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. 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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. 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PubMed Scopus Google Scholar, G. S. P. J. Med. 1999; PubMed Scopus Google Scholar). In to all MAP kinase including ERK, JNK and p38, in peritoneal macrophages isolated from both and in the activation of these kinases the MKP-1 was also induced by in the primary macrophages from both of protein among all was by the of the by these results that was in the used in the MKP-1 induction and inactivation of JNK and p38, we a to MKP-1 and the of MKP-1 MAP kinase dephosphorylation. have found that a diterpenoid J. 1999; Full Text Full Text PDF PubMed Scopus Google MKP-1 induction by P. J. J. J. Immunol. 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In to p38 and JNK, also from not with an against p38 protein among all the results for a critical role of MKP-1 in the of p38 and JNK the to Overexpression of MKP-1 TNF-α by has been that TNF-α in macrophages in to stimulation is a mediated by MAP including ERK, JNK, and p38 P.R. J. PubMed Scopus Google Scholar, M. F. G. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C. K. C. G. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the of MAP kinases in TNF-α production by macrophages, RAW264.7 cells with the the JNK or the p38 to stimulation with TNF-α to the by the RAW264.7 cells was by using potently induced the production of TNF-α in the from cells was than whereas TNF-α in from cells with was of the cells with resulted in a in TNF-α the TNF-α by the p38 also attenuated the production of TNF-α, TNF-α by the JNK potently TNF-α in a in TNF-α Recently, it has been that can a of protein kinases in J. M. P. J. 2003; PubMed Scopus Google the of the JNK to TNF-α we found that at a of p38 activation the in RAW264.7 macrophages not the of JNK in TNF-α production by PepG, a was used to JNK, it has been that of JNK or p38 M. J.S. J. A. Science. 1997; PubMed Scopus Google Scholar). RAW264.7 cells with a for the protein with an or the expression cells isolated and for cells with for and and TNF-α production was by with cells with an cells produced less TNF-α these results that MAP kinases are critical in the leading to TNF-α production in macrophages. increased MKP-1 expression the production of pro-inflammatory cytokines after RAW264.7 cells with a expression MKP-1 or an with a cells in containing and and for expression by using a against the these as and found to of their to that in cells an with PepG, and the TNF-α by these was by with the both and produced substantially of TNF-α a of protein and produced about of the of TNF-α by more protein than cells about less TNF-α with of MKP-1 by RNA TNF-α by MKP-1 as a major or a factor in the of pro-inflammatory cytokines, a small interfering RNA was used to MKP-1 a in the of MKP-1 was to a siRNA, and the was RAW264.7 in isolated and for MKP-1 induction after stimulation. was found to substantially less MKP-1 protein to the after both stimulation not and treatment with a than in MKP-1 protein by after stimulation and a than in MKP-1 after the MKP-1 not to in as not these the of MKP-1 knockdown the of p38 cells from both and with a and p38 activation was by stimulation resulted in a transient p38 activation in and the of p38 substantially after In in cells the inactivation of p38 after the was In p38 at was with that at after it also to to by Most interestingly, p38 a at in not in a role of MKP-1 in the inflammatory the of MKP-1 TNF-α cells from both and with or for and was and TNF-α was by cells the MKP-1 produced of TNF-α than the cells an in TNF-α production the at both and with both of with the cells the MKP-1 produced more TNF-α at the and about more at the TNF-α, is also a critical inflammatory produced by macrophages. p38 has been to play a role in mediating the of the in macrophages J.J. J. Immunol. 1999; Google Scholar, M. P. J. Immunol. 2000; PubMed Scopus Google Scholar). the of MKP-1 cells from both the and the with for and in the by was at after treatment and to the with the cells a cells the MKP-1 produced substantially of these results that MKP-1 a role in the signaling the production of pro-inflammatory cytokines in macrophages. MKP-1 with of p38 and JNK in the role of MKP-1 in the inactivation of MAP kinases Gram-positive bacterial infection in primary macrophages, peritoneal macrophages isolated from of MAP kinase activation and the induction of MKP-1 following stimulation in these primary macrophages by MAP kinase in to PepG, their in about JNK and p38 and their to to after whereas was with the that MKP-1 is for the inactivation of JNK and p38 coincided with MKP-1 MKP-1 protein was in MKP-1 protein and at about of the with the role of MKP-1 in MAP kinase regulation the primary macrophages with of to MKP-1 of MAP kinase by In the of MKP-1 was coincided with inactivation of JNK and p38 and a of blocked MKP-1 induction by and the of all MAP kinases in a MKP-1 also to play an important role in the attenuation of MAP kinases in primary macrophages. mediating the initiation of the of TNF-α and pro-inflammatory cytokines are B. Curr. Opin. Immunol. 2000; PubMed Scopus Google Scholar, S. Curr. Opin. Immunol. 2003; PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, K. J. 2000; PubMed Scopus Google Scholar). However, the mechanisms for the production of these cytokines are poorly understood. In this study, we have the role that MKP-1 in macrophages the responses to stimulation. the of a we that the used in not a was further the primary macrophages isolated from in the Our that MKP-1 a role in mediating the of the JNK and p38 MAP kinase the responses to PepG, and to the the of pro-inflammatory the MKP-1 was potently induced in to PepG, and protein with of both JNK and p38 and MKP-1 induction by to a prolonged activation of both JNK and p38 after MKP-1 expression by an MKP-1 expression resulted in attenuated TNF-α production of MKP-1 expression production of both TNF-α and and we that MKP-1 induction is associated with inactivation of a kinase critical for the production of inflammatory cytokines stimulation A. A. C. R. C. M. 1999; PubMed Scopus Google Scholar). an important by macrophages the signaling mediating production the responses to Gram-positive bacterial infection. we have that MKP-1 induction with inactivation of both p38 and JNK and that of MKP-1 the production of TNF-α in RAW264.7 macrophages. that MKP-1 play a role in pro-inflammatory of JNK and p38 P. J. J. J. Immunol. PubMed Scopus Google Scholar). However, not the to MKP-1 mediated these In are at Curr. Opin. 2000; PubMed Scopus Google Scholar, M. A. S. J. 2000; PubMed Scopus Google Scholar). In RAW264.7 macrophages, at have been to increased expression in to LPS, including and PubMed Scopus Google Scholar). Most interestingly, for MAP MKP-1 p38 and JNK J. 1997; Full Text Full Text PDF PubMed Scopus Google whereas a JNK PubMed Scopus Google and and p38 R. K. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In to these is also in RAW264.7 macrophages, expression was not by inflammatory not is that to the MAP kinase signaling and the production of In the present study, we an MKP-1 to the expression of MKP-1 that MKP-1 substantially attenuated the in MKP-1 MKP-1 protein by As a p38 inactivation was to cells that not MKP-1 In p38 was substantially decreased by following stimulation. However, in the cells MKP-1 siRNA, p38 was at at Moreover, MKP-1 knockdown not the of p38 also resulted in an in p38 at the TNF-α production triggered by in these MKP-1 cells increased by more than and was also substantially results suggest that MKP-1 as a critical in both TNF-α and in macrophages. not the that in to also contribute to the of the the of pro-inflammatory cytokines in macrophages. Although MAP kinases play an important role in TNF-α production in both and macrophages, the of MAP kinase to the process to has been that the not play a role in mediating TNF-α production in RAW264.7 macrophages with J. 2000; PubMed Scopus Google Scholar, J. Immunol. 168: PubMed Scopus Google whereas both JNK and p38 are for TNF-α in these cells J.L. 1997; PubMed Google Scholar, A. K. K. K. M. S. PubMed Scopus Google Scholar, M. G. J. 2000; PubMed Scopus Google Scholar). found in to JNK and p38, also a role in TNF-α production by RAW264.7 TNF-α in RAW264.7 macrophages was by the of these MAP kinase the in responses by the bacterial the most in this is that MKP-1 induction is associated with of JNK and p38 in primary macrophages. found that MKP-1 was potently induced stimulation in primary peritoneal macrophages isolated from all of including and and of MKP-1 protein after treatment is associated with inactivation of JNK and p38. the that MKP-1 for p38 and JNK, we found that MKP-1 induction by the inactivation of these protein that also an also that MKP-1 play a role in the In of the function of MKP-1 in the of MAP kinases pro-inflammatory the that of MKP-1 expression a to the treatment of sepsis and inflammatory and for with and for are to for