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β in TLR4 and TLR2 dependent activation of NF-κB
Cellular Signalling 17 (2005) 385 – 394 www.elsevier.com/locate/cellsig
Involvement of PKCa/h in TLR4 and TLR2 dependent activation of NF-nB Karim Asehnounea,b, Derek Strassheima, Sanchayita Mitraa, Jae Yeol Kima, Edward Abrahama,* a
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Box C272, 4200 East 9th Ave, Denver, CO 80262, USA b Service d’Anesthesie-Re´animation et Unite´ Propre de Recherche de l’Enseignement Superieur-Equipe d’Accueil (UPRES-EA 3540), Hoˆpital de Biceˆtre, Le Kremlin Biceˆtre, France Received 4 June 2004; received in revised form 18 August 2004; accepted 19 August 2004 Available online 16 September 2004
Abstract Protein kinase C (PKC)a/h dependent signaling events downstream of TLR4 or TLR2 were investigated in neutrophils stimulated with LPS or PGN. Pretreatment of neutrophils with the structurally distinct PKCa/h inhibitors Gf6976 or GF109203X decreased nuclear translocation of NF-nB and production of the proinflammatory cytokine TNF-a. Inhibition of PKCa/h also prevented LPS or PGN induced phosphorylation of IKKa/h, phosphorylation and degradation of InB-a, as well as phosphorylation of the p65 subunit of NF-nB. Activation of p38, JNK, and ERK 1/2 in response to TLR2 engagement was diminished in neutrophils in which PKCa/h was inhibited. However, no alteration in the activation of these kinases was found in TLR4 stimulated neutrophils when PKCa/h was blocked. Such results indicate that distinct intracellular signalling pathways leading to MAPK activation are induced by TLR4 and TLR2 stimulation. PKCa/h can regulate NFnB dependent transcription in neutrophils both by enhancing nuclear translocation of NF-nB and also by stimulating phosphorylation of the p65 subunit. D 2004 Elsevier Inc. All rights reserved. Keywords: PKC; Neutrophils; NF-nB; TLR; Cell signalling; Kinases
1. Introduction The protein kinase C (PKC) family of serine/threonine kinases mediate essential cellular signals required for activation, proliferation, differentiation, and survival [1]. There are at least 12 different PKC isoforms. Among the PKC family, three different general groupings can be characterized: (1) classical PKC isotypes (a, h, g) are diacylglycerol and calcium dependent, (2) novel PKC isotypes (y, q) are diacylglycerol dependent but calcium independent, and (3) atypical PKC isotypes (~, E) are diacylglycerol and calcium independent [2]. The transcriptional regulatory factor NF-nB is a central participant in modulating the expression of many immunor-
* Corresponding author. Tel.: +1 303 315 6025; fax: +1 303 315 5632. E-mail address: [email protected] (E. Abraham). 0898-6568/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2004.08.005
egulatory mediators involved in acute inflammatory response [3]. Signalling pathways initiated by engagement of Toll-like receptors (TLRs), such as TLR2 and TLR4, by bacterial products lead to nuclear accumulation of NF-nB and enhanced transcription of genes responsible for the expression of cytokines, chemokines, adhesion molecules, and other mediators of the inflammatory response associated with bacterial infection. Association of NF-nB with the inhibitory protein InB-a in the cytoplasm blocks the nuclear localization sequence (NLS) of NF-nB, inhibiting its movement into the nucleus [4]. Exposure of cells to inflammatory stimuli, including LPS, peptidoglycan (PGN), and proinflammatory cytokines (such as TNF-a or IL-1h), results in phosphorylation of InB-a on serines 32 and 36, leading to its subsequent ubiquitinylation and degradation by the 26S proteosome. Phosphorylation of InB-a is mediated by the kinases IKKa and IKKh, which are catalytically active components of the IKK complex [5].
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Previous studies have shown that PKCs are involved in the regulation of NF-nB in different cell types [1,2,6]. Moreover, it was demonstrated that the classical PKC family controls nuclear translocation of NF-nB through activation of IKKs and degradation of InB-a [7,8]. Neutrophils play a central role in the inflammatory responses such as acute lung injury induced by LPS or infection [3]. The role of PKCa/h in TLR2 and TLR4induced NF-nB activation has not been delineated. Similarly, a role for PKCa/h in modulating activation of NF-nB in neutrophils has not been described. In the present experiments, we explored these issues and found that PKCa/h was involved in enhancing nuclear translocation of NF-nB in TLR2 and TLR4 stimulated neutrophils, and also enhanced IKKa/h activation as well as phosphorylation of the p65 subunit of NF-nB.
2. Material and methods 2.1. Mice Male BALB/c mice, 8 to 12 weeks of age, were purchased from Harlan Sprague–Dawley (Indianapolis, IN). The mice were kept on a 12-h light/dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review boardapproved protocols. 2.2. Reagents and antibodies Isoflurane was obtained from Abbott Laboratories (Chicago, IL). Escherichia coli 0111:B4 endotoxin highly purified with phenol extraction (LPS, lipopolysaccharide) was purchased from Sigma (St. Louis, MO), and peptidoglycan (PGN) was purchased from InvivoGen (San Diego, CA). GF109203X was purchased from Calbiochem (San Diego, CA). RPMI 1640/25 mM HEPES/l-glutamine was obtained from Bio-Whittaker Products (Walkersville, MD). Fetal calf serum (FCS) and penicillin/streptomycin were purchased from Gemini Bioproducts (Calabasas, CA). Bicinchoninic acid (BCA) protein assay reagent was purchased from Pierce (Rockford, IL). Activation specific antibodies for phospho-Thr202/Tyr204 ERK1, phosphoThr183/Tyr185 ERK2, phospho-Thr180/Tyr182 p38, phospho-Thr183/Tyr185 JNK, phospho-Ser536 p65, phosphoSer180/Ser181 IKKa/h phospho-Ser32/Ser36 InB-a, phospho Thr638/641 PKCa/h and total ERK1/2, p38, JNK, InBa, IKKa/h p65 were purchased from Cell Signaling Technologies (Beverly, MA). Horseradish peroxidaselabeled anti-rabbit antibodies, and ECL reagents were purchased from Bio-Rad (Hercules, CA). All other reagents were purchased from Sigma unless otherwise noted in the text. Custom cocktail antibodies and columns for neutrophil isolation were purchased from Stem Cell Technologies (Vancouver, BC).
2.3. Isolation and culture of bone marrow derived mouse neutrophils Bone marrow neutrophils were isolated as described previously [9]. To obtain the bone marrow cell suspension, the femur and tibia of a mouse were flushed with RPMI 1640. Tissue fragments were removed by rapid filtration through a glass wool column and cells collected by centrifugation. The cell pellets were resuspended in RPMI 1640, 2% FCS and then incubated with primary Abs specific for the cell surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min at 4 8C. This custom mixture (StemCell Technologies) is specific for T and B cells, RBC, monocytes, and macrophages. After 15 min, 100 Al of antibiotin tetrameric Ab complexes were added and the cells incubated for a further 15 min at 4 8C. Following this, 60 Al of colloidal magnetic dextran iron particles were added to the suspension and incubated for 15 min at 4 8C. The entire cell suspension was then placed into a column surrounded by a magnet. The T cells, B cells, RBC, monocytes, and macrophages were captured in the column, allowing the neutrophils to pass through by negative-selection methods. Viability, as determined by trypan blue exclusion, was consistently greater than 98%. Neutrophil purity, as determined by Wright’s-stained cytospin preparations, was greater than 97%. Less than 0.3% of the purified cell population consisted of mononuclear cells. Bone marrow neutrophils (2106/0.5 ml) were cultured in RPMI 1640, 0.1% FCS, with or without drugs as described in figure legends. LPS, PGN, Gf6976 and GF109203X were made fresh for each experiment. In each experiment, Gf6976 or GF109203X was added to the cell cultures 15 min before stimulation with LPS or PGN. 2.4. Cytokine ELISA Immunoreactive TNF-a was quantitated using commercially available ELISA kits (R&D Systems, Minneapolis, MN), according to manufacturer’s instructions and as described previously [9]. 2.5. EMSA Nuclear extracts were prepared and assayed by EMSA as previously described [9]. For analysis of NF-nB, the nBDNA sequence of the Ig gene was used. Synthetic doublestranded sequences (with enhancer motifs underlined) were filled in and labeled with [a-32 P]dATP using Sequenase DNA polymerase as follows: nB, 5V-TTTTCGAGCTCGGGACTTTCCGAGC-3Vand 3V-GCTCGAGCCCTGAAAGGCTCGTTTT-5V. 2.6. Western blot analysis Western blots for phosphorylated and total kinases were performed as described previously [9]. Parallel samples for
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total protein kinase were run with samples for activation specific phosphorylation analysis. Densitometry was performed using chemiluminescence system and analysis software (BioRad, Hercules, CA) to determine the ratio between phosphorylated and total kinase. Densitometry data for each kinase are shown in graphic form, with baseline phosphorylation levels before LPS stimulation (i.e. at 0 min) assigned a value of 100%. 2.7. Statistical analysis Values are expressed as the meanFS.E.M. Data were analysed by analysis of variance, and differences between means were determined using the Bonferroni multiple comparison test. Significance is defined as pb0.05.
3. Results 3.1. Inhibition of PKCa/ß blocks proinflammatory cytokine secretion by neutrophils after TLR2 or TLR4 activation To assess the role of PKCa/h in TLR2 and TLR4induced cellular activation, neutrophils were incubated with LPS or PGN and varying concentrations of two specific, structurally distinct PKCa/h inhibitors, Gf6976 or GF109203X. As expected, concentrations of TNF-a were increased in supernatants of neutrophils stimulated with LPS (Fig. 1A and B) or PGN (Fig. 1C and D). The release of TNF-a from LPS and PGN-stimulated neutrophils was
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inhibited in a dose dependent manner by both Gf6976 and GF109203X (Fig. 1). 3.2. Nuclear translocation of NF-jB is dependent on PKCa/ß in TLR2 and TLR4-stimulated neutrophils Increased concentrations of NF-nB in the nucleus were present within 20 min of LPS or PGN exposure and were maximal 40 min after addition of LPS and 60 min after addition of PGN to the cultures (data not shown). Pretreatment of neutrophils with either Gf6976 and GF109203X prevented LPS (Fig. 2A and B) or PGN (Fig. 2C and D) induced NF-nB activation. 3.3. Activation of PKCa/ß in TLR2 and TLR4-stimulated neutrophils The time course for PGN and LPS-mediated activation of PKCa/h in neutrophils is shown in Fig. 3A and C. Pretreatment of neutrophils with Gf6976 prevented PGN or LPS induced PKCa/h activation (Fig. 3B and D, respectively). 3.4. Blockade of PKCa/ß inhibits TLR2 and TLR4 induced activation of IKKa/ß A potential mechanism for the role that PKCa/h exerts on TLR2 and TLR4 induced nuclear translocation of NF-nB could be through enhancing activation of the IKK complex. IKK mediated phosphorylation of InB-a on serines 32 and
Fig. 1. PKCa/h blockade with Gf6976 or GF109203X inhibit TNF-a production in LPS or PGN stimulated neutrophils. Neutrophils were incubated with control media or with the indicated concentrations of Gf6976 or GF109203X for 15 min, and then cultured with or without LPS (1 Ag/ml) in A and B, with or without PGN (10 Ag/ml) in C and D. After 90 min, the supernatants were collected and assayed for concentrations of TNF-a. Data are combined from three independent experiments. *pb0.05 and **pb0.01 versus neutrophils cultured with LPS or PGN alone.
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tively). To confirm that PKCa/h also affected functional downstream effects of IKK activation, we examined cytoplasmic levels of InB-a in neutrophils treated with LPS or PGN alone or with LPS and PGN plus Gf6976 (Fig. 5). In neutrophils challenged with LPS or PGN, time dependent increases in phosphorylation of InB-a were observed (Fig. 5A and C) while total InB-a underwent concomitant degradation (Fig. 5B and D). However, in neutrophils incubated with Gf6976 and LPS or PGN, phosphorylation of InB-a was diminished compared to stimulation with PGN or LPS alone (Fig. 5A and C) and no degradation of total InB-a was apparent (Fig. 5B and D). Fig. 2. Inhibition of PKCa/h prevents nuclear translocation of NF-nB in LPS or PGN-stimulated neutrophils. Culture of neutrophils with LPS (1 Ag/ ml) or PGN (10 Ag/ml) induces nuclear accumulation of NF-nB (A and B or C and D, respectively), which is inhibited by pretreatment with Gf6976 (A and C) or GF109203X (B and D). In A and C, the neutrophils were incubated with Gf6976 (100 nM), in B and D the neutrophils were incubated with GF109203X (1 AM) for 15 min before LPS or PGN were added to the cultures. Neutrophils not treated with Gf6976, GF109203X, LPS, or PGN were included as controls (C). Nuclear extracts were obtained after 40 min of LPS stimulation or 60 min after PGN stimulation, time points when nuclear translocation of NF-nB was found to be maximal after these stimuli. Two additional experiments provided similar results.
3.5. Blockade of PKCa/b inhibits TLR2 or TLR4 induced phosphorylation of p65
36 leads to subsequent ubiquitinylation and degradation of InB-a, with removal of its tonic inhibitory effects on nuclear translocation of NF-nB [5]. In these experiments, activation of IKK was assessed by determining phosphorylation of serine 180 in IKKa, and serine 181 in IKKh. As shown in Fig. 4A, stimulation of neutrophils with PGN resulted in phosphorylation of IKK that peaked after 45 min. Following LPS stimulation, maximal activation of IKK was found after 30 min (Fig. 4C). Blockade of PKCa/h inhibited PGN and LPS-induced IKK phosphorylation (Fig. 4B and D, respec-
3.6. Differential roles of PKCa/b in TLR2 or TLR4-induced activation of p38, ERK, and JNK
NF-nB transcriptional activity is modulated through phosphorylation of the p65 subunit [10], an event mediated by IKK as well as other kinases [11]. Incubation of neutrophils with either LPS or PGN induced the phosphorylation of p65 (Fig. 6A and C). Inhibition of PKCa/h with Gf6976 abolished p65 phosphorylation after PGN or LPS stimulation (Fig. 6B and D).
Exposure of neutrophils to TLR2 or TLR4 agonists has been shown to activate mitogen activated protein kinases (MAPK), including p38 [12], ERK 1/2 [13], and JNK [9], all of which participate in enhancing nuclear translocation of NF-nB [3]. To examine the role of PKCa/h in modulating activation of these kinases, we determined levels of phosphorylated kinases in LPS or PGN stimulated neutro-
Fig. 3. Gf6976 prevents PKCa/h activation in LPS or PGN-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with PGN (10 Ag/ml) or LPS (1 Ag/ml). Culture of neutrophils with PGN or LPS induced activation of PKCa/h (A and C, respectively), that was inhibited by pretreatment with Gf6976 (B and D). At the indicated times after addition of PGN or LPS, the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting and levels of phosphorylated PKCa/h (p-PKCa/h) were determined. Densitometry data are shown in graphic form, with baseline PKCa/h phosphorylation before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
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Fig. 4. Inhibition of PKCa/h prevents LPS and PGN-induced IKKa/h phosphorylation. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting using antibodies specific for Ser180/Ser181 IKKa/h (p-IKKa/h) and for total kinases. Densitometry data are shown in graphic form, with baseline phosphorylation of Ser180/Ser181 IKK before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
phils and in those pretreated with Gf6976. Inhibition of PKCa/h in neutrophils prevented PGN-induced activation of p38 and ERK1/2, and strongly diminished that of JNK 1/ 2 (Figs. 7, 8 and 9, respectively). In contrast, blockade of PKCa/h did not inhibit LPS-induced activation of p38 or ERK1/2 (Figs. 7 and 8, respectively). Activation of JNK 1/2 in LPS treated neutrophils was too weak to observe any effect of PKCa/h inhibition (data not shown).
4. Discussion Previous studies have suggested that PKCs are involved in the activation of NF-nB (see Ref. [1] for review). Evidence linking PKCs to NF-nB comes from complementary observations demonstrating that nuclear translocation of NF-nB can be enhanced by exposure of cells to PKC activators and prevented by selective PKC inhibitors [1,2]. Such reports, which have primarily utilized lymphocytes,
have provided new insights into the role of PKC on NF-nB activation. However, the assumption that PKC plays a general role in the activation of NF-nB has been the subject of considerable debate. In part, this controversy arises from reports regarding the role of each PKC member during intracellular signalling, complicated by the cell type used, the initial stimulus, as well as the PKC isoform investigated [14]. Little is known about regulation of NF-nB activity in neutrophils [6], and the role of PKC isoforms in affecting NF-nB activity in this cell population through TLR signalling pathways has not previously been examined. TLRs play a role in innate defense by recognizing different molecular patterns associated with microbial pathogens [15]. Stimulation of TLR by bacterial products activates NF-nB and results in enhanced generation of cytokines and other proinflammatory mediators whose transcription is dependent on NF-nB [3]. Among the TLR family, TLR4 is involved in the recognition of LPS from gram-negative bacteria and TLR2 in the response to
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Fig. 5. PKCa/h blockade inhibits LPS or PGN-induced InB-a phosphorylation and degradation. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting and levels of phosphorylated InB-a (pInB-a) and total InB-a were determined. Densitometry data are shown in graphic form, with baseline InB-a phosphorylation before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
products of gram-positive bacteria, such as PGN [16]. After homodimerization, TLR4 induces intracellular signaling through pathways utilizing MyD88, MyD88-like/Toll-IL1R domain-containing adapter proteins, and IL-1R-associated kinases. Signaling via TLR2 is at least partly dependent upon dimerization of this receptor with either TLR1 or TLR6 [17,18]. While a large body of literature indicates that the members of the TLR family activate nearly identical cytoplasmic signaling programs, it was recently suggested that there are distinct differences in the response elicited by TLR2 or TLR4 agonists [19].
Neutrophils have been shown to contain the classical PKCs a and h, which are both DAG and Ca2+ dependent, as well as PKCy, which does not require either DAG or Ca2+ [20,21]. With respect to NF-nB activation, PKCa/h isoforms were shown to have a central role in lymphocytes [1,22]. Among PKC isoforms, only PKCa/h are specifically inhibited by Gf6976 and GF109203X [23–27]. Thus, the present experiments demonstrate that PKCa/h is essential for NF-nB activation and the expression of NFnB dependent cytokines such as TNF-a, in neutrophils [3,28].
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Fig. 6. PKCa/h participates in LPS and PGN-induced p65 phosphorylation. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting using antibodies specific for Ser536 p65 (p-p65) and for total p65. Densitometry data are shown in graphic form, with baseline phosphorylation of Ser536 p65 before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
Fig. 7. Involvement of PKCa/h in p38 activation in PGN or LPS-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), cellular extracts were obtained and levels of phosphorylated p38 (p-p38) and total p38 determined. Representative gels are shown. Densitometry data are presented with the baseline kinase phosphorylation before PGN or LPS addition to cultures (i.e. at time 0 min) given a value of 100%. The data shown are representative of three independent experiments.
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Fig. 8. Involvement of PKCa/h on ERK1/2 activation in PGN or LPS-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), cellular extracts were obtained and levels of phosphorylated (p-ERK1/2) and total kinase were determined. Representative gels are shown. Densitometry data are presented with the baseline kinase phosphorylation before PGN or LPS addition to cultures (i.e. at time 0 min) given a value of 100%. The data shown are representative of three independent experiments.
Although PKCs can affect signaling pathways leading to NF-nB activation [1,22], the specific intracellular events modulated by PKCa/h have not been completely defined. Kinases shown to be activated by PKCs include JNK, p38, ERK, and the IKK complex [7,8,29–31]. It was recently demonstrated in lymphocytes that PKCh induces NF-nB activation through IKK mediated phosphorylation of InBa [7,8]. Although PKCs have been shown to activate p38 MAPK [32], JNK, and ERK1/2 [29,30] in some cell
populations, their effect on these kinases in neutrophils has not been previously reported. Several studies have shown that pathways including MEK/ERK/p38 kinases participate in the regulation of NFnB [33]. ERK1/2 lies immediately downstream of and is activated by the dual-specificity kinases MEK1/2. MEK 1/2 represent major targets of the serine/threonine kinase Raf-1 [31] and several studies demonstrated that Raf-1 acts as a MAPK kinase kinase (see Ref. [34] for review). Raf-1 is
Fig. 9. Involvement of PKCa/h on JNK activation in PGN-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B), cellular extracts were obtained and levels of phosphorylated (p-JNK) and total kinase were determined. Representative gels are shown. Densitometry data are presented with the baseline kinase phosphorylation before PGN addition to cultures (i.e. at time 0 min) given a value of 100%. The data shown are representative of three independent experiments.
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itself activated by PKC [35]. Consequently, it is presumed that inhibition of PKC would reduce basal activity and/or prevent ERK activation. We [9,36] and others [12,13,37] have demonstrated that stimulation of neutrophils with LPS results in activation of members of the MAPK family, including ERK 1/2 and p38. TLR2 agonists, such as PGN, are also potent activators of the MAPK family [19,38]. In the present experiments, inhibition of PKCa/h blocked phosphorylation of ERK 1/2, JNK, and p38 in neutrophils cultured with PGN, but not with LPS. Such results suggest that distinct intracellular signalling pathways leading to MAPK activation and involving PKCa/h are induced by TLR2 and TLR4 stimulation. Activation of NF-nB is critically dependent on the phosphorylation and degradation of the cytoplasmic inhibitor InB-a. One of the critical regulatory steps modulating degradation of InB-a is activation of the InB-a kinase (IKK) complex, which consists of the catalytic subunits, IKKa and IKKh, and the regulatory subunit NEMO [5]. Recent studies in lymphocytes demonstrated that PKCa/h was involved in IKK activation and the degradation of InBa [7,8]. Vancurova et al. [6] found that PKCy promoted NFnB activation in TNF-a-stimulated neutrophils through inducing InB-a degradation. We show here for the first time that PKCa/h plays a vital role in enhancing nuclear translocation of NF-nB by inducing IKKa/h activation in TLR2 or TLR4 activated neutrophils. Upon stimulus-induced InB-a degradation, NF-nB complexes move to the nucleus and initiate NF-nB-dependent transcription. Phosphorylation events involving NF-nB subunits also participate in transcriptional regulation. In particular, NF-nB dependent transcription has been demonstrated to be modulated by the phosphorylation of the p65 subunit [10]. Previous studies showed that IKKa/h, ERK, and p38 were involved in the phosphorylation of the p65 subunit [39,40]. The PKC family also appears to participate in this process (see Ref. [34] for review) since the Nterminal domain of p65 can be phosphorylated by PKC~ [41]. In PKC~-deficient mice, NF-nB is inactive and phosphorylation of p65 is severely impaired [42]. However, PKC~ was not involved in p65 phosphorylation in intestinal epithelial cells infected with E. coli [43]. To obtain further insight into the regulation of PKCa/h-induced phosphorylation of p65, we blocked PKCa/h with Gf6976 and found that phosphorylation of p65 in TLR2 or TLR4 stimulated neutrophils was severely impaired. Our data therefore show that PKCa/h can regulate NF-nB dependent transcription in neutrophils both by enhancing nuclear translocation of NFnB and also by increasing phosphorylation of the p65 subunit.
Acknowledgement This work was supported by NIH grants HL 62221 and PO1 HL-68743 (to E. Abraham), French Ministry of Foreign
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Affairs (LAVOISIER program) and the Societe Francaise d’Anesthesie et de Reanimation (to K. Asehnoune).
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Involvement of PKCa/h in TLR4 and TLR2 dependent activation of NF-nB Karim Asehnounea,b, Derek Strassheima, Sanchayita Mitraa, Jae Yeol Kima, Edward Abrahama,* a
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Box C272, 4200 East 9th Ave, Denver, CO 80262, USA b Service d’Anesthesie-Re´animation et Unite´ Propre de Recherche de l’Enseignement Superieur-Equipe d’Accueil (UPRES-EA 3540), Hoˆpital de Biceˆtre, Le Kremlin Biceˆtre, France Received 4 June 2004; received in revised form 18 August 2004; accepted 19 August 2004 Available online 16 September 2004
Abstract Protein kinase C (PKC)a/h dependent signaling events downstream of TLR4 or TLR2 were investigated in neutrophils stimulated with LPS or PGN. Pretreatment of neutrophils with the structurally distinct PKCa/h inhibitors Gf6976 or GF109203X decreased nuclear translocation of NF-nB and production of the proinflammatory cytokine TNF-a. Inhibition of PKCa/h also prevented LPS or PGN induced phosphorylation of IKKa/h, phosphorylation and degradation of InB-a, as well as phosphorylation of the p65 subunit of NF-nB. Activation of p38, JNK, and ERK 1/2 in response to TLR2 engagement was diminished in neutrophils in which PKCa/h was inhibited. However, no alteration in the activation of these kinases was found in TLR4 stimulated neutrophils when PKCa/h was blocked. Such results indicate that distinct intracellular signalling pathways leading to MAPK activation are induced by TLR4 and TLR2 stimulation. PKCa/h can regulate NFnB dependent transcription in neutrophils both by enhancing nuclear translocation of NF-nB and also by stimulating phosphorylation of the p65 subunit. D 2004 Elsevier Inc. All rights reserved. Keywords: PKC; Neutrophils; NF-nB; TLR; Cell signalling; Kinases
1. Introduction The protein kinase C (PKC) family of serine/threonine kinases mediate essential cellular signals required for activation, proliferation, differentiation, and survival [1]. There are at least 12 different PKC isoforms. Among the PKC family, three different general groupings can be characterized: (1) classical PKC isotypes (a, h, g) are diacylglycerol and calcium dependent, (2) novel PKC isotypes (y, q) are diacylglycerol dependent but calcium independent, and (3) atypical PKC isotypes (~, E) are diacylglycerol and calcium independent [2]. The transcriptional regulatory factor NF-nB is a central participant in modulating the expression of many immunor-
* Corresponding author. Tel.: +1 303 315 6025; fax: +1 303 315 5632. E-mail address: [email protected] (E. Abraham). 0898-6568/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2004.08.005
egulatory mediators involved in acute inflammatory response [3]. Signalling pathways initiated by engagement of Toll-like receptors (TLRs), such as TLR2 and TLR4, by bacterial products lead to nuclear accumulation of NF-nB and enhanced transcription of genes responsible for the expression of cytokines, chemokines, adhesion molecules, and other mediators of the inflammatory response associated with bacterial infection. Association of NF-nB with the inhibitory protein InB-a in the cytoplasm blocks the nuclear localization sequence (NLS) of NF-nB, inhibiting its movement into the nucleus [4]. Exposure of cells to inflammatory stimuli, including LPS, peptidoglycan (PGN), and proinflammatory cytokines (such as TNF-a or IL-1h), results in phosphorylation of InB-a on serines 32 and 36, leading to its subsequent ubiquitinylation and degradation by the 26S proteosome. Phosphorylation of InB-a is mediated by the kinases IKKa and IKKh, which are catalytically active components of the IKK complex [5].
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Previous studies have shown that PKCs are involved in the regulation of NF-nB in different cell types [1,2,6]. Moreover, it was demonstrated that the classical PKC family controls nuclear translocation of NF-nB through activation of IKKs and degradation of InB-a [7,8]. Neutrophils play a central role in the inflammatory responses such as acute lung injury induced by LPS or infection [3]. The role of PKCa/h in TLR2 and TLR4induced NF-nB activation has not been delineated. Similarly, a role for PKCa/h in modulating activation of NF-nB in neutrophils has not been described. In the present experiments, we explored these issues and found that PKCa/h was involved in enhancing nuclear translocation of NF-nB in TLR2 and TLR4 stimulated neutrophils, and also enhanced IKKa/h activation as well as phosphorylation of the p65 subunit of NF-nB.
2. Material and methods 2.1. Mice Male BALB/c mice, 8 to 12 weeks of age, were purchased from Harlan Sprague–Dawley (Indianapolis, IN). The mice were kept on a 12-h light/dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review boardapproved protocols. 2.2. Reagents and antibodies Isoflurane was obtained from Abbott Laboratories (Chicago, IL). Escherichia coli 0111:B4 endotoxin highly purified with phenol extraction (LPS, lipopolysaccharide) was purchased from Sigma (St. Louis, MO), and peptidoglycan (PGN) was purchased from InvivoGen (San Diego, CA). GF109203X was purchased from Calbiochem (San Diego, CA). RPMI 1640/25 mM HEPES/l-glutamine was obtained from Bio-Whittaker Products (Walkersville, MD). Fetal calf serum (FCS) and penicillin/streptomycin were purchased from Gemini Bioproducts (Calabasas, CA). Bicinchoninic acid (BCA) protein assay reagent was purchased from Pierce (Rockford, IL). Activation specific antibodies for phospho-Thr202/Tyr204 ERK1, phosphoThr183/Tyr185 ERK2, phospho-Thr180/Tyr182 p38, phospho-Thr183/Tyr185 JNK, phospho-Ser536 p65, phosphoSer180/Ser181 IKKa/h phospho-Ser32/Ser36 InB-a, phospho Thr638/641 PKCa/h and total ERK1/2, p38, JNK, InBa, IKKa/h p65 were purchased from Cell Signaling Technologies (Beverly, MA). Horseradish peroxidaselabeled anti-rabbit antibodies, and ECL reagents were purchased from Bio-Rad (Hercules, CA). All other reagents were purchased from Sigma unless otherwise noted in the text. Custom cocktail antibodies and columns for neutrophil isolation were purchased from Stem Cell Technologies (Vancouver, BC).
2.3. Isolation and culture of bone marrow derived mouse neutrophils Bone marrow neutrophils were isolated as described previously [9]. To obtain the bone marrow cell suspension, the femur and tibia of a mouse were flushed with RPMI 1640. Tissue fragments were removed by rapid filtration through a glass wool column and cells collected by centrifugation. The cell pellets were resuspended in RPMI 1640, 2% FCS and then incubated with primary Abs specific for the cell surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min at 4 8C. This custom mixture (StemCell Technologies) is specific for T and B cells, RBC, monocytes, and macrophages. After 15 min, 100 Al of antibiotin tetrameric Ab complexes were added and the cells incubated for a further 15 min at 4 8C. Following this, 60 Al of colloidal magnetic dextran iron particles were added to the suspension and incubated for 15 min at 4 8C. The entire cell suspension was then placed into a column surrounded by a magnet. The T cells, B cells, RBC, monocytes, and macrophages were captured in the column, allowing the neutrophils to pass through by negative-selection methods. Viability, as determined by trypan blue exclusion, was consistently greater than 98%. Neutrophil purity, as determined by Wright’s-stained cytospin preparations, was greater than 97%. Less than 0.3% of the purified cell population consisted of mononuclear cells. Bone marrow neutrophils (2106/0.5 ml) were cultured in RPMI 1640, 0.1% FCS, with or without drugs as described in figure legends. LPS, PGN, Gf6976 and GF109203X were made fresh for each experiment. In each experiment, Gf6976 or GF109203X was added to the cell cultures 15 min before stimulation with LPS or PGN. 2.4. Cytokine ELISA Immunoreactive TNF-a was quantitated using commercially available ELISA kits (R&D Systems, Minneapolis, MN), according to manufacturer’s instructions and as described previously [9]. 2.5. EMSA Nuclear extracts were prepared and assayed by EMSA as previously described [9]. For analysis of NF-nB, the nBDNA sequence of the Ig gene was used. Synthetic doublestranded sequences (with enhancer motifs underlined) were filled in and labeled with [a-32 P]dATP using Sequenase DNA polymerase as follows: nB, 5V-TTTTCGAGCTCGGGACTTTCCGAGC-3Vand 3V-GCTCGAGCCCTGAAAGGCTCGTTTT-5V. 2.6. Western blot analysis Western blots for phosphorylated and total kinases were performed as described previously [9]. Parallel samples for
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total protein kinase were run with samples for activation specific phosphorylation analysis. Densitometry was performed using chemiluminescence system and analysis software (BioRad, Hercules, CA) to determine the ratio between phosphorylated and total kinase. Densitometry data for each kinase are shown in graphic form, with baseline phosphorylation levels before LPS stimulation (i.e. at 0 min) assigned a value of 100%. 2.7. Statistical analysis Values are expressed as the meanFS.E.M. Data were analysed by analysis of variance, and differences between means were determined using the Bonferroni multiple comparison test. Significance is defined as pb0.05.
3. Results 3.1. Inhibition of PKCa/ß blocks proinflammatory cytokine secretion by neutrophils after TLR2 or TLR4 activation To assess the role of PKCa/h in TLR2 and TLR4induced cellular activation, neutrophils were incubated with LPS or PGN and varying concentrations of two specific, structurally distinct PKCa/h inhibitors, Gf6976 or GF109203X. As expected, concentrations of TNF-a were increased in supernatants of neutrophils stimulated with LPS (Fig. 1A and B) or PGN (Fig. 1C and D). The release of TNF-a from LPS and PGN-stimulated neutrophils was
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inhibited in a dose dependent manner by both Gf6976 and GF109203X (Fig. 1). 3.2. Nuclear translocation of NF-jB is dependent on PKCa/ß in TLR2 and TLR4-stimulated neutrophils Increased concentrations of NF-nB in the nucleus were present within 20 min of LPS or PGN exposure and were maximal 40 min after addition of LPS and 60 min after addition of PGN to the cultures (data not shown). Pretreatment of neutrophils with either Gf6976 and GF109203X prevented LPS (Fig. 2A and B) or PGN (Fig. 2C and D) induced NF-nB activation. 3.3. Activation of PKCa/ß in TLR2 and TLR4-stimulated neutrophils The time course for PGN and LPS-mediated activation of PKCa/h in neutrophils is shown in Fig. 3A and C. Pretreatment of neutrophils with Gf6976 prevented PGN or LPS induced PKCa/h activation (Fig. 3B and D, respectively). 3.4. Blockade of PKCa/ß inhibits TLR2 and TLR4 induced activation of IKKa/ß A potential mechanism for the role that PKCa/h exerts on TLR2 and TLR4 induced nuclear translocation of NF-nB could be through enhancing activation of the IKK complex. IKK mediated phosphorylation of InB-a on serines 32 and
Fig. 1. PKCa/h blockade with Gf6976 or GF109203X inhibit TNF-a production in LPS or PGN stimulated neutrophils. Neutrophils were incubated with control media or with the indicated concentrations of Gf6976 or GF109203X for 15 min, and then cultured with or without LPS (1 Ag/ml) in A and B, with or without PGN (10 Ag/ml) in C and D. After 90 min, the supernatants were collected and assayed for concentrations of TNF-a. Data are combined from three independent experiments. *pb0.05 and **pb0.01 versus neutrophils cultured with LPS or PGN alone.
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tively). To confirm that PKCa/h also affected functional downstream effects of IKK activation, we examined cytoplasmic levels of InB-a in neutrophils treated with LPS or PGN alone or with LPS and PGN plus Gf6976 (Fig. 5). In neutrophils challenged with LPS or PGN, time dependent increases in phosphorylation of InB-a were observed (Fig. 5A and C) while total InB-a underwent concomitant degradation (Fig. 5B and D). However, in neutrophils incubated with Gf6976 and LPS or PGN, phosphorylation of InB-a was diminished compared to stimulation with PGN or LPS alone (Fig. 5A and C) and no degradation of total InB-a was apparent (Fig. 5B and D). Fig. 2. Inhibition of PKCa/h prevents nuclear translocation of NF-nB in LPS or PGN-stimulated neutrophils. Culture of neutrophils with LPS (1 Ag/ ml) or PGN (10 Ag/ml) induces nuclear accumulation of NF-nB (A and B or C and D, respectively), which is inhibited by pretreatment with Gf6976 (A and C) or GF109203X (B and D). In A and C, the neutrophils were incubated with Gf6976 (100 nM), in B and D the neutrophils were incubated with GF109203X (1 AM) for 15 min before LPS or PGN were added to the cultures. Neutrophils not treated with Gf6976, GF109203X, LPS, or PGN were included as controls (C). Nuclear extracts were obtained after 40 min of LPS stimulation or 60 min after PGN stimulation, time points when nuclear translocation of NF-nB was found to be maximal after these stimuli. Two additional experiments provided similar results.
3.5. Blockade of PKCa/b inhibits TLR2 or TLR4 induced phosphorylation of p65
36 leads to subsequent ubiquitinylation and degradation of InB-a, with removal of its tonic inhibitory effects on nuclear translocation of NF-nB [5]. In these experiments, activation of IKK was assessed by determining phosphorylation of serine 180 in IKKa, and serine 181 in IKKh. As shown in Fig. 4A, stimulation of neutrophils with PGN resulted in phosphorylation of IKK that peaked after 45 min. Following LPS stimulation, maximal activation of IKK was found after 30 min (Fig. 4C). Blockade of PKCa/h inhibited PGN and LPS-induced IKK phosphorylation (Fig. 4B and D, respec-
3.6. Differential roles of PKCa/b in TLR2 or TLR4-induced activation of p38, ERK, and JNK
NF-nB transcriptional activity is modulated through phosphorylation of the p65 subunit [10], an event mediated by IKK as well as other kinases [11]. Incubation of neutrophils with either LPS or PGN induced the phosphorylation of p65 (Fig. 6A and C). Inhibition of PKCa/h with Gf6976 abolished p65 phosphorylation after PGN or LPS stimulation (Fig. 6B and D).
Exposure of neutrophils to TLR2 or TLR4 agonists has been shown to activate mitogen activated protein kinases (MAPK), including p38 [12], ERK 1/2 [13], and JNK [9], all of which participate in enhancing nuclear translocation of NF-nB [3]. To examine the role of PKCa/h in modulating activation of these kinases, we determined levels of phosphorylated kinases in LPS or PGN stimulated neutro-
Fig. 3. Gf6976 prevents PKCa/h activation in LPS or PGN-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with PGN (10 Ag/ml) or LPS (1 Ag/ml). Culture of neutrophils with PGN or LPS induced activation of PKCa/h (A and C, respectively), that was inhibited by pretreatment with Gf6976 (B and D). At the indicated times after addition of PGN or LPS, the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting and levels of phosphorylated PKCa/h (p-PKCa/h) were determined. Densitometry data are shown in graphic form, with baseline PKCa/h phosphorylation before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
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Fig. 4. Inhibition of PKCa/h prevents LPS and PGN-induced IKKa/h phosphorylation. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting using antibodies specific for Ser180/Ser181 IKKa/h (p-IKKa/h) and for total kinases. Densitometry data are shown in graphic form, with baseline phosphorylation of Ser180/Ser181 IKK before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
phils and in those pretreated with Gf6976. Inhibition of PKCa/h in neutrophils prevented PGN-induced activation of p38 and ERK1/2, and strongly diminished that of JNK 1/ 2 (Figs. 7, 8 and 9, respectively). In contrast, blockade of PKCa/h did not inhibit LPS-induced activation of p38 or ERK1/2 (Figs. 7 and 8, respectively). Activation of JNK 1/2 in LPS treated neutrophils was too weak to observe any effect of PKCa/h inhibition (data not shown).
4. Discussion Previous studies have suggested that PKCs are involved in the activation of NF-nB (see Ref. [1] for review). Evidence linking PKCs to NF-nB comes from complementary observations demonstrating that nuclear translocation of NF-nB can be enhanced by exposure of cells to PKC activators and prevented by selective PKC inhibitors [1,2]. Such reports, which have primarily utilized lymphocytes,
have provided new insights into the role of PKC on NF-nB activation. However, the assumption that PKC plays a general role in the activation of NF-nB has been the subject of considerable debate. In part, this controversy arises from reports regarding the role of each PKC member during intracellular signalling, complicated by the cell type used, the initial stimulus, as well as the PKC isoform investigated [14]. Little is known about regulation of NF-nB activity in neutrophils [6], and the role of PKC isoforms in affecting NF-nB activity in this cell population through TLR signalling pathways has not previously been examined. TLRs play a role in innate defense by recognizing different molecular patterns associated with microbial pathogens [15]. Stimulation of TLR by bacterial products activates NF-nB and results in enhanced generation of cytokines and other proinflammatory mediators whose transcription is dependent on NF-nB [3]. Among the TLR family, TLR4 is involved in the recognition of LPS from gram-negative bacteria and TLR2 in the response to
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Fig. 5. PKCa/h blockade inhibits LPS or PGN-induced InB-a phosphorylation and degradation. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting and levels of phosphorylated InB-a (pInB-a) and total InB-a were determined. Densitometry data are shown in graphic form, with baseline InB-a phosphorylation before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
products of gram-positive bacteria, such as PGN [16]. After homodimerization, TLR4 induces intracellular signaling through pathways utilizing MyD88, MyD88-like/Toll-IL1R domain-containing adapter proteins, and IL-1R-associated kinases. Signaling via TLR2 is at least partly dependent upon dimerization of this receptor with either TLR1 or TLR6 [17,18]. While a large body of literature indicates that the members of the TLR family activate nearly identical cytoplasmic signaling programs, it was recently suggested that there are distinct differences in the response elicited by TLR2 or TLR4 agonists [19].
Neutrophils have been shown to contain the classical PKCs a and h, which are both DAG and Ca2+ dependent, as well as PKCy, which does not require either DAG or Ca2+ [20,21]. With respect to NF-nB activation, PKCa/h isoforms were shown to have a central role in lymphocytes [1,22]. Among PKC isoforms, only PKCa/h are specifically inhibited by Gf6976 and GF109203X [23–27]. Thus, the present experiments demonstrate that PKCa/h is essential for NF-nB activation and the expression of NFnB dependent cytokines such as TNF-a, in neutrophils [3,28].
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Fig. 6. PKCa/h participates in LPS and PGN-induced p65 phosphorylation. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting using antibodies specific for Ser536 p65 (p-p65) and for total p65. Densitometry data are shown in graphic form, with baseline phosphorylation of Ser536 p65 before PGN or LPS stimulation (i.e. at 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.
Fig. 7. Involvement of PKCa/h in p38 activation in PGN or LPS-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), cellular extracts were obtained and levels of phosphorylated p38 (p-p38) and total p38 determined. Representative gels are shown. Densitometry data are presented with the baseline kinase phosphorylation before PGN or LPS addition to cultures (i.e. at time 0 min) given a value of 100%. The data shown are representative of three independent experiments.
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Fig. 8. Involvement of PKCa/h on ERK1/2 activation in PGN or LPS-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with LPS (1 Ag/ml) or PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B) or LPS (C and D), cellular extracts were obtained and levels of phosphorylated (p-ERK1/2) and total kinase were determined. Representative gels are shown. Densitometry data are presented with the baseline kinase phosphorylation before PGN or LPS addition to cultures (i.e. at time 0 min) given a value of 100%. The data shown are representative of three independent experiments.
Although PKCs can affect signaling pathways leading to NF-nB activation [1,22], the specific intracellular events modulated by PKCa/h have not been completely defined. Kinases shown to be activated by PKCs include JNK, p38, ERK, and the IKK complex [7,8,29–31]. It was recently demonstrated in lymphocytes that PKCh induces NF-nB activation through IKK mediated phosphorylation of InBa [7,8]. Although PKCs have been shown to activate p38 MAPK [32], JNK, and ERK1/2 [29,30] in some cell
populations, their effect on these kinases in neutrophils has not been previously reported. Several studies have shown that pathways including MEK/ERK/p38 kinases participate in the regulation of NFnB [33]. ERK1/2 lies immediately downstream of and is activated by the dual-specificity kinases MEK1/2. MEK 1/2 represent major targets of the serine/threonine kinase Raf-1 [31] and several studies demonstrated that Raf-1 acts as a MAPK kinase kinase (see Ref. [34] for review). Raf-1 is
Fig. 9. Involvement of PKCa/h on JNK activation in PGN-stimulated neutrophils. Neutrophils were pretreated with Gf6976 (100 nM) or left untreated for 15 min, and then were cultured with PGN (10 Ag/ml). At the indicated times after addition of PGN (A and B), cellular extracts were obtained and levels of phosphorylated (p-JNK) and total kinase were determined. Representative gels are shown. Densitometry data are presented with the baseline kinase phosphorylation before PGN addition to cultures (i.e. at time 0 min) given a value of 100%. The data shown are representative of three independent experiments.
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itself activated by PKC [35]. Consequently, it is presumed that inhibition of PKC would reduce basal activity and/or prevent ERK activation. We [9,36] and others [12,13,37] have demonstrated that stimulation of neutrophils with LPS results in activation of members of the MAPK family, including ERK 1/2 and p38. TLR2 agonists, such as PGN, are also potent activators of the MAPK family [19,38]. In the present experiments, inhibition of PKCa/h blocked phosphorylation of ERK 1/2, JNK, and p38 in neutrophils cultured with PGN, but not with LPS. Such results suggest that distinct intracellular signalling pathways leading to MAPK activation and involving PKCa/h are induced by TLR2 and TLR4 stimulation. Activation of NF-nB is critically dependent on the phosphorylation and degradation of the cytoplasmic inhibitor InB-a. One of the critical regulatory steps modulating degradation of InB-a is activation of the InB-a kinase (IKK) complex, which consists of the catalytic subunits, IKKa and IKKh, and the regulatory subunit NEMO [5]. Recent studies in lymphocytes demonstrated that PKCa/h was involved in IKK activation and the degradation of InBa [7,8]. Vancurova et al. [6] found that PKCy promoted NFnB activation in TNF-a-stimulated neutrophils through inducing InB-a degradation. We show here for the first time that PKCa/h plays a vital role in enhancing nuclear translocation of NF-nB by inducing IKKa/h activation in TLR2 or TLR4 activated neutrophils. Upon stimulus-induced InB-a degradation, NF-nB complexes move to the nucleus and initiate NF-nB-dependent transcription. Phosphorylation events involving NF-nB subunits also participate in transcriptional regulation. In particular, NF-nB dependent transcription has been demonstrated to be modulated by the phosphorylation of the p65 subunit [10]. Previous studies showed that IKKa/h, ERK, and p38 were involved in the phosphorylation of the p65 subunit [39,40]. The PKC family also appears to participate in this process (see Ref. [34] for review) since the Nterminal domain of p65 can be phosphorylated by PKC~ [41]. In PKC~-deficient mice, NF-nB is inactive and phosphorylation of p65 is severely impaired [42]. However, PKC~ was not involved in p65 phosphorylation in intestinal epithelial cells infected with E. coli [43]. To obtain further insight into the regulation of PKCa/h-induced phosphorylation of p65, we blocked PKCa/h with Gf6976 and found that phosphorylation of p65 in TLR2 or TLR4 stimulated neutrophils was severely impaired. Our data therefore show that PKCa/h can regulate NF-nB dependent transcription in neutrophils both by enhancing nuclear translocation of NFnB and also by increasing phosphorylation of the p65 subunit.
Acknowledgement This work was supported by NIH grants HL 62221 and PO1 HL-68743 (to E. Abraham), French Ministry of Foreign
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Affairs (LAVOISIER program) and the Societe Francaise d’Anesthesie et de Reanimation (to K. Asehnoune).
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