D. Holtmann H. Kollias G. Gaestel M. J. Biol. Chem. 2002; 277: 3065-3068Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). As MK2 is a downstream target of p38, we examined whether M