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The Journal of Immunology, 2000, 164: 29-37.
Copyright © 2000 by The American Association of Immunologists

Protein Kinase C{epsilon} Is Required for the Induction of Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages1

Annabel F. Valledor, Jordi Xaus, Mònica Comalada, Concepció Soler and Antonio Celada2

Departament de Fisiologia (Biologia del Macròfag), Facultat de Biologia and Fundació August Pi i Sunyer, Campus Bellvitge, Universitat de Barcelona, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LPS induces in bone marrow macrophages the transient expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). Because MKP-1 plays a crucial role in the attenuation of different MAPK cascades, we were interested in the characterization of the signaling mechanisms involved in the control of MKP-1 expression in LPS-stimulated macrophages. The induction of MKP-1 was blocked by genistein, a tyrosine kinase inhibitor, and by two different protein kinase C (PKC) inhibitors (GF109203X and calphostin C). We had previously shown that bone marrow macrophages express the isoforms PKCßI, {epsilon}, and {zeta}. Of all these, only PKCßI and {epsilon} are inhibited by GF109203X. The following arguments suggest that PKC{epsilon} is required selectively for the induction of MKP-1 by LPS. First, in macrophages exposed to prolonged treatment with PMA, MKP-1 induction by LPS correlates with the levels of expression of PKC{epsilon} but not with that of PKCßI. Second, Gö6976, an inhibitor selective for conventional PKCs, including PKCßI, does not alter MKP-1 induction by LPS. Last, antisense oligonucleotides that block the expression of PKC{epsilon}, but not those selective for PKCßI or PKC{zeta}, inhibit MKP-1 induction and lead to an increase of extracellular-signal regulated kinase activity during the macrophage response to LPS. Finally, in macrophages stimulated with LPS we observed significant activation of PKC{epsilon}. In conclusion, our results demonstrate an important role for PKC{epsilon} in the induction of MKP-1 and the subsequent negative control of MAPK activity in macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages perform critical functions in the immune system. They act as regulators of homeostasis and as effector cells in infection, tumor growth, and healing of wounds (1). In contrast to other cells of the immune system, macrophages show a marked duality in their biological responses; they either proliferate (e.g., in response to the specific growth factor M-CSF) or become activated, undergo a growth arrest, and start performing their specialized functions in the context of the immune response (2). LPS or endotoxin, a major component of the outer membrane of Gram-negative bacteria, activates macrophages and induces the secretion of arachidonic acid metabolites (e.g., PGs, leukotrienes, and platelet-activating factor), nitrogen intermediates, and cytokines such as TNF-{alpha}, IL-1, and IL-6 (3, 4), which play important roles in the immune response.

In macrophages, LPS triggers the activation of several signal transduction pathways involving G proteins, tyrosine kinases, phospholipase C (PLC),3 protein kinase A (PKA), and protein kinase C (PKC) (5, 6, 7, 8). The serine/threonine kinase PKC family consists of several isoforms that are distributed in three main groups (conventional, novel, and atypical PKCs) based on their primary structure and activation requirements (9, 10). Conventional PKCs, which include {alpha}, ßI, ßII, and {gamma}, need both Ca2+ and diacylglycerol (DAG)/phorbol esters for activation and phosphatidylserine as a cofactor. Novel PKCs, which include {delta}, {epsilon}, {theta}, {eta}, and µ, need DAG/phorbol esters and phosphatidylserine, but do not require Ca2+ for activation. Atypical PKCs, including {zeta} and {iota}v, cannot be activated by Ca2+ or DAG/phorbol esters, but are regulated by phosphatidylinositol (3, 4, 5)-trisphosphate, ceramide, and phosphatidic acid (11, 12, 13).

LPS also activates different mitogen-activated protein kinase (MAPK) cascades, including the extracellular-signal regulated protein kinase (ERK) (14, 15), the c-Jun N-terminal protein kinase, and the p38 MAPK/reactivating kinase pathways (16). For general MAPK activity, phosphorylation in both tyrosine and threonine residues is required (17). Active ERKs phosphorylate and regulate several cellular proteins, including additional protein kinases, cytoskeletal components, phospholipase A2, and nuclear transcription factors, such as Elk1/TCF and c-Jun, which regulate the expression of immediate early genes (reviewed in Ref. 18).

MAPK phosphatase-1 (MKP-1) is a member of a family of inducible dual-specificity tyrosine phosphatases (19, 20, 21). MKP-1 selectively dephosphorylates tyrosine and threonine residues on ERK-1/2 (22, 23), thus allowing the inactivation of these kinases. Recent reports have also shown the capability of MKP-1 to dephosphorylate and inactivate c-Jun N-terminal kinase/stress-activated protein kinase and p38/reactivating kinase (24).

At the present, little is known about the mechanisms involved in the induction of MKP-1 by LPS. Because the study of the events that account for the negative control of MAPK activity is of great interest, in this report we have characterized the signaling pathway(s) that lead to MKP-1 expression in response to LPS. We have found that MKP-1 expression was not dependent on the activation of the MAPKs ERK-1/2 in response to LPS. Instead, the induction of this phosphatase required the activation of tyrosine kinases and PKC isozyme {epsilon}. This suggests that, in macrophages, PKC{epsilon} plays a key role in the negative control of general MAPK activity through the induction of the phosphatase MKP-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Macrophages

Bone marrow-derived macrophages were obtained from 6- to 10-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA) as described (25). Macrophages were cultured in DMEM (Sigma, St. Louis, MO) supplemented with 20% FBS (Sigma) and 30% L cell-conditioned medium as a source of M-CSF. Once macrophages were 80% confluent, normally after 6 days of culture, they were deprived of L cell-conditioned medium for 16–18 h and treated with LPS (Sigma) in the presence or absence of selective inhibitors/activators. All treatments were not toxic for the cells as determined by flow cytometry analysis.

Reagents

Genistein, bisindolylmaleimyde I (GF109203X), calphostin C, PMA, PLC from Bacillus cereus, KT5720, and KT5823 were purchased from Calbiochem (San Diego, CA). Ionomycin was purchased from ICN Pharmaceuticals (Costa Mesa, CA). 8-Bromo-cAMP (8-Br-cAMP) and 8-bromo-cyclic GMP (8-Br-cGMP) were obtained from Fluka Biochemika (Buchs, Switzerland). Sphingomyelinase from B. cereus and wortmannin were obtained from Sigma. PD98059 was purchased from New England Biolabs (Beverly, MA). Gö6976 was a kind gift from Dr. A. García de Herreros (Institut Municipal d’Investigació Mèdica, Barcelona, Spain). All reagents were used following the manufacturer’s recommendations.

Antisense oligonucleotides

Antisense phosphorothioated-oligonucleotides specific for PKC isoforms ßI/II, {epsilon}, and {zeta} were used to block the expression of specific PKC isoforms. The cells were incubated for 34 h in DMEM with 5% FBS containing 12 µM thioated-oligonucleotides (Biognostik, Göttingen, Germany). Oligonucleotides specific for mouse PKCßI/II corresponded to the reverse complement of a target sequence as described (C. L. Ashendel, unpublished observations) (accession no. X53532). Oligonucleotides against mouse PKC{zeta} were designed as described (26). Oligonucleotides specific for mouse PKC{epsilon} corresponded to the sequence: 5'-GCTCACCGCCTCGCAGATTT-3'.

RNA extraction and Northern blot analysis

The cells were washed twice in cold PBS, and extraction of total RNA was performed as described (27). Total RNA samples (15 µg) were separated on 1.2% agarose gels containing formaldehyde and transferred to nylon membranes (Genescreen; NEN Life Science Products, Boston, MA). For MKP-1 mRNA detection, we obtained the full-length cDNA fragment of MKP-1 following purification from a HindIII digestion of the plasmid pBluescript KS/MKP-1 (kindly provided by Dr. R. Bravo, Bristol-Myers Squibb Pharmacology Research Institute, Princeton, NJ). For TNF-{alpha} mRNA detection, we used the EcoRI/HindIII fragment of pSP65/TNF-{alpha} (kindly supplied by Dr. M. Nabholz, Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland). To study the expression of IL-1ß, we obtained a probe by digesting the construct pGEM1/IL-1ß (kindly provided by Dr. R. Wilson, Glaxo Research and Development Limited, Greenford, U.K.) with EcoRI/PstI. For ß-actin mRNA detection, we obtained the PstI fragment of pSP65/ß-actin (28). To detect the L32 transcript, we used the EcoRI/HindIII fragment of pGEM1/L32 as a probe (29). All probes were labeled with [{alpha}-32P]dCTP (ICN Pharmaceuticals). After incubating in hybridization solution (20% formamide, 5x Denhardt’s, 5x SSC, 10 mM EDTA, 1% SDS, 25 mM Na2HPO4, 25 mM NaH2PO4, and 0.2 mg/ml salmon sperm DNA) at 65°C, the membranes were washed and exposed to Kodak X-AR films (Kodak, Rochester, NY). Besides, bands of interest were quantified with a Molecular Analyst (Bio-Rad, Richmond, CA).

Protein extraction and Western blot analysis

The cells were washed twice in cold PBS and lysed on ice with lysis solution (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml iodoacetamide, 1 mM PMSF). When inhibition of the activity of tyrosine phosphatases was required, 1 mM sodium orthovanadate was included. For PKC detection experiments, 1 mM EGTA and 2 mM EDTA were added to the lysis solution. Insoluble material was removed by centrifugation at 13,000 x g for 8 min at 4°C. The protein concentration of the samples was determined by the Bio-Rad protein assay. The proteins from cell lysates (50–100 µg) were boiled at 95°C in Laemli SDS-loading buffer and separated by 10% SDS-PAGE (unless otherwise stated) and electrophoretically transferred to nitrocellulose membranes (Hybond-ECL; Amersham, Arlington Heights, IL). The membranes were blocked in 2% BSA in TBS-0.1% Tween 20 (TBS-T) for 3 h at room temperature and then incubated with the primary Ab. For MKP-1 immunoblotting, incubation was performed for 1 h at room temperature with rabbit IgG anti-mouse MKP-1 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA). For the recognition of PKC isozymes, incubation was done overnight at 4°C with rabbit IgG selective for each mouse PKC isoform (1:1000) in the presence or absence of specific competitor peptides (1:1000) (Abs and peptides were kindly provided by Dr. P. J. Parker, Imperial Cancer Research Fund, London, U.K.). For some experiments, we used equivalent Abs purchased from Life Technologies (Grand Island, NY). After three washes of 15 min each in TBS-T, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG Ab (Cappel, Durham, NC) (1:5000) for 1 h. After three washes of 15 min with TBS-T, enhanced chemiluminescence detection was performed (Amersham Life Science, London, U.K.) and the membranes were exposed to x-ray films (Amersham).

Determination of ERK activity by in-gel-kinase assay

This assay was performed as previously described (30). Briefly, 50 µg of total protein were separated by 12.5% SDS-PAGE in the presence of 0.1 mg/ml of myelin basic protein (Sigma) copolymerized in the gel. After electrophoresis, SDS was removed by washing the gel with two changes of 20% 2-propanol in 50 mM Tris-HCl, pH 8.0, for 1 h at room temperature. The gel was then incubated with 50 mM Tris-HCl, pH 8.0, containing 5 mM 2-ME (buffer A) for 1 h at room temperature. The proteins were denatured by incubating the gel with two changes of 6 M guanidine-HCl for 1 h at room temperature and then renatured by incubating with five changes of buffer A containing 0.04% Tween 20 for 16 h at 4°C. To perform the phosphorylation assay, the gel was first equilibrated in 40 mM HEPES-NaOH, pH 7.4, containing 2 mM DTT, 0.1 mM EGTA, 15 mM MgCl2, and 300 µM sodium orthovanadate for 30 min at 25°C and then incubated for 1 h in the same solution containing 50 µM ATP and 100 µCi [{gamma}-32P]ATP (ICN). The reaction was stopped by washing the gel with 5% TCA containing 10 mM sodium pyrophosphate to inhibit phosphatase activity. The gel was dried, exposed to x-ray films (Kodak), and quantified with a Bio-Rad molecular analyst.

Determination of PKC translocation

PKC translocation was determined as described previously (31) with some modifications. The cells were lysed by scraping in cold hypotonic buffer T10 (10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 10 mM NaCl), containing inhibitors of proteases (1 µg/ml aprotinin, 1 µg/ml leupeptin, 86 µg/ml iodoacetamide, 1 mM PMSF) and 1 mM sodium orthovanadate. The cell lysates were centrifuged at 100,000 x g for 30 min at 4°C and the supernatants were collected (cytosol fraction). The pellets were resuspended in cold buffer T10 containing 1% Triton X-100 and homogenized on ice (15–20 strokes with a Dounce homogenizer). To allow PKC extraction from cell membranes, the samples were left for 1 h at 4°C and then centrifuged at 100,000 x g for 30 min at 4°C. The supernatants were collected (plasma membrane fraction) and the pellets were resuspended in cold T10 containing 1% SDS, passed through a 19-gauge needle five times, and boiled at 100°C for 5 min. Insoluble material was removed by centrifugation at 13,000 x g for 10 min, and the supernatants were collected (cytoskeleton fraction). Samples from each fraction (25 µg of protein) were boiled at 95°C in loading buffer and separated by 8% SDS-PAGE. The proteins were electrophoretically transferred to Hybond-ECL membranes (Amersham) and immunoblotted with anti-PKC isoforms Abs in the presence or absence of competitor peptide.

Measurement of PKC{epsilon} activity

This assay was performed as previously described (32) with modifications. Specific Abs against PKC{epsilon} (Life Technologies) were used to immunoprecipitate this isoform from subcellular fractions (2 µg of Ab per 150 µg of total protein in a total volume of 300 µl). Incubation was conducted for 2 h at 4°C. Immunocomplexes were separated by addition of 75 µl of 20% protein A-Sepharose slurry, incubated 2 h at 4°C, and pelleted. The pellets were washed twice with RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 2 mM EDTA, 1 mM EGTA) supplemented with protease inhibitors and 1 mM sodium orthovanadate, once with prereaction buffer (50 mM ß-glycerophosphate, 10 mM MgCl2, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, protease inhibitors, 1 mM sodium orthovanadate), and then resuspended in reaction buffer (prereaction buffer supplemented with 100 µM ATP, 33 µM 1, 2-sn-dioleoylglycerol, 40 µg/ml L-{alpha}-phosphatidylserine, 1 µM 25Ser-PKC, and 5 µCi [{gamma}-32P]ATP). A 25Ser-substituted peptide obtained from the pseudosubstrate region of PKC (Calbiochem) was used as the substrate for the phosphorylation assay because it represents an appropiate substrate for measuring PKC{epsilon} activity (33). The reaction was conducted for 10 min at 30°C. Each sample was spotted on a phosphocellulose filter (Whatman 3 MM) and subjected to five washes of 30 min each in 5% TCA, 10 mM sodium pyrophosphate. Radioactivity was counted by liquid scintillation using a Packard Tri-Carb 1400 scintillation counter (Meriden, CT).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The present study was conducted with bone marrow-derived macrophages because they represent an homogeneous population of primary macrophages. In these cells, LPS induced the transient expression of the phosphatase MKP-1. The induction of the MKP-1 transcript could be detected after 20–30 min of LPS treatment (Fig. 1GoA). Maximal expression was observed at 45–60 min and then it gradually decayed to basal levels within 2–3 h. Synthesis of the MKP-1 protein tightly correlated with the time course of MKP-1 mRNA expression (Fig. 1GoB). The expression of MKP-1 was also analyzed after the stimulation of macrophages with different concentrations of LPS for 45 min (Fig. 1Go, C and D). The induction of MKP-1 by LPS was dose dependent and reached a plateau at 100 ng/ml of LPS. Therefore, in additional experiments we used 100 ng/ml and 1 ng/ml of LPS for saturant and subsaturant conditions, respectively.



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  		FIGURE 1. MKP-1 expression is induced by LPS in bone marrow macrophages in a time- and dose-dependent manner. A, MKP-1 mRNA expression was assessed by Northern blotting (15 µg of total RNA per lane). Total RNA extracts were obtained after treating quiescent bone marrow macrophages with LPS (100 ng/ml) for the indicated periods of time. B, The expression of the MKP-1 protein was analyzed by Western blotting (100 µg of total cell extracts per lane). Cell extracts were obtained after the treatment with LPS (100 ng/ml) for the indicated periods of time. The expression of ß-actin mRNA (A) and protein (B) was analyzed to check for differences in sample loading and transfer efficiency. C, Northern blotting was performed with total RNA extracts (15 µg per lane) from cells treated with the indicated concentrations of LPS for 45 min. D, Values of MKP-1 expression from C were normalized with the L32 control and represented as percentages of maximal expression. These images are representative of two independent experiments.

 
Because the members of the MKP-1 family play a crutial role in the inactivation of MAPK cascades, we were next interested in determining the signal transduction pathway that leads to the induction of this phosphatase after the stimulation of macrophages with LPS. We first studied whether the induction of MKP-1 by LPS was mediated, as a negative feedback mechanism, by the activation of the MAPKs ERK-1/2. Fig. 2Go shows that the blockage of ERK-1/2 activation by PD98059, a specific inhibitor of MAPK kinase (MEK)-1, did not alter the LPS-induced expression of MKP-1 at either the mRNA or protein levels. This indicates that LPS induces the expression of MKP-1 through a mechanism that is independent of the activation of the late elements of the ERK cascade (MEK1 and ERK-1/2).



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  		FIGURE 2. MEK-1, ERK-1, and ERK-2 do not mediate the induction of MKP-1 after LPS stimulation. A, Activation of ERK-1/2 by LPS was blocked by the inhibitor PD98059. The cells were untreated or preincubated with either PD98059 (50 µM) or vehicle (0.1% DMSO) for 1 h and then stimulated with LPS (100 ng/ml) for 15 min. ERK-1/2 activity was analyzed by an in-gel-kinase assay. B and C, Macrophages were preincubated for 1 h with either PD98059 (50 µM) or vehicle (0.1% DMSO) and then stimulated or not with LPS (100 ng/ml) for 45 min. The expression of MKP-1 was analyzed by Northern blotting (15 µg of total RNA per sample) (B) and by Western blotting (100 µg of total protein per lane) (C). These assays were repeated twice with identical results.

 
Maximal induction of MKP-1 was obtained at a concentration of 100 ng/ml of LPS (Fig. 1Go, C and D), a dose that saturates the binding of LPS to its high-affinity receptor, the molecule CD14 (34). Although other molecules may also transduce signals at very high doses of LPS (1–10 µg/ml), our results suggest that the signaling pathway leading to MKP-1 induction is triggered after the recognition of LPS by CD14. The fact that the induction of MKP-1 by LPS was dependent on the presence of serum (data not shown) supports this hypothesis. In fact, the recognition of LPS by CD14 requires the previous association of LPS with the serum protein LBP (LPS-binding protein) (35). Binding of LPS to CD14 results in the tyrosine phosphorylation of several cellular proteins (8, 36). To determine the involvement of tyrosine kinases in the induction of MKP-1 by LPS, the cells were preincubated with genistein, a general inhibitor of this type of kinases (Fig. 3Go). A significant decrease of the expression of MKP-1 mRNA was observed. As a control of the effect of genistein, we also analyzed the LPS-induced expression of TNF-{alpha} and IL-1ß. As previously reported (37), the expression of these cytokines was inhibited by this treatment (Fig. 3Go). Thus, our results suggest that the LPS-induced expression of MKP-1 is dependent on the activation of tyrosine kinases.



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  		FIGURE 3. The induction of MKP-1 by LPS depends on the activation of tyrosine kinases. The cells were untreated or preincubated with either genistein (100 µM) or vehicle (0.1% DMSO) for 1 h and then stimulated with LPS (100 ng/ml) for 45 min. A, Total RNA (15 µg) was analyzed by Northern blotting. B, Values of gene expression were normalized with the L32 control. The mean of three independent experiments is represented.

 
cAMP-response elements (CREs) are the binding sites for transcription factors that belong to the family of CRE binding proteins. Following the elevation of intracellular cAMP levels, these proteins are phosphorylated and thus activated by PKA (38). Because LPS is able to activate PKA in macrophages (39) and a CRE element has been described at position -137 bp in the promoter of the MKP-1 gene (21), we analyzed the involvement of cAMP-dependent pathways in the induction of MKP-1 by LPS. We first studied whether an increase in intracellular cAMP levels alone was able to induce the expression of MKP-1 in macrophages. The cells were treated with 8-Br-cAMP, which is a membrane-permeable and phosphodiesterase-resistant analogue of cAMP. However, the expression of MKP-1 was not detected after treating macrophages with this compound (Fig. 4GoA). Besides, 8-Br-cAMP was unable to increase the induction of MKP-1 when macrophages were stimulated with a subsaturant concentration of LPS. Because sustained activation of PKA has been associated with repression of the LPS-induced expression of TNF-{alpha} in macrophages (40), the expression of TNF-{alpha} mRNA was determined as a control of the activation of PKA by 8-Br-cAMP. Our results show that the addition of 8-Br-cAMP blocked the LPS-induced expression of TNF-{alpha} (Fig. 4GoA). Furthermore, the activation of PKA was inhibited by pretreating the cells with KT5720. This treatment did not affect the induction of MKP-1 mRNA and protein by either saturant or subsaturant concentrations of LPS (Fig. 4Go, B and C). The implication of a cGMP-dependent pathway was also tested. However, 8-Br-cGMP neither induced the expression of MKP-1 nor increased its induction by a subsaturant dose of LPS (Fig. 4GoA). Besides, the inhibition of c-GMP-dependent protein kinase G (PKG) with KT5823 did not alter the induction of MKP-1 by LPS (Fig. 4Go, B and C). Our results allow us to conclude that the LPS signaling events that lead to MKP-1 induction are not dependent on the activation of PKA or PKG.



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  		FIGURE 4. The LPS-induced expression of MKP-1 is not mediated by either PKA or PKG. A, Macrophages were either left untreated or incubated for 45 min with 8-Br-cAMP (100 µM), 8-Br-cGMP (100 µM), or with LPS (1 ng/ml) in the presence or absence of 8-Br-cAMP (100 µM) or 8-Br-cGMP (100 µM). Total RNA was extracted and the expression of MKP-1 was analyzed by Northern blotting (15 µg per sample). The expression of TNF-{alpha} was studied as a control of the effect of 8-Br-cAMP. B, Cells were pretreated or not with either the PKA inhibitor KT5720 (100 nM) or the PKG inhibitor KT5823 (400 nM) for 1 h and then stimulated with LPS (100 ng/ml) for 45 min. The expression of MKP-1 was studied by Northern blotting (15 µg per sample). C, Macrophages were pretreated or not with KT5720 (100 nM) or KT5823 (400 nM) for 1 h and then stimulated with a subsaturant dose of LPS (1 ng/ml) for 1 h. The expression of MKP-1 protein was analyzed by Western blot. All the experiments shown were performed twice with identical results.

 
A number of LPS-induced processes have been described to be dependent on PKC activation in macrophages (6, 7, 8). The MKP-1 gene promoter contains a PMA-responsive element at position -450 bp (21). PMA response element sites are recognized by the transcriptional complex AP-1 (41), whose activation may be mediated by PKC (42). For this reason, we studied whether the activation of PKC was sufficient to induce the expression of MKP-1 in bone marrow macrophages. The cells were treated with the phorbol ester PMA, a direct activator of PKC. Treatment with PMA alone induced the expression of MKP-1 mRNA and the effect of PMA could be reversed by preincubating the cells with a specific PKC inhibitor, GF109203X (43) (Fig. 5GoA). The cells were also treated with two natural activators of PKC, PLC and sphyngomyelinase. Fig. 5GoB shows that the expression of MKP-1 was induced in macrophages by both treatments. All these results indicate that the activation of PKC is sufficient to induce the expression of MKP-1 in macrophages. The cells were also treated with ionomycin, an ionophore that promotes the mobilization of intracellular Ca2+, thus inducing the activation of Ca2+-dependent PKC isoforms. However, this treatment neither induced the expression of MKP-1 nor increased the induction of this phosphatase by PMA, thus suggesting that Ca2+ mobilization alone does not induce the expression of MKP-1 in macrophages (Fig. 5GoA).



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  		FIGURE 5. PKC mediates the expression of MKP-1 by LPS. A, Macrophages were treated with PMA (100 ng/ml) in the presence or absence of GF109203X (GF) (1 µM), with ionomycin (Io) (500 nM) or with both PMA and ionomycin for 45 min. A subset of cells were stimulated with LPS (1 ng/ml) (positive control) or were not treated (negative control). B, The cells were treated for 1 h with either PLC from B. cereus (5 U/ml), sphingomyelinase (Smase) from B. cereus (1 U/ml), or LPS (100 ng/ml). C, The cells were preincubated during 2 h with the indicated concentrations of GF109203X and then stimulated with LPS (100 ng/ml) for 45 min. A subset of cells were stimulated with LPS in the presence of vehicle (0.1% DMSO) (positive control) or were left untreated (negative control). Northern blot analysis for MKP-1 was performed with total RNA extracts (15 µg per lane) from A, B, and C. D, The values of MKP-1 mRNA expression from C were normalized with the values of L32 expression. The graphic represents the mean of three independent experiments. E, The cells were preincubated with calphostin C (100 nM) for 1 h and then stimulated with LPS (100 ng/ml) for 45 min. The expression of MKP-1 was analyzed by Northern blot. All the experiments were reproduced at least twice. F, The pattern of ERK-1/2 activation is extended in GF109203X-treated macrophages. The cells were preincubated with GF109203X (5 µM) or vehicle for 2 h and then stimulated with LPS (100 ng/ml) for the indicated periods of time. ERK activity was measured by an in-gel-kinase assay.

 
Because the activation of PKC was sufficient to induce the expression of MKP-1 in macrophages, we determined whether PKC mediated the induction of this phosphatase by LPS. When the cells were preincubated with the PKC inhibitor GF109203X (1–5 µM) before the addition of LPS, we observed a dose-dependent inhibition of the expression of MKP-1 mRNA in response to LPS (Fig. 5Go, C and D). GF109203X also appears to block the activation of Rsk-2 and p70 S6 kinase (44). However, it is unlikely that the blockage of MKP-1 induction is due to the inhibition of any of these molecules. In fact, Rsk-2 is activated by ERK-1 and -2 (45), and, as we have shown above, specific blockage of this cascade with PD98059 did not inhibit the MKP-1 induction by LPS. In contrast, rapamycin, a selective inhibitor for p70 S6 kinase (46), did not alter MKP-1 expression in response to LPS (data not shown). Besides, calphostin C, another PKC inhibitor not related to GF109203X, also inhibited the LPS-induced expression of MKP-1 (Fig. 5GoE), further supporting the involvement of PKC in this process.

We further explored the effect of the inhibition of PKC on the pattern of ERK activation. In bone marrow macrophages, LPS induced the activation of ERK-1/2 within the first 15 min. ERK activity remained high until 30 min of LPS stimulation and decayed drastically thereafter (Fig. 5GoF). The major part of ERK-1/2 inactivation correlated with the time course of expression of the MKP-1 protein (Fig. 1Go). Interestingly, the inhibition of PKC with GF109203X lead to a significant prolongation of ERK activity during the macrophage response to LPS (Fig. 5GoF), correlating with the ability of the drug to inhibit MKP-1 expression (Fig. 5Go, C and D). These results reinforce the importance of MKP-1 as a negative regulator of ERK activity in LPS-stimulated macrophages. We were also interested in determining which PKC isoform was responsible for MKP-1 induction in LPS-stimulated macrophages. At least 11 PKC isoforms have been described, including {alpha}, ßI, ßII, {gamma}, {delta}, {epsilon}, {eta}, {theta}, µ, {iota}/{lambda}, and {zeta} (9, 10). However, only a subset of PKC isozymes are expressed in a given tissue and at a certain stage of differentiation (10). In previous studies, we used specific Abs to determine the expression of PKC isozymes in bone marrow-derived macrophages. We found that only PKCßI, {epsilon}, and {zeta} were present in these cells, whereas the rest of the isozymes were not detected (47). Among the three isoforms expressed in bone marrow macrophages, PKC{zeta} is not effectively inhibited by treating the cells with GF109203X at the doses used in our experiments (48), thus suggesting that the LPS-induced expression of MKP-1 is not mediated by this isoform. In support of this hypothesis, MKP-1 induction was not affected by wortmannin (data not shown), an inhibitor of phosphatidylinositol 3-kinase. This enzyme catalyzes the production of phosphatidylinositol (3, 4, 5)-trisphosphate, an activator of PKC{zeta} (13).

To determine which of the PKC isoforms, ßI or {epsilon}, regulated the expression of MKP-1, macrophages were treated with PMA for 12 h before the addition of LPS. Prolonged exposure to PMA completely down-regulated PKCßI but had only a partial down-modulating effect on PKC{epsilon} (Fig. 6GoA). Accordingly, this treatment had only a low inhibitory effect on the induction of MKP-1 mRNA by LPS (Fig. 6GoB). Furthermore, Gö6976, a selective inhibitor of conventional PKC isoforms, including PKCßI (48), did not alter the LPS-induced expression of this phosphatase (Fig. 6GoC). Taken together, these results indicate that the activation of PKCßI is not necessary for the induction of MKP-1 by LPS and thus suggest that PKC{epsilon} is the main PKC isoform involved in this process.



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  		FIGURE 6. The induction of MKP-1 by LPS is not dependent on PKCßI. A, Prolonged exposure of macrophages to PMA completely down-regulates PKCßI. The cells were treated or not with PMA (100 ng/ml) for 12 h and PKC expression was assessed by Western blot. B, Prolonged exposure to PMA does not block the induction of MKP-1 by LPS. The cells were treated with either PMA (100 ng/ml) or vehicle (0.1% DMSO) for 12 h before the addition of LPS (100 ng/ml). MKP-1 expression was analyzed by Northern blot. C, The PKCßI inhibitor Gö6976 does not block the induction of MKP-1 by LPS. The cells were preincubated with Gö6976 (2 µM) or with vehicle (0.1% DMSO) for 1 h and then stimulated with LPS (100 ng/ml) for 45 min. MKP-1 expression was analyzed by Northern blot. All the experiments were reproduced twice.

 
To further confirm this hypothesis, we studied the intracellular localization of PKCßI, {epsilon}, and {zeta} in response to LPS. Fig. 7Go shows that PKCßI and {zeta} were mainly present in the cytosol in unstimulated cells. Whereas PMA induced the translocation of PKCßI to the membrane and cytoskeleton fractions (Fig. 7GoB), LPS, at the concentrations used in our experiments, did not induce the translocation of either PKCßI or {zeta} to any of these compartments (Fig. 7GoA). In contrast, PKC{epsilon} was present in the membrane fraction of both unstimulated and LPS-treated cells, thus being able to interact with its cofactors once a peak of DAG is generated in response to LPS.



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  		FIGURE 7. PKC{epsilon} is membrane-bound in unstimulated and LPS-treated bone marrow macrophages. The cells were either not treated or stimulated for 10 min with LPS (100 ng/ml) (A) or PMA (100 ng/ml) (B). Fractions corresponding to cytosol, membrane (triton-soluble), and cytoskeleton (SDS-soluble) were obtained and proteins (25 µg) from each fraction were separated by 8% SDS-PAGE and immunoblotted for the indicated PKC isozymes. The images are representative of two independent experiments with identical results. C, LPS induces the activation of membrane-bound PKC{epsilon}. PKC{epsilon} was immunoprecipitated from the membrane fractions of macrophages stimulated for different periods of time. PKC{epsilon} activity was measured as described in Materials and Methods, and the mean of three independent experiments is represented.

 
To study whether LPS was able to efficiently activate membrane-bound PKC{epsilon}, we immunoprecipitated PKC{epsilon} from the membrane fraction of macrophages stimulated or not with LPS and analyzed its capability to phosphorylate the specific susbtrate 25Ser-substituted PKC peptide in vitro. The PKC{epsilon} activity detected in the membrane fraction of nonstimulated macrophages was equivalent to the background signal detected in the cytosolic fraction of these cells (data not shown). Fig. 7GoC shows that LPS induced significant activation of PKC{epsilon} within the first 15 min of stimulation. These results further correlate PKC{epsilon} activation with the induction of MKP-1 in bone marrow macrophages.

Moreover, the cells were treated with antisense oligonucleotides specific for each of the three PKC isoforms ßI, {epsilon}, or {zeta}. Antisense oligonucleotides directed against PKCßI or {zeta}, although blocking the expression of these isoforms (Fig. 8GoA), did not inhibit the induction of MKP-1 by LPS (Fig. 8GoB). In contrast, oligonucleotides specific for PKC{epsilon} efficiently blocked the expression of this isoform and significantly inhibited the induction of MKP-1 in response to LPS. Besides, this treatment lead to an increase in the levels of ERK-1/2 activity after 70 min of exposure to LPS in comparison to those observed in control cells and in cells treated with specific oligonucleotides against PKCßI or {zeta} (Fig. 8GoD). These results indicate that PKC{epsilon} is specifically involved in the induction of the phosphatase MKP-1 and thus in the negative regulation of ERK activity in LPS-stimulated macrophages.



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  		FIGURE 8. PKC{epsilon} is required for the induction of MKP-1 and the negative regulation of ERK-1/2 during the macrophage response to LPS. Macrophages were treated during 34 h with antisense oligonucleotides (12 µM) specific for the indicated PKC isoforms. A, The blocking efficiency of each oligonucleotide was analyzed by Western blot with Abs specific to each PKC isoform. Bands corresponding to each isozyme are shown. B, The expression of MKP-1 was determined by Western blot analysis. The expression of the ß-actin protein was assessed to detect differences in loading and transfer. C, Normalized values of MKP-1 expression are represented as percentage of maximal induction and represent the mean of two independent assays. D, The state of activation of ERK-1/2 after the treatment with LPS (100 ng/ml) for 70 min was analyzed by an in-gel-kinase assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MKP-1 is a tyrosine/threonine-phosphatase that dephosphorylates different members of the MAPK superfamily (22, 23, 24). In bone marrow macrophages, we found that LPS induces the transient expression of both MKP-1 mRNA and protein. This agrees with a previous report that described the accumulation of MKP-1 mRNA in the macrophagic cell line RAW 264.7 after stimulation with LPS (49). The study of the mechanisms that induce the expression of MKP-1 is critical to understand how the cell machinery controls the duration of MAPK activity. Like many other immediate early genes, the expression of MKP-1 is regulated mainly at the transcriptional level. Both mRNA and protein have a very short half life, and no mechanisms of posttranslational control have been described (19, 21, 22). We have explored the signal transduction pathway that leads to the expression of this phosphatase in response to LPS.

In fibroblasts, the activation of ERK-1/2 is sufficient to induce the expression of MKP-1, thus promoting the attenuation of their own cascade in a direct negative feedback loop (50). However, in bone marrow macrophages, the induction of MKP-1 by LPS takes place even when ERK activation is blocked. Thus, LPS seems to activate two independent pathways, one leading to ERK-1/2 activation and the other one determining the expression of their specific phosphatase. Also in contrast to what has been reported in fibroblasts (21, 50), a cAMP-dependent pathway is not involved in the induction of MKP-1 in macrophages.

Our results suggest that LPS leads to MKP-1 induction through a pathway that involves the activation of intracellular tyrosine kinases and PKC. Although we have not identified the tyrosine kinases involved in this response, the members of the src family of tyrosine kinases are likely candidates for two reasons. First, p53/56lyn is associated with CD14 in macrophages (51). Second, p53/56lyn, p58/64hck, and p59c-fgr are all transiently activated upon stimulation of macrophages with LPS (15, 51). However, some LPS responses occur in the absence of Hck, Fgr, and Lyn (52), which suggests that their activity can be replaced either by other members of the same family or by totally distinct tyrosine kinases.

In bone marrow macrophages, PKC activation is sufficient to induce the expression of MKP-1. This has been also described in some other cell types (21, 53, 54). By using two unrelated PKC inhibitors, we have shown that PKC is also involved in the induction of MKP-1 by LPS. Although PKC has been extensively implicated in the control of several LPS-induced events (6, 7, 8, 55, 56, 57, 58, 59), it is still unclear which isoform(s) are involved in each of these effects. In previous studies, we found that bone marrow macrophages express PKC isoforms ßI, {epsilon}, and {zeta} (47). Although LPS shows high structural similarities with ceramide (60), a second messenger that activates PKC{zeta} (12), and despite the fact that MKP-1 expression is induced in macrophages stimulated with sphyngomyelinase, an upstream activator of PKC{zeta}, it is unlikely that this isoform mediates the induction of MKP-1 by LPS for several reasons. First, the PKC inhibitor that blocks MKP-1 expression, GF109203X, was used at doses that inhibit conventional and novel PKCs, but not the atypical isoforms, including PKC{zeta} (48). Second, the use of wortmannin to inhibit PI3K, an upstream regulator of PKC{zeta}, did not affect MKP-1 induction by LPS. Third, we could not detect translocation of PKC{zeta} in response to LPS. And finally, oligonucleotides against PKC{zeta} did not block MKP-1 induction by LPS. All these observations suggest that PKC{zeta} is not involved in the LPS signaling pathway that leads to MKP-1 expression.

Several observations support the involvement of PKC{epsilon} rather than PKCßI in the induction of MKP-1 by LPS. First, GF109203X inhibits conventional PKC isoforms better than novel ones (48). Concentrations of up to 1 µM of GF109203X completely inhibit the activation of conventional PKCs, including ßI, whereas concentrations of up to 5 µM are needed to completely block novel PKC isoforms, including {epsilon}. In our hands, maximal inhibition of MKP-1 was reached at concentrations of 3–5 µM of GF109203X. Second, prolonged treatment of macrophages with PMA causes only a low inhibition of MKP-1 induction by LPS. In bone marrow macrophages, this treatment leads to a complete depletion of PKCßI, but not of PKC{epsilon}. Similarly, in three macrophagic cell lines and in several other cell systems, PKC{epsilon} has been also shown to be resistant to prolonged PMA treatment (6, 61, 62, 63, 64). Third, Gö6976, a selective inhibitor of conventional PKCs, does not block MKP-1 induction by LPS. And finally, treatment of these cells with antisense oligonucleotides specific for PKC{epsilon}, and not with those specific for PKCßI, significantly inhibits the induction of MKP-1 by LPS. All these results suggest that, in bone marrow macrophages, PKC{epsilon} is specifically involved in the induction of the phosphatase MKP-1 by LPS. In previous studies, we had observed that MKP-1 expression was also induced in macrophages stimulated with M-CSF (47). A strong parallelism has been observed between the mechanism used by M-CSF and LPS to induce MKP-1 expression, although both agents trigger totally opposed effects in macrophage biology (proliferation vs activation). During the macrophage response to M-CSF, MKP-1 induction was similarly mediated through a PKC-dependent pathway, and, of all the PKC isoforms detected in bone marrow macrophages, PKC{epsilon} was the main candidate to mediate MKP-1 expression. These, together with the data shown in the present report, suggest that, in bone marrow macrophages, PKC{epsilon} plays a major role in the control of MKP-1 expression.

We have also shown a significant prolongation of the time course of ERK activity in macrophages treated with the PKC inhibitor GF109203X or with antisense oligonucleotides against PKC{epsilon}, correlating with the capability of these compounds to inhibit MKP-1 expression. Although we cannot discard the involvement of other phosphatases different to MKP-1, specially because partial inactivation of ERK-1/2 still took place in those experimental conditions, our results allow us to conclude that PKC{epsilon} has an important function in the control of the pattern of ERK activity by inducing the expression of MKP-1.

PKC activation has been frequently associated to its translocation to the membrane or the cytoskeleton (9, 10). Surprisingly, we have detected PKC{epsilon} at the membranal fraction of unstimulated macrophages. This confirms previous reports in which the constitutive association of PKC{epsilon} to the membrane was also described in other cell types and in a macrophagic cell line (10, 65). However, our results reveal that the presence of PKC{epsilon} at this compartment does not mean that PKC{epsilon} is constitutively active in unstimulated macrophages. In fact, treatment with LPS is required to detect significant activation of membrane-bound PKC{epsilon}, thus correlating the activation of this isoform with the induction of MKP-1 in LPS-stimulated macrophages.

Although we cannot exclude the activation of PKC isoforms other than PKC{epsilon}, studies with blocking Abs showed that activation of PKC{epsilon} accounts for the majority of PKC activity in murine peritoneal macrophages treated with LPS (66). Moreover, stimulation of human monocytes with LPS results in the specific activation of a Ca2+-independent isoform of PKC (7). These and our observations predict a crutial role for PKC{epsilon} in the biology of macrophages. Several reports suggest that generation of DAG by LPS takes place through the action of a phosphatydilcholine-specific form of PLC (67). Phosphatydilcholine-PLC activity does not generate IP3, a second messenger that induces the release of Ca2+ from intracellular storage sites (68). In fact, in accordance with previously published work (69, 70, 71), we have not detected mobilization of intracellular Ca2+ in bone marrow macrophages treated with 1–100 ng/ml of LPS (A.F.V. et al., manuscript in preparation). Because conventional PKCs require the presence of Ca2+ for full activation (9, 10), the absence of Ca2+ mobilization would explain the lack of translocation of PKCßI from the cytosolic to particulate fractions in response to the doses of LPS used in our experiments.

Although we can assume that PKC{epsilon} is required for the induction of MKP-1 by LPS, we cannot discard the involvement of other signaling molecules that have not been analyzed in this report. In fact, we did not get a total inhibition of MKP-1 expression in macrophages treated with either specific inhibitors or with oligonucleotides against PKC{epsilon}. Besides, the induction of this phosphatase by PMA was substantially lower than that mediated by LPS. These observations suggest that PKC{epsilon} is required for this process but some other mechanism may participate in the induction of MKP-1 by LPS. We are currently analyzing the involvement of other pathways that regulate MKP-1 expression.

In this report, we have studied the mechanisms that control the expression of MKP-1 in bone marrow macrophages stimulated with LPS. It should be emphasized that LPS induces the expression of MKP-1 in an ERK-independent manner. Therefore, in macrophages stimulated with LPS, ERK-1/2 do not attenuate their own activation by inducing MKP-1. LPS ensures this negative control by triggering an alternative pathway that is activated via the CD14 molecule and specifically requires the action of tyrosine kinases and PKC{epsilon}. The results of this report demonstrate the key role of PKC{epsilon} in the control of the duration of MAPK activity during the macrophage response to LPS.


   Acknowledgments
 
We thank Dr. Antonio García de Herreros (Institut Municipal d’Investigació Médica, Barcelona, Spain) for discussing the experiments regarding PKC and for some reagents. We also thank Dr. Rich A. Maki (The Burnham Institute, La Jolla, CA), Dr. Jorge Moscat and Dr. Teresa Díaz-Meco (CBM, Madrid, Spain) for some reagents and for helpful advice about signal transduction, Dr. Peter Parker (Imperial Cancer Research Fund, London, U.K.) for the anti-PKC Abs, Dr. Michael Weber (University of Virginia, Charlottesville, VA) for the anti-ERK1/2 Abs, Dr. Rodrigo Bravo (Bristol-Myers, Princeton, NJ) for the plasmid containing the full-length MKP-1 cDNA, Dr. Markus Nabholz (Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland) for the plasmid containing TNF-{alpha} cDNA, Dr. Rose Wilson (Glaxo Research and Development Limited, Greenford, U.K.) for the IL-1ß-containing plasmid, and Dr. José A. García-Sanz (CNB, Madrid, Spain) for the plasmid containing ß-actin and for helpful advice about some protocols. We also thank Martin Cullell-Young for reviewing the manuscript.


   Footnotes
 
1 This work was supported by grants from the Comision Interministerial de Ciencia y Tecnologia (SAF98/102 and PM98/0200; to A.C.). A.F.V. and J.X. were recipients of a fellowship from the Comissió Interdepartamental de Recerca i Innovació Tecnològica, Generalitat de Catalunya. Back

2 Address correspondence and reprint requests to Dr. Antonio Celada, Departament de Fisiologia, Facultat de Biologia, Av. Diagonal 645, 08028 Barcelona, Spain. E-mail: Back

3 Abbreviations used in this paper: PLC, phospholipase C; CRE, cAMP response element; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MKP-1, MAPK phosphatase-1; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; 8-Br-cAMP. 8-bromo-cAMP; 8-Br-cGMP, 8-bromo-cyclic GMP. Back

Received for publication June 16, 1999. Accepted for publication October 8, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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