SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Procyk, K. J. Articles by Baccarini, M. Search for Related Content PubMed PubMed Citation Articles by Procyk, K. J. Articles by Baccarini, M. Related Collections Immunobiology Phagocytes Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 October 2000, Vol. 96, No. 7, pp. 2592-2598 PHAGOCYTES Lipopolysaccharide induces Jun N-terminal kinase activation in macrophages by a novel Cdc42/Rac-independent pathway involving sequential activation of protein kinase C  and phosphatidylcholine-dependent phospholipase C Katarzyna J. Procyk, Maria Rita Rippo, Roberto Testi, Fred Hofmann, Peter J. Parker, and Manuela Baccarini From the Section of Cell Biology and Microbiology, Institute of Microbiology and Genetics, Vienna Biocenter, Vienna, Austria; Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata," Rome, Italy; Institut für Pharmakologie und Toxikologie, Freiburg, Germany; Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.     Abstract Top Abstract Introduction Materials and methods Results Discussion References The activation of kinases of the mitogen-activated protein kinase superfamily initiated by lipopolysaccharide (LPS) plays an important role in transducing inflammatory signals. The pathway leading to the induction of stress-activated protein kinases in macrophages stimulated with LPS was investigated. The activation of Jun N-terminal kinases (JNK) by LPS is herbimycin sensitive. Using specific inhibitors, it was shown that the pathway involves the activation of phosphoinositide 3-kinase (PI 3-K). However, in contrast to previous reports, the small GTPases Cdc42 and Rac are not required downstream of PI 3-K for JNK activation. Instead, the phosphoinositides produced by PI 3-K stimulate protein kinase C (PKC)  activation through PDK1. In turn, activation of this atypical PKC leads to the stimulation of phosphatidylcholine phospholipase C (PC-PLC) and acidic sphingomyelinase (ASMase). It is therefore proposed that PKC regulates the PC-PLC/ASMase pathway, and it is hypothesized that the resultant ceramide accumulation mediates the activation of the SEK/JNK module by LPS. (Blood. 2000;96:2592-2598) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Lipopolysaccharide (LPS), a conserved component of the gram-negative bacteria cell wall, serves as a potent modulator of macrophage activity. It triggers the activation of multiple intracellular signaling cascades, resulting in the release of immunoregulatory molecules such as TNF, IL-1, IL-6, and arachidonic acid metabolites. These mediators are essential for the recruitment and activation of immunocompetent cells that concur in fighting bacterial infection. The precise molecular mechanism by which LPS induces these events is not entirely understood. It is known that LPS complexed with a serum protein (LPS-binding protein) binds to the surface molecule CD14.1 This GPI-anchored protein lacks a cytosolic domain and was therefore postulated to interact with a co-receptor that transduces the signal across the plasma membrane. The molecule performing this function is Tlr-4 (toll-like receptor 4), recently identified as the product of the Lpsd gene.2 Among the first steps in LPS signal transduction is the rapid phosphorylation of various proteins on tyrosyl residues. Signal transduction cascades initiated in this way result in the activation of mitogen-activated protein kinases (MAPK).3-6 The MAPK subgroup JNK is activated preferentially by cellular stress signals such as irradiation, heat shock, osmotic stress, and protein synthesis inhibitors,7 but stimulation by growth factors has also been reported.8,9 Relevant to our study, this pathway is also activated by inflammatory stimuli (LPS3-6; IL-1, TNF-7) and on infection of cultured cells by various pathogens, including gram-negative bacteria.10,11 Targeted disruption of the JNK-kinase, stress and extracellular signal-activated kinase 1 (SEK1), causes defects in the transcriptional activity of AP-1,12 a transcription factor implicated in the regulation of cytokine genes.13 Recent data also implicate JNK in the stabilization14,15 and translation16 of cytokine mRNA. Consistently, T-cell differentiation is defective in Jnk-1-17 and Jnk-2-deficient mice.18 The process of JNK activation by LPS in macrophages depends on tyrosine kinase activity.3,6 Among the best-characterized JNK activators that act downstream of tyrosine kinases are members of the PI 3-K family. Thus, it has been reported that PI 3-K participates in endothelial growth factor-mediated activation of the JNK pathway in epithelial cells8 and that it plays a crucial role in JNK activation mediated by c-Kit in bone marrow-derived mast cells.9 In the latter instance, the pathway requires Rac1 as an intermediate. Small GTPases of the Rho-family modulate JNK activity,19,20 and their function has been reported to be necessary for JNK activation in several systems.21-23 The connection between PI 3-K and members of Cdc42/Rac is supported by the fact that the PI 3-K inhibitor, wortmannin, and dominant-negative forms of PI 3-K block cytoskeletal reorganization mediated by Rac.24-27 PI 3-K can interact physically with small GTPases,28 and the phospholipid products of PI 3-K stimulate GDP/GTP exchange on the members of Rho family GTPases directly29 or indirectly through the guanine nucleotide exchange factor Vav.30 Another prominent downstream target of PI 3-K is phosphoinositide-dependent kinase 1 (PDK1), which in turn regulates PKB,31,32 p70S6K,33,34 and protein kinase C (PKC) with its isoenzymes , , , , and .35,36 Both PKC and  are activated in monocytes and macrophages stimulated with LPS.37,38 In particular, PKC activation occurs in a PI 3-K-dependent manner.37 However, the downstream targets of this signaling pathway remained unidentified. The lipid second messenger ceramide has also been shown to participate in JNK activation.39-41 Notably, low but consistent amounts of ceramide are produced in the course of macrophage stimulation by LPS.42 Here we show that LPS stimulates the JNK pathway by the successive activation of tyrosine kinases, PI 3-K, PKC, and PC-PLC/ASMase. We propose that this cascade generates ceramide that can stimulate the SEK1/JNK module. Unexpectedly, the involvement of Rac, Rho, or Cdc42, which are common mediators in JNK activation in many cell types, is not required in macrophages.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Cell culture, stimulation, and pretreatment BAC-1.2F5 cells43 were cultured in Dulbecco modified Eagle's medium supplemented with 10% fetal calf serum and 20% L-cell-conditioned medium as a source of CSF-1. Confluent cells (approximately 5 × 106 cells per 100-mm diameter tissue culture dish) were cultured for 16 hours in medium without CSF-1 and then stimulated with 1.5 µg/mL bacterial LPS (from Salmonella typhimurium; Sigma, Vienna, Austria) for the indicated times. In selected experiments, macrophages were treated with PC-PLC (from Bacillus cereus; 50 U/mL, 60 minutes; Boehringer Mannheim), C2 ceramide, or the inactive analog dihydro-C2 ceramide (250 µmol/L for 30 minutes, unless otherwise indicated; both from Calbiochem, Darmstadt, Germany) to monitor the effect of these signal transducers on JNK activation. Tyrosine kinases were inhibited by pretreatment with herbimycin A (4 µg/mL, 4 hours; Sigma). Activation of PI 3-K was blocked by pretreatment with wortmannin (100 nmol/L, 20 minutes; Sigma). Inhibition of Rho-family small GTPases (RhoA, Rac1, and Cdc42) was performed by a 60-minute preincubation with toxin B (from Clostridium difficile)44 at a final concentration of 100 ng/mL. Inhibition of PC-PLC activity was performed by preincubating the cells for 60 minutes with 10 µmol/L xanthogenate tricyclodecan-9-yl (D609; Alexis Biochemicals, Laufelfingen, Switzerland). PKC was inhibited by treating the cells with 10 µmol/L bisindoleylmaleimide I (BIM; Calbiochem) for 60 minutes before stimulation.45 Down-regulation of diacylglycerol (DAG)-dependent PKC isoforms was performed by 24-hour treatment with 5 µmol/L tetradecanoyl-phorbol 13-acetate (TPA; Sigma) in dimethyl sulfoxide (DMSO; 14 mmol/L final concentration). Cell lysis, immunoprecipitation, and Western blotting Cells were lysed in solubilization buffer (10 mmol/L Tris-base, 50 mmol/L sodium chloride, 30 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 1% Triton X-100, pH 7.0) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 100 µmol/L sodium vanadate, 1 mmol/L dithiothreitol, and protease inhibitors (aprotinin, 3 µg/mL; pepstatin and leupeptin, 0.5 µg/mL each). For immunoblotting, 30-40 µg whole cell extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. For immunoprecipitation, 500-600 µg whole cell extracts were incubated in the presence of protein A beads (Amersham, Pharmacia, Freiburg, Germany) with anti-PKC (Santa Cruz Biotechnology, Santa Cruz, CA) for 16 to 18 hours at 4°C. After incubation, beads were collected and washed 3 times with lysis buffer. The immunocomplexes were eluted by boiling in SDS sample buffer and subjected to Western blot analysis. Membranes were blocked for 8 to 16 hours at 4°C in TTBS (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.1% Tween-20) supplemented with 4% bovine serum albumin (BSA, fraction V; Sigma) and probed with the appropriate primary antibodies diluted in 1% BSA in TTBS before incubation with peroxidase-conjugated secondary antibodies and detection by the enhanced chemiluminescence system (Pierce). The primary antibodies used in this study recognize selectively the phosphorylated forms of JNK1/2 (anti-phJNK, T183/Tyr185), SEK1 (anti-phSEK, T223; all from New England BioLabs, Schwalbach, Germany), and PKC (recognizing the PDK1 phosphorylation site T410 within the C-loop)36 or their unmodified forms (Santa Cruz Biotechnology). In vitro glucosylation of Rho-family GTPases Cells were lysed by sonication in hypotonic buffer (25 mmol/L HEPES, pH 7.5, 2 mmol/L MgCl2, 100 µmol/L PMSF, 40 µg/mL aprotinin, 25 µg/mL leupeptin, 80 µg/mL benzamidin). After the removal of nuclei and debris by centrifugation, lysates of untreated or toxin B-stimulated BAC-1.2F5 cells (100 µg) were incubated with Clostridium difficile toxin B (fragment CDB546; 100 nmol/L) in 20 µL glucosylation buffer (50 mmol/L HEPES, pH 7.5, 100 mmol/L KCl, 2 mmol/L MgCl2, 1 mmol/L MnCl2, 100 µg/mL BSA) supplemented with 20 µmol/L [14C]-UDP-glucose for 60 minutes at 37°C. Recombinant GST-Rac (1µg) was used as a positive control. Labeled proteins were analyzed by SDS-PAGE and subsequently by phosphorimaging (Molecular Dynamics, Freiburg, Germany). Measurement of PC-PLC and ASMase activity PC-PLC and ASMase activity of whole cell extracts was determined as previously described.46 Briefly, cells (2.5 × 106) were scraped in 2-mL ice-cold phosphate-buffered saline and centrifuged for 10 minutes at 400 rpm, 4°C. Three hundred microliters of Triton X-100 (0.01% for PC-PLC, 0.2% for ASMase activity measurements) was added to the pellet, and the samples were incubated on ice for 10 minutes before sonication. Fifteen micrograms of lysate was incubated for 2 hours at 37°C either in PC-PLC buffer (50 mmol/L Tris-Cl, pH 7.3, 6.3 mmol/L CaCl2, 150 mmol/L ammonium sulfate, plus 50 nCi L-3-phosphatidyl[N-methyl-14C]choline, [14C]PC; 80 µL total volume) or in ASMase buffer (250 mmol/L sodium acetate, pH 5.0, 0.2% Triton X-100, plus 50 nCi methyl-[14C]sphingomyelin; 50 µL total volume). Labeled lipids were from Amersham. The PC-PLC assay was terminated by extracting the lipids with CHCl3:CH3OH (1:2 vol/vol, 180 µL), 0.9% NaCl (60 µL), and CHCl3 (60 µL). The aqueous and organic phases containing [14C]phosphocholine ([14C]PCho) and [14C]PC, respectively, were separated and quantitated by liquid scintillation. The ASMase assay was terminated by extracting the lipids with CHCl3:CH3OH (1:1 vol/vol, 400 µL) and water (180 µL). The amount of [14C]PCho produced was quantitated by liquid scintillation counting. PC-PLC and ASMase activity were expressed as percentages of substrate hydrolyzed.     Results Top Abstract Introduction Materials and methods Results Discussion References Herbimycin and wortmannin block LPS-induced activation of SEK and JNK Quiescent BAC-1.2F5 cells were stimulated with LPS for different time periods. Activation states of the relevant kinases were assessed in whole cell extracts by immunoblotting with antibodies that specifically recognize the phosphorylated, activated form of each enzyme (Figure 1A). The identity of the isoforms detected by the phosphospecific antibodies was determined by immunoblotting lysates of LPS-treated, bone marrow-derived macrophages from JNK1/ or JNK2/ mice (gift of Dr Erwin Wagner, I.M.P., Vienna, Austria; data not shown). As a control for equal loading, the membranes were stripped and reprobed with antibodies against unmodified SEK1/JNK. SEK/JNK were phosphorylated with activation/inactivation kinetics comparable to those published for JNK.3 Peak activation occurred after 25 minutes and then decayed. Inactivation was complete by 1 hour, and no further changes were observed over a period of 4 hours (data not shown). These kinetics of activation resembled those of the other MAPK subfamily, ERK (extracellularly regulated kinases).4 View larger version (34K): [in this window] [in a new window]   Figure 1. Herbimycin- and wortmannin-sensitive activation of SEK and JNK is an early event in LPS-induced signal transduction. (A) Quiescent BAC-1.2F5 cells were stimulated with 1.5 µg/mL LPS at 37°C for different times before solubilization. The presence of the phosphorylated, active forms of SEK (phSEK) and JNK (phJNK) and of their unmodified forms was detected by immunoblotting with the corresponding antibodies. The band labeled phJNK1 + 2 consists of co-migrating, phosphorylated forms of both kinases. (B) Inhibition of tyrosine kinases was performed by treating the cells with herbimycin A (4 µg/mL for 4 hours) before stimulation with LPS (15 minutes). (C) Cells were incubated with wortmannin (WM, 100 nmol/L, 20 minutes) before a 15-minute stimulation with 1.5 µg/mL LPS. Herbimycin-dependent kinases have previously been implicated in the activation of JNK3,6 by LPS. We could confirm the inhibition of LPS-mediated JNK activation by herbimycin A; in addition, SEK activation was also herbimycin sensitive (Figure 1B). PI 3-K-dependent JNK activation has been reported after the stimulation of tyrosine kinase8,9,47 and of G-protein-coupled receptors.22 To test whether the activation of JNK by LPS was similarly regulated in BAC-1.2F5 macrophages, the cells were pretreated with wortmannin (100 nmol/L) before LPS stimulation. Wortmannin severely inhibited LPS-mediated activation of SEK and JNK (Figure 1C). Identical results were obtained using a second PI 3-K inhibitor, LY294002 (data not shown). We therefore postulate that PI 3-K acts as a downstream effector of LPS-stimulated tyrosine kinase(s) in the pathway to JNK activation. JNK and SEK activation is a Cdc42/Rac-independent process PI 3-K-mediated JNK activation has been shown to be dependent on Rac, a member of Rho-family small GTPases.9,22 To test whether Cdc42, Rac, or Rho participated in LPS-mediated JNK activation, we pretreated BAC-1.2F5 cells with toxin B, which glucosylates and inactivates these GTPases.44 In BAC-1.2F5 cells, toxin B inhibits phagocytosis completely4 and, on longer incubation, reduces the adherence of these macrophages to the substrate. Therefore, toxin B perturbs Rho-family-dependent cytoskeletal rearrangements in these cells. However, toxin B pretreatment did not prevent SEK or JNK activation by LPS (Figure 2A). We could not corroborate the toxin B result by overexpressing dominant-negative alleles of CDC42 and Rac in BAC-1.2F5 cells because of the extremely low transfection efficiency that can be achieved in this cell line. We therefore tested directly whether toxin B efficiently glucosylated Cdc42 and Rac in BAC-1.2F5 macrophages in vivo. To this end, we performed in vitro glucosylation assays using lysates from untreated or toxin B-treated cells as substrates. In this assay, [14C]-UDP-glucose is transferred to the Rho-type GTPases present in cell lysates by toxin B in vitro. Rho-family GTPases in lysates of untreated cells were efficiently glucosylated in vitro; in contrast, toxin B failed to glucosylate in vitro Cdc42, Rac, or Rho in the lysates of toxin B-pretreated cells (Figure 2B). These data indicate that Rho-type GTPases had been successfully glucosylated (and therefore inactivated) by the in vivo treatment of macrophages with toxin B. Hence, unexpectedly, a novel pathway independent of functional Cdc42/Rac operates in macrophages stimulated with LPS. View larger version (52K): [in this window] [in a new window]   Figure 2. Toxin B efficiently glucosylates Cdc42 and Rac without affecting LPS-mediated SEK and JNK activation. (A) The Rho-family GTPase inhibitor toxin B does not affect JNK activation by LPS; quiescent BAC-1.2F5 cells were incubated with toxin B (Tox B, 100 ng/mL) for 60 minutes before stimulation with 1.5 µg/mL LPS for 15 minutes. The presence of phosphorylated forms of SEK and JNK, and of JNK1 as a loading control, was detected by immunoblotting with the corresponding antibodies. (B) In vivo treatment with toxin B efficiently glucosylates Rho subtype proteins in BAC-1.2F5 cells. Cells were either left untreated or pretreated with toxin B (100 ng/mL for 60 minutes). Lysates (100 µg) or recombinant GST-Rac (1 µg) were incubated in vitro with recombinant toxin B (fragment CDB546) and UDP-[14C]glucose (20 µmol/L) for 60 minutes at 37°C. Glucosylated proteins were analyzed by SDS-PAGE and phosphorimaging. The identity of the GTPases was confirmed by immunoblotting. Atypical PKC is involved in LPS-mediated activation of the SEK/JNK pathway Results obtained with toxin B excluded Cdc42/Rac as potential downstream effectors of PI 3-K. Because various PKC isoforms have been shown to be regulated by PI 3-K in vitro and in vivo,35,36 we examined whether PKCs were involved in LPS-mediated SEK/JNK activation. BAC-1.2F5 cells express the novel DAG-dependent PKC isoforms  and  and the atypical PKC,45 all of which can be inhibited by BIM.48 Activation of SEK and JNK was completely suppressed after pretreatment with BIM (Figure 3A). Sustained treatment (up to 24 hours) with 5 µmol/L TPA, which causes the effective degradation of the DAG-dependent PKC isoforms  and  (Figure 3B), did not affect SEK and JNK activation (Figure 3C). Therefore, the effect of BIM on JNK activation must result from the inhibition of an atypical PKC. View larger version (31K): [in this window] [in a new window]   Figure 3. Involvement of PKC in LPS-initiated SEK and JNK activation. (A) The PKC inhibitor BIM inhibits SEK/JNK activation by LPS; quiescent BAC-1.2F5 cells were treated with the PKC inhibitor BIM (10 µmol/L, 60 minutes) before stimulation with 1.5 µg/mL LPS for 15 minutes. The presence of phosphorylated SEK/JNK and of unmodified JNK1 was detected with the corresponding antibodies. (B) Prolonged TPA treatment of BAC-1.2F5 cells down-regulates DAG-dependent PKCs; cells were either left untreated or incubated with TPA (5 µmol/L in DMSO) or with DMSO alone for 24 hours. PKC , , and  were detected by immunoblotting. (C) Down-regulation of DAG-dependent PKC does not affect SEK/JNK activation by LPS; down-regulation of DAG-dependent PKCs was performed as described in B before stimulation with 1.5 µg/mL LPS for 15 minutes, cell lysis, and immunodetection of phosphorylated SEK/JNK and of unmodified JNK1. In fact, atypical PKCs would be a natural target of the phosphoinositides generated by PI 3-K. PKC, the only atypical PKC expressed in BAC-1.2F5 macrophages (data not shown), can be activated by PI(3,4,5)P3, PI(1,4,5)P3, and PI(3,4)P2 directly49 and indirectly through PDK1.35,36 PKC activation was monitored by immunoblotting with antibodies that specifically recognize phosphorylation of the PDK1 site T410 in the C-loop of the PKC isoform.36 Western blot analysis of whole cell extracts showed that the T410 phosphorylation was stimulated by LPS, reached a maximum after 10 minutes of stimulation, and slowly decayed (Figure 4A). We confirmed the identity of the PKC isoform activated by immunoprecipitating PKC and analyzing its phosphorylation status (Figure 4B). T410 is the PKC residue phosphorylated by the PI-3K target PDK1.35,36 Its phosphorylation on LPS treatment of macrophages confirms the previously reported involvement of PI-3K in LPS signaling.50 Consistently, LPS-mediated PKC phosphorylation/activation, like JNK activation, was abolished by wortmannin pretreatment (Figure 4C). Taken together, the data implicate PKC as the enzyme involved downstream of PI 3-K in relaying the LPS signal to the JNK module and indicate that its activation is mediated by PDK1. View larger version (35K): [in this window] [in a new window]   Figure 4. Wortmannin-sensitive activation of PKC is an early event in LPS-induced signal transduction. (A, B) LPS stimulates the phosphorylation of PKC on the PDK1 target residue T410; quiescent BAC-1.2F5 cells were stimulated with 1.5 µg/mL LPS for the indicated time points. The presence of phosphorylated PKC was assessed by immunoblot analysis of either whole cell extracts (A) or anti-PKC immunoprecipitates (B). (C) Wortmannin affects LPS-stimulated PKC phosphorylation. BAC-1.2F5 cells were incubated with wortmannin (WM, 100 nmol/L, 20 minutes) before a 15-minute stimulation with 1.5 µg/mL LPS. Anti-PKC blots of whole cell extracts (A, C) or immunoprecipitates (B) are shown as a loading control. Involvement of PC-PLC and ASMase in signaling to JNK High concentrations of the PC-PLC inhibitor D609 decrease LPS-mediated stimulation of Raf, MEK, and ERK.45 To investigate whether phospholipase activation was important for the stimulation of SEK and JNK by LPS, we treated quiescent BAC-1.2F5 cells with D609 (10 µmol/L) before LPS stimulation. The inhibitor severely blunted kinase activation (Figure 5A). PC-PLC was activated by LPS with kinetics consistent with those observed for SEK/JNK activation (Figure 5B), and the addition of bacterial PC-PLC to macrophages caused the activation of JNK, confirming that this signal transducer is in principle able to stimulate the JNK pathway (Figure 5C). Like SEK/JNK activation, stimulation of PC-PLC by LPS could be blocked completely by pretreating the cells with the PKC inhibitor BIM but was completely resistant to down-regulation of DAG-dependent PKCs by sustained TPA treatment (Figure 5D). These data indicate that PC-PLC is involved in JNK activation by LPS. As previously reported in other systems (reviewed in Exton51), in LPS-stimulated macrophages PC-PLC is a downstream target of PKC. View larger version (26K): [in this window] [in a new window]   Figure 5. Involvement of PC-PLC in LPS-induced JNK activation. (A) PC-PLC inhibitor affects SEK/JNK activation; quiescent BAC-1.2F5 cells were pretreated with the PC-PLC inhibitor D609 (10 µmol/L, 60 minutes) before stimulation with 1.5 µg/mL LPS for 15 minutes. The presence of phosphorylated SEK and JNK was detected by immunoblotting with the corresponding antibodies. The inhibitor did not have any effect on the basal level of kinase phosphorylation. An anti-JNK1 blot is shown as loading control. (B) LPS stimulates PC-PLC; quiescent BAC-1.2F5 cells were stimulated with 1.5 µg/mL LPS for different times before solubilization. PC-PLC activity was determined in whole cell extracts as described in "Materials and methods." Results are expressed as the percentage increase with respect to the control values. The plot in B represents the mean of 3 independent experiments, and vertical bars represent the standard errors of the mean. (C) Bacterial PC-PLC activates JNK in macrophages; quiescent BAC-1.2F5 cells were stimulated with 50 U/mL bacterial PC-PLC for 60 minutes before solubilization. The presence of phosphorylated JNK was detected by immunoblotting with the corresponding antibodies. An anti-JNK1 blot is shown as a loading control. (D) BIM pretreatment, but not down-regulation of DAG-dependent PKC, affects the activation of PC-PLC by LPS. Quiescent BAC-1.2F5 cells were left untreated (black bars) or were treated with BIM (10 µmol/L, 60 minutes; gray bars); alternatively, down-regulation of DAG-dependent PKC was performed as described in Figure 3B (white bars). Cells were stimulated with LPS for 15 minutes before lysis and determination of PC-PLC activity in whole cell extracts. Results are expressed as the percentage increase with respect to the control values. LPS treatment of BAC-1.2F5 cells also increased the activity of ASMase, the PC-PLC downstream target responsible for ceramide generation.52 ASMase activity increased steadily during the first 30 minutes of LPS stimulation, and the extent of activation that was achieved correlated well with the small but significant increase in ceramide observed by others in LPS-treated macrophages.42 Like SEK/JNK stimulation, ASMase activation was abrogated by pretreatment with the PC-PLC inhibitor D609 (Figure 6A) and by treatment with the PKC inhibitor BIM (Figure 6B). As in SEK/JNK and PC-PLC activation, down-regulation of DAG-dependent PKCs by prolonged TPA treatment did not affect LPS-mediated ASMase activation (Figure 6B). Taken together, the data in Figures 5 and 6 imply that LPS stimulation of macrophages generates ceramide42 by a PC-PLC/ASMase pathway operating downstream of PKC, and they strongly suggest that this lipid second messenger is responsible for SEK/JNK activation. Exogenous C2 ceramide, but not dihydro-C2 ceramide, was capable of stimulating JNK phosphorylation when added to BAC-1.2F5 cells, though to a lesser extent than LPS (Figure 6C). Ceramide-mediated JNK stimulation was insensitive to pretreatment with D609 or BIM, demonstrating that these inhibitors do not generally prevent JNK stimulation but act specifically on the LPS-induced JNK activation. View larger version (28K): [in this window] [in a new window]   Figure 6. Involvement of ASMase and ceramide in JNK activation by LPS. (A) LPS stimulates ASMase; quiescent BAC-1.2F5 cells were either left untreated or pretreated with D609 (10 µmol/L, 60 minutes) to block PC-PLC before stimulation with 1.5 µg/mL LPS for different times before solubilization. ASMase activity was determined in whole cell extracts as described in "Materials and methods." Results are expressed as the percentage increase with respect to the control values. The plot represents the mean of 2 independent experiments, and vertical bars represent the range of the samples. (B) BIM pretreatment, but not down-regulation of DAG-dependent PKC, affects the activation of ASMase by LPS. Quiescent BAC-1.2F5 cells were left untreated (black bars) or were treated with BIM (10 µmol/L, 60 minutes; gray bars); alternatively, down-regulation of DAG-dependent PKC was performed as described in Figure 3B (white bars). Cells were stimulated with LPS for 15 minutes before lysis and determination of ASMase activity in whole cell extracts. Results are expressed as the percentage increase with respect to the control values. (C) Exogenous ceramide stimulates JNK in macrophages; BAC-1.2F5 cells were left untreated or were pretreated with D609 (10 µmol/L, 60 minutes) to block PC-PLC or with BIM (10 µmol/L, 60 minutes) to inhibit PKC. Cells were then stimulated with C2 ceramide (C2, 250 µmol/L) or with the inactive analog dihydro-C2 ceramide (DH-C2, 250 µmol/L), as indicated. The presence of phosphorylated JNK was detected by immunoblotting with the corresponding antibodies. An anti-JNK1 blot is shown as a loading control.     Discussion Top Abstract Introduction Materials and methods Results Discussion References Bacterial LPS is one of the most potent immunomodulatory substances known. Macrophages represent the main cellular target of LPS in the body, and their response mediates the positive and the negative pathophysiological effects of this powerful stimulus. In this paper we concentrated on describing the cascade that leads to the activation of the SEK/JNK module after LPS stimulation in macrophages. We find that a pathway involving the sequential activation of PI 3-K, PKC, PC-PLC, and ASMase is responsible for SEK/JNK activation. Unexpectedly, stimulation of these kinases is independent of the function of Cdc42/Rac. Herbimycin- and wortmannin-sensitive pathway mediates Cdc42/Rac-independent SEK/JNK activation by LPS Herbimycin-sensitive kinases have been implicated in PI 3-K50 and JNK3,6 activation by LPS in macrophages. Herbimycin A and PI 3-K inhibitors severely blunted LPS-mediated activation of SEK/JNK. PI 3-K has been reported previously to mediate JNK activation by tyrosine kinase and G-protein-coupled receptors.8,9,22 However, in all cases in which this has been investigated, dominant-negative forms of Cdc42/Rac blocked PI 3-K-dependent JNK activation. In contrast, we show that toxin B, an efficient bacterial inhibitor of these GTPases, does not prevent the stimulation of the SEK/JNK module by LPS (Figure 2). Both toxin B and wortmannin completely blocked phagocytosis by BAC-1.2F5 cells4 and, on longer incubations, reduced their adherence to the substrate (data not shown). Furthermore, a pathway involving Cdc42/Rac and PI 3-K mediates CD14-dependent, LPS-stimulated monocyte adherence.53 Thus, these signal transducers act in concert to regulate the cytoskeleton of monocytes/macrophages, whereas they do not cooperate to implement JNK activation. It is possible that macrophages contain different PI 3-K isoforms, which are all stimulated by LPS but operate in distinct pathways to accomplish different outcomes. PKC-mediated activation of PC-PLC/ASMase is responsible for SEK/JNK activation by LPS Conventional, novel, and atypical isoforms of PKC have been implicated in MAPK stimulation.54-57 PI 3-K participates in the activation of PKC isoenzymes.35,36 In the course of LPS stimulation, DAG-dependent PKC isoforms are in fact activated as a result of the PI 3-K-mediated stimulation of PLD-dependent phosphatidylcholine hydrolysis, and they act as intermediates in ERK activation.4 Here, we show that the atypical PKC is stimulated by LPS in a wortmannin-sensitive manner. PI 3-K-dependent activation of PKC is well documented in the literature.35-37,57 It may occur through the direct binding of PI 3-K-generated phosphoinositides to PKC49 or through the phosphorylation of T410 in the activation loop by PDK1.35,36 We demonstrate directly that LPS stimulates phosphorylation of the PDK1 site (Figure 4). Therefore, the latter mechanism operates in LPS-stimulated macrophages. Activated PKC participates in the activation of the SEK/JNK module. This is interesting because, though this enzyme has been shown to modulate the JNK pathway target AP-1,58,59 activation of this isoform has mostly been connected with the stimulation of the ERK pathway.54,55,57,60 The regulation of phosphatidylcholine breakdown is one of the functions of PKC enzymes (reviewed in Exton51). More recent studies point to a role of ceramide generated through the PC-PLC/ASMase pathway as an activator of PKC,61,62 though this is somewhat controversial.63 In LPS-stimulated macrophages, both PC-PLC and ASMase are activated (Figures 5B, 6A), and their stimulation is inhibited by BIM but not by prolonged TPA treatment (Figures 5D, 6B). Thus, in macrophages PKC appears to function in the stimulation of the PC-PLC/ASMase system. The generation of ceramide in LPS-treated macrophages has been reported,42 and the modest increases observed are in good agreement with the level of ASMase activation detected in our study. It should be kept in mind that these relatively small fluctuations were detected in whole cell lysates, and they might be much more impressive in the context of the specific subcellular microenvironment in which activation occurs. In this context, sphingomyelin hydrolysis reportedly occurs in caveolae.64 In further support of our hypothesis, the inhibition of ASMase with D609 correlates with the lack of SEK/JNK activation by LPS, and the treatment of macrophages with exogenous bacterial PC-PLC and with exogenous ceramide is sufficient to cause JNK activation. This demonstrates that both signal-transducing molecules are, in principle, able to couple to JNK in macrophages. We thus propose a model in which the PI 3-K-mediated activation of PKC causes PC-PLC/ASMase activation. This results in the stimulation of the SEK/JNK module, likely through ceramide generation (Figure 7). Ceramide generated by PC-PLC/ASMase might also feed back into the stimulation of PKC, thus amplifying the pathway (dotted line in Figure 7). Although the actual link between ceramide generation and SEK/JNK activation remains unidentified, the TAK1 kinase, a SEK1 activator stimulated by endogenous and exogenous ceramides,65 would be a suitable candidate. Interestingly, activation of the JNK target AP-1 by the CD28 stimulation of T cells has been suggested to depend on both PI 3-K and ASMase.66 It is therefore possible that a pathway similar to the one described here operates in other systems as well. View larger version (20K): [in this window] [in a new window]   Figure 7. Schematic representation of LPS-mediated signal transduction pathways leading to JNK activationa working model. LPS-stimulated tyrosine kinase(s) activate PI 3-K, which in turn generates the phosphoinositides PI(3,4)P2 and PI(3,4,5)P3. Activation of the downstream target PKC occurs through PDK1-mediated phosphorylation of T410 in the C-loop of PKC. The possibility of an allosteric activation of PKC by PI(3,4)P2, PI(3,4,5)P3, or both is indicated by a dotted arrow. PKC stimulates PC-PLC, which generates DAG required for the activation of ASMase. This in turn results in the production of the second messenger ceramide and in SEK/JNK activation. A candidate link between PC-PLC/ASMase pathway and SEK/JNK module is TAK1, a ceramide-stimulative SEK1 kinase. Speculative steps are indicated by question marks, and possible feedback mechanisms are depicted by dotted lines. The data reported here extend our understanding of LPS signal transduction and show for the first time that a pathway comprising PI 3-K, PKC, and PC-PLC/ASMase leads to JNK activation independently of CDC42/Rac.     Acknowledgments We thank Klaus Aktories (University of Freiburg) for support, Pavel Kovarik for constructive discussion and continuous support, and Thomas Decker (Vienna Biocenter) for critically reading this manuscript.     Footnotes This work was supported by grants #P10766-MED and #P13252-MOB of the Austrian Research Fund (M.B.) and by a grant of the Associazione Italiana per la Ricerca sul Cancro (R.T.). Submitted September 28, 1999; accepted June 5, 2000. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Manuela Baccarini, Section of Cell Biology and Microbiology, Institute of Microbiology and Genetics, Vienna Biocenter, Dr-Bohrgasse 9, 1030 Vienna, Austria; e-mail: manuela{at}gem.univie.ac.at.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990;249:1431-1433[Abstract/Free Full Text]. 2. Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998;282:2085-2088[Abstract/Free Full Text]. 3. Hambleton J, Weinstein SL, Lem L, DeFranco AL. Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci U S A. 1996;93:2774-2778[Abstract/Free Full Text]. 4. Procyk KJ, Kovarik P, von Gabain A, Baccarini M. Salmonella typhimurium and lipopolysaccharide stimulate extracellularly regulated kinase activation in macrophages by a mechanism involving phosphatidylinositol 3-kinase and phospholipase D as novel intermediates. Infect Immun. 1999;67:1011-1017[Abstract/Free Full Text]. 5. Reimann T, Buscher D, Hipskind RA, Krautwald S, Lohmann-Matthes ML, Baccarini M. Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway: a putative role for Raf-1 in the induction of the IL-1 beta and the TNF- alpha genes. J Immunol. 1994;153:5740-5749[Abstract]. 6. Sanghera JS, Weinstein SL, Aluwalia M, Girn J, Pelech SL. Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages. J Immunol. 1996;156:4457-4465[Abstract]. 7. Kyriakis JM, Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays. 1996;18:567-577[Medline] [Order article via Infotrieve]. 8. Logan SK, Falasca M, Hu P, Schlessinger J. Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway. Mol Cell Biol. 1997;17:5784-5790[Abstract]. 9. Timokhina I, Kissel H, Stella G, Besmer P. Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation. EMBO J. 1998;17:6250-6262[Medline] [Order article via Infotrieve]. 10. Hobbie S, Chen LM, Davis RJ, Galan JE. Involvement of mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal epithelial cells. J Immunol. 1997;159:5550-5559[Abstract]. 11. Palmer LE, Pancetti AR, Greenberg S, Bliska JB. YopJ of yersinia spp. is sufficient to cause downregulation of multiple mitogen-activated protein kinases in eukaryotic cells. Infect Immun. 1999;67:708-716[Abstract/Free Full Text]. 12. Yang D, Tournier C, Wysk M, et al. Targeted disruption of the MKK4 gene causes embryonic death, inhibition of c-Jun NH2-terminal kinase activation, and defects in AP-1 transcriptional activity. Proc Natl Acad Sci U S A. 1997;94:3004-3009[Abstract/Free Full Text]. 13. Foletta VC, Segal DH, Cohen DR. Transcriptional regulation in the immune system: all roads lead to AP-1. J Leukoc Biol. 1998;63:139-152[Abstract]. 14. Chen CY, Del Gatto-Konczak F, Wu Z, Karin M. Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science. 1998;280:1945-1949[Abstract/Free Full Text]. 15. Ming XF, Kaiser M, Moroni C. c-jun N-terminal kinase is involved in AUUUA-mediated interleukin-3 mRNA turnover in mast cells. EMBO J. 1998;17:6039-6048[Medline] [Order article via Infotrieve]. 16. Swantek JL, Cobb MH, Geppert TD. Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alpha (TNF-alpha) translation: glucocorticoids inhibit TNF-alpha translation by blocking JNK/SAPK. Mol Cell Biol. 1997;17:6274-6282[Abstract]. 17. Dong C, Yang DD, Wysk M, Whitmarsh AJ, Davis RJ, Flavell RA. Defective T cell differentiation in the absence of Jnk1. Science. 1998;282:2092-2095[Abstract/Free Full Text]. 18. Yang DD, Conze D, Whitmarsh AJ, et al. Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Immunity. 1998;9:575-585[Medline] [Order article via Infotrieve]. 19. Coso OA, Chiariello M, Yu JC, et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995;81:1137-1146[Medline] [Order article via Infotrieve]. 20. Minden A, Lin A, Claret FX, Abo A, Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 1995;81:1147-1157[Medline] [Order article via Infotrieve]. 21. Hashimoto A, Okada H, Jiang A, et al. Involvement of guanosine triphosphatases and phospholipase C-2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor. J Exp Med. 1998;188:1287-1295[Abstract/Free Full Text]. 22. Lopez-Ilasaca M, Gutkind JS, Wetzker R. Phosphoinositide 3-kinase gamma is a mediator of G-dependent Jun kinase activation. J Biol Chem. 1998;273:2505-2508[Abstract/Free Full Text]. 23. Davis W, Stephens LR, Hawkins PT, Saklatvala J. Synergistic activation of JNK/SAPK by interleukin-1 and platelet-derived growth factor is independent of Rac and Cdc42. Biochem J. 1999;338:387-392. 24. Hawkins PT, Eguinoa A, Qiu RG, et al. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr Biol. 1995;5:393-403[Medline] [Order article via Infotrieve]. 25. Keely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV. Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature. 1997;390:632-636[Medline] [Order article via Infotrieve]. 26. Ma AD, Metjian A, Bagrodia S, Taylor S, Abrams CS. Cytoskeletal reorganization by G protein-coupled receptors is dependent on phosphoinositide 3-kinase gamma, a Rac guanosine exchange factor, and Rac. Mol Cell Biol. 1998;18:4744-4751[Abstract/Free Full Text]. 27. Reif K, Nobes CD, Thomas G, Hall A, Cantrell DA. Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways. Curr Biol. 1996;6:1445-1455[Medline] [Order article via Infotrieve]. 28. Tolias KF, Cantley LC, Carpenter CL. Rho family GTPases bind to phosphoinositide kinases. J Biol Chem. 1995;270:17656-17659[Abstract/Free Full Text]. 29. Missy K, Van Poucke V, Raynal P, et al. Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and stimulate GDP dissociation. J Biol Chem. 1998;273:30279-30286[Abstract/Free Full Text]. 30. Han J, Luby-Phelps K, Das B, et al. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science. 1998;279:558-560[Abstract/Free Full Text]. 31. Alessi DR, James SR, Downes CP, et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B. Curr Biol. 1997;7:261-269[Medline] [Order article via Infotrieve]. 32. Stephens L, Anderson K, Stokoe D, et al. Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science. 1998;279:710-714[Abstract/Free Full Text]. 33. Alessi DR, Deak M, Casamayor A, et al. 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol. 1997;7:776-789[Medline] [Order article via Infotrieve]. 34. Pullen N, Dennis PB, Andjelkovic M, et al. Phosphorylation and activation of p70s6k by PDK1. Science. 1998;279:707-710[Abstract/Free Full Text]. 35. Chou MM, Hou W, Johnson J, et al. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr Biol. 1998;8:1069-1077[Medline] [Order article via Infotrieve]. 36. Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science. 1998;281:2042-2045[Abstract/Free Full Text]. 37. Herrera-Velit P, Knutson KL, Reiner NE. Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-zeta in bacterial lipopolysaccharide-treated human monocytes. J Biol Chem. 1997;272:16445-16452[Abstract/Free Full Text]. 38. St-Denis A, Chano F, Tremblay P, St-Pierre Y, Descoteaux A. Protein kinase C-alpha modulates lipopolysaccharide-induced functions in a murine macrophage cell line. J Biol Chem. 1998;273:32787-32792[Abstract/Free Full Text]. 39. Brenner B, Koppenhoefer U, Weinstock C, Linderkamp O, Lang F, Gulbins E. Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153. J Biol Chem. 1997;272:22173-22181[Abstract/Free Full Text]. 40. Huang C, Ma W, Ding M, Bowden GT, Dong Z. Direct evidence for an important role of sphingomyelinase in ultraviolet-induced activation of c-Jun N-terminal kinase. J Biol Chem. 1997;272:27753-27757[Abstract/Free Full Text]. 41. Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 1996;380:75-79[Medline] [Order article via Infotrieve]. 42. MacKichan ML, DeFranco AL. Role of ceramide in lipopolysaccharide (LPS)-induced signaling: LPS increases ceramide rather than acting as a structural homolog. J Biol Chem. 1999;274:1767-1775[Abstract/Free Full Text]. 43. Morgan C, Pollard JW, Stanley ER. Isolation and characterization of a cloned growth factor dependent macrophage cell line, BAC1.2F5. J Cell Physiol. 1987;130:420-427[Medline] [Order article via Infotrieve]. 44. Aktories K. Rho proteins: targets for bacterial toxins. Trends Microbiol. 1997;5:282-288[Medline] [Order article via Infotrieve]. 45. Buscher D, Hipskind RA, Krautwald S, Reimann T, Baccarini M. Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages. Mol Cell Biol. 1995;15:466-475[Abstract]. 46. De Maria R, Rippo MR, Schuchman EH, Testi R. Acidic sphingomyelinase (ASM) is necessary for fas-induced GD3 ganglioside accumulation and efficient apoptosis of lymphoid cells. J Exp Med. 1998;187:897-902[Abstract/Free Full Text]. 47. Lopez-Ilasaca M, Li W, Uren A, et al. Requirement of phosphatidylinositol-3 kinase for activation of JNK/SAPKs by PDGF. Biochem Biophys Res Commun. 1997;232:273-277[Medline] [Order article via Infotrieve]. 48. Martiny-Baron G, Kazanietz MG, Mischak H, et al. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J Biol Chem. 1993;268:9194-9197[Abstract/Free Full Text]. 49. Nakanishi H, Brewer KA, Exton JH. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993;268:13-16[Abstract/Free Full Text]. 50. Herrera-Velit P, Reiner NE. Bacterial lipopolysaccharide induces the association and coordinate activation of p53/56lyn and phosphatidylinositol 3-kinase in human monocytes. J Immunol. 1996;156:1157-1165[Abstract]. 51. Exton JH. Phosphatidylcholine breakdown and signal transduction. Biochim Biophys Acta. 1994;1212:26-42[Medline] [Order article via Infotrieve]. 52. Schutze S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, Kronke M. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell. 1992;71:765-776[Medline] [Order article via Infotrieve]. 53. Hmama Z, Knutson KL, Herrera-Velit P, Nandan D, Reiner NE. Monocyte adherence induced by lipopolysaccharide involves CD14, LFA-1, and cytohesin-1: regulation by Rho and phosphatidylinositol 3-kinase. J Biol Chem. 1999;274:1050-1057[Abstract/Free Full Text]. 54. Berra E, Diaz-Meco MT, Lozano J, et al. Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta. EMBO J. 1995;14:6157-6163[Medline] [Order article via Infotrieve]. 55. Schonwasser DC, Marais RM, Marshall CJ, Parker PJ. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol. 1998;18:790-798[Abstract/Free Full Text]. 56. Soh JW, Lee EH, Prywes R, Weinstein IB. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol. 1999;19:1313-1324[Abstract/Free Full Text]. 57. Takeda H, Matozaki T, Takada T, et al. PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a G protein-coupled receptor. EMBO J. 1999;18:386-395[Medline] [Order article via Infotrieve]. 58. Rooney JW, Hodge MR, McCaffrey PG, Rao A, Glimcher LH. A common factor regulates both Th1- and Th2-specific cytokine gene expression. EMBO J. 1994;13:625-633[Medline] [Order article via Infotrieve]. 59. Ways DK, Posekany K, deVente J, et al. Overexpression of protein kinase C-zeta stimulates leukemic cell differentiation. Cell Growth Differ. 1994;5:1195-1203[Abstract]. 60. Diaz-Meco MT, Dominguez I, Sanz L, et al. zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro. EMBO J. 1994;13:2842-2848[Medline] [Order article via Infotrieve]. 61. Lozano J, Berra E, Municio MM, et al. Protein kinase C zeta isoform is critical for kappa B-dependent promoter activation by sphingomyelinase. J Biol Chem. 1994;269:19200-19202[Abstract/Free Full Text]. 62. Monick MM, Carter AB, Gudmundsson G, Mallampalli R, Powers LS, Hunninghake GW. A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages. J Immunol. 1999;162:3005-3012[Abstract/Free Full Text]. 63. Huwiler A, Fabbro D, Pfeilschifter J. Selective ceramide binding to protein kinase C-alpha and -delta isoenzymes in renal mesangial cells. Biochemistry. 1998;37:14556-14562[Medline] [Order article via Infotrieve]. 64. Shaul PW, Anderson RG. Role of plasmalemmal caveolae in signal transduction. Am J Physiol. 1998;275:L843-L851[Abstract/Free Full Text]. 65. Shirakabe K, Yamaguchi K, Shibuya H, et al. TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase. J Biol Chem. 1997;272:8141-8144[Abstract/Free Full Text]. 66. Edmead CE, Patel YI, Wilson A, et al. Induction of activator protein (AP)-1 and nuclear factor-B by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals. J Immunol. 1996;157:3290-3297[Abstract]. © 2000 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: J. P. Thaler, S. J. Choi, M. P. Sajan, K. Ogimoto, H. T. Nguyen, M. Matsen, S. C. Benoit, B. E. Wisse, R. V. Farese, and M. W. Schwartz Atypical Protein Kinase C Activity in the Hypothalamus Is Required for Lipopolysaccharide-Mediated Sickness Responses Endocrinology, December 1, 2009; 150(12): 5362 - 5372. [Abstract] [Full Text] [PDF] E. Rovida, E. Spinelli, S. Sdelci, V. Barbetti, A. Morandi, S. Giuntoli, and P. Dello Sbarba ERK5/BMK1 Is Indispensable for Optimal Colony-Stimulating Factor 1 (CSF-1)-Induced Proliferation in Macrophages in a Src-Dependent Fashion J. Immunol., March 15, 2008; 180(6): 4166 - 4172. [Abstract] [Full Text] [PDF] C. Moulakakis, S. Adam, U. Seitzer, A. B. Schromm, M. Leitges, and C. Stamme Surfactant Protein A Activation of Atypical Protein Kinase C {zeta} in I{kappa}B-{alpha}-Dependent Anti-Inflammatory Immune Regulation J. Immunol., October 1, 2007; 179(7): 4480 - 4491. [Abstract] [Full Text] [PDF] S. Falcone, C. Perrotta, C. De Palma, A. Pisconti, C. Sciorati, A. Capobianco, P. Rovere-Querini, A. A. Manfredi, and E. Clementi Activation of Acid Sphingomyelinase and Its Inhibition by the Nitric Oxide/Cyclic Guanosine 3',5'-Monophosphate Pathway: Key Events in Escherichia coli-Elicited Apoptosis of Dendritic Cells J. Immunol., October 1, 2004; 173(7): 4452 - 4463. [Abstract] [Full Text] [PDF] P. G. Arndt, N. Suzuki, N. J. Avdi, K. C. Malcolm, and G. S. Worthen Lipopolysaccharide-induced c-Jun NH2-terminal Kinase Activation in Human Neutrophils: ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE AND Syk-MEDIATED PATHWAYS J. Biol. Chem., March 19, 2004; 279(12): 10883 - 10891. [Abstract] [Full Text] [PDF] S. Okugawa, Y. Ota, T. Kitazawa, K. Nakayama, S. Yanagimoto, K. Tsukada, M. Kawada, and S. Kimura Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages Am J Physiol Cell Physiol, August 1, 2003; 285(2): C399 - C408. [Abstract] [Full Text] [PDF] A. Castrillo, P. G. Traves, P. Martin-Sanz, S. Parkinson, P. J. Parker, and L. Bosca Potentiation of Protein Kinase C {zeta} Activity by 15-Deoxy-{Delta}12,14-Prostaglandin J2 Induces an Imbalance between Mitogen-Activated Protein Kinases and NF-{kappa}B That Promotes Apoptosis in Macrophages Mol. Cell. Biol., February 15, 2003; 23(4): 1196 - 1208. [Abstract] [Full Text] [PDF] X. Ye and S. F. Liu Lipopolysaccharide Down-regulates Sp1 Binding Activity by Promoting Sp1 Protein Dephosphorylation and Degradation J. Biol. Chem., August 23, 2002; 277(35): 31863 - 31870. [Abstract] [Full Text] [PDF] B. W. Jones, T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle, and M. J. Fenton Different Toll-like receptor agonists induce distinct macrophage responses J. Leukoc. Biol., June 1, 2001; 69(6): 1036 - 1044. [Abstract] [Full Text] [PDF] A. Castrillo, B. de las Heras, S. Hortelano, B. Rodriguez, A. Villar, and L. Bosca Inhibition of the Nuclear Factor kappa B (NF-kappa B) Pathway by Tetracyclic Kaurene Diterpenes in Macrophages. SPECIFIC EFFECTS ON NF-kappa B-INDUCING KINASE ACTIVITY AND ON THE COORDINATE ACTIVATION OF ERK AND p38 MAPK J. Biol. Chem., May 4, 2001; 276(19): 15854 - 15860. [Abstract] [Full Text] [PDF] A. Castrillo, M. Mojena, S. Hortelano, and L. Bosca Peroxisome Proliferator-activated Receptor-gamma -independent Inhibition of Macrophage Activation by the Non-thiazolidinedione Agonist L-796,449. COMPARISON WITH THE EFFECTS OF 15-DEOXY-Delta 12,14-PROSTAGLANDIN J2 J. Biol. Chem., August 31, 2001; 276(36): 34082 - 34088. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Procyk, K. J. Articles by Baccarini, M. Search for Related Content PubMed PubMed Citation Articles by Procyk, K. J. Articles by Baccarini, M. Related Collections Immunobiology Phagocytes Signal Transduction Social Bookmarking                   What's this?

	Copyright © 2000 by American Society of Hematology         Online ISSN: 1528-0020