Protein Kinase Cε Is Required for the Induction of Mitogen-Activated

Protein Kinase Cε Is Required for the Induction of Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages This information is current as of October 1, 2021. Annabel F. Valledor, Jordi Xaus, Mònica Comalada, Concepció Soler and Antonio Celada J Immunol 2000; 164:29-37; ; doi: 10.4049/jimmunol.164.1.29

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Protein Kinase C⑀ Is Required for the Induction of Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages1

Annabel F. Valledor, Jordi Xaus, Mo`nica Comalada, Concepcio´Soler, and Antonio Celada2

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 charac- terization 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 PKC␤I, ⑀, and ␨. Of all these, only PKC␤I and ⑀ are inhibited by GF109203X. The following arguments suggest that PKC⑀ 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 Downloaded from correlates with the levels of expression of PKC⑀ but not with that of PKC␤I. Second, Go¨6976, an inhibitor selective for conventional PKCs, including PKC␤I, does not alter MKP-1 induction by LPS. Last, antisense oligonucleotides that block the expression of PKC⑀, but not those selective for PKC␤IorPKC␨, 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⑀. In conclusion, our results demonstrate an important role for PKC⑀ in the induction of MKP-1 and the subsequent negative control of MAPK activity in macrophages. The Journal of Immunology, 2000, 164: 29–37. http://www.jimmunol.org/

acrophages perform critical functions in the immune sists of several isoforms that are distributed in three main groups system. They act as regulators of homeostasis and as (conventional, novel, and atypical PKCs) based on their primary effector cells in infection, tumor growth, and healing structure and activation requirements (9, 10). Conventional PKCs, M 2ϩ of wounds (1). In contrast to other cells of the immune system, which include ␣, ␤I, ␤II, and ␥, need both Ca and diacylglycerol macrophages show a marked duality in their biological responses; (DAG)/phorbol esters for activation and phosphatidylserine as a they either proliferate (e.g., in response to the speciﬁc growth fac- cofactor. Novel PKCs, which include ␦, ⑀, ␪, ␩, and ␮, need DAG/ tor M-CSF) or become activated, undergo a growth arrest, and start phorbol esters and phosphatidylserine, but do not require Ca2ϩ for performing their specialized functions in the context of the im- activation. Atypical PKCs, including ␨ and ␫v, cannot be activated by guest on October 1, 2021 mune response (2). LPS or endotoxin, a major component of the by Ca2ϩ or DAG/phorbol esters, but are regulated by phosphati- outer membrane of Gram-negative bacteria, activates macrophages dylinositol (3,4,5)-trisphosphate, ceramide, and phosphatidic acid and induces the secretion of arachidonic acid metabolites (e.g., (11–13). PGs, leukotrienes, and platelet-activating factor), nitrogen inter- LPS also activates different mitogen-activated protein kinase mediates, and cytokines such as TNF-␣, IL-1, and IL-6 (3, 4), (MAPK) cascades, including the extracellular-signal regulated which play important roles in the immune response. protein kinase (ERK) (14, 15), the c-Jun N-terminal protein kinase, In macrophages, LPS triggers the activation of several signal and the p38 MAPK/reactivating kinase pathways (16). For general transduction pathways involving G proteins, tyrosine kinases, MAPK activity, phosphorylation in both tyrosine and threonine phospholipase C (PLC),3 protein kinase A (PKA), and protein ki- residues is required (17). Active ERKs phosphorylate and regulate nase C (PKC) (5–8). The serine/threonine kinase PKC family con- several cellular proteins, including additional protein kinases,

cytoskeletal components, phospholipase A2, and nuclear transcrip- Departament de Fisiologia (Biologia del Macro`fag), Facultat de Biologia and Fun- tion factors, such as Elk1/TCF and c-Jun, which regulate the dacioÁugust Pi i Sunyer, Campus Bellvitge, Universitat de Barcelona, Barcelona, expression of immediate early genes (reviewed in Ref. 18). Spain MAPK phosphatase-1 (MKP-1) is a member of a family of in- Received for publication June 16, 1999. Accepted for publication October 8, 1999. ducible dual-specificity tyrosine phosphatases (19–21). MKP-1 se- The costs of publication of this article were defrayed in part by the payment of page lectively dephosphorylates tyrosine and threonine residues on charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ERK-1/2 (22, 23), thus allowing the inactivation of these kinases. 1 This work was supported by grants from the Comision Interministerial de Ciencia Recent reports have also shown the capability of MKP-1 to de- y Tecnologia (SAF98/102 and PM98/0200; to A.C.). A.F.V. and J.X. were recipients phosphorylate and inactivate c-Jun N-terminal kinase/stress-acti- of a fellowship from the ComissioÍnterdepartamental de Recerca i Innovacio´Tec- vated protein kinase and p38/reactivating kinase (24). nolo`gica, Generalitat de Catalunya. At the present, little is known about the mechanisms involved in 2 Address correspondence and reprint requests to Dr. Antonio Celada, Departament de Fisiologia, Facultat de Biologia, Av. Diagonal 645, 08028 Barcelona, Spain. E-mail: the induction of MKP-1 by LPS. Because the study of the events [email protected] that account for the negative control of MAPK activity is of great 3 Abbreviations used in this paper: PLC, phospholipase C; CRE, cAMP response interest, in this report we have characterized the signaling path- element; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; MAPK, way(s) that lead to MKP-1 expression in response to LPS. We have 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; found that MKP-1 expression was not dependent on the activation 8-Br-cAMP. 8-bromo-cAMP; 8-Br-cGMP, 8-bromo-cyclic GMP. of the MAPKs ERK-1/2 in response to LPS. Instead, the induction

of this phosphatase required the activation of tyrosine kinases and 8 min at 4°C. The protein concentration of the samples was determined by PKC isozyme ⑀. This suggests that, in macrophages, PKC⑀ plays the Bio-Rad protein assay. The proteins from cell lysates (50–100 ␮g) a key role in the negative control of general MAPK activity were boiled at 95°C in Laemli SDS-loading buffer and separated by 10% SDS-PAGE (unless otherwise stated) and electrophoretically transferred to through the induction of the phosphatase MKP-1. nitrocellulose membranes (Hybond-ECL; Amersham, Arlington Heights, IL). The membranes were blocked in 2% BSA in TBS-0.1% Tween 20 Materials and Methods (TBS-T) for3hatroom temperature and then incubated with the primary Macrophages Ab. For MKP-1 immunoblotting, incubation was performed for1hatroom temperature with rabbit IgG anti-mouse MKP-1 (1:500) (Santa Cruz Bio- Bone marrow-derived macrophages were obtained from 6- to 10-wk-old technology, Santa Cruz, CA). For the recognition of PKC isozymes, incu- BALB/c mice (Charles River Laboratories, Wilmington, MA) as described bation was done overnight at 4°C with rabbit IgG selective for each mouse (25). Macrophages were cultured in DMEM (Sigma, St. Louis, MO) sup- PKC isoform (1:1000) in the presence or absence of specific competitor plemented with 20% FBS (Sigma) and 30% L cell-conditioned medium as peptides (1:1000) (Abs and peptides were kindly provided by Dr. P. J. a source of M-CSF. Once macrophages were 80% confluent, normally after Parker, Imperial Cancer Research Fund, London, U.K.). For some exper- 6 days of culture, they were deprived of L cell-conditioned medium for iments, we used equivalent Abs purchased from Life Technologies (Grand 16–18 h and treated with LPS (Sigma) in the presence or absence of se- Island, NY). After three washes of 15 min each in TBS-T, the membranes lective inhibitors/activators. All treatments were not toxic for the cells as were incubated with peroxidase-conjugated anti-rabbit IgG Ab (Cappel, determined by flow cytometry analysis. Durham, NC) (1:5000) for 1 h. After three washes of 15 min with TBS-T, enhanced chemiluminescence detection was performed (Amersham Life Reagents Science, London, U.K.) and the membranes were exposed to x-ray films (Amersham). Genistein, bisindolylmaleimyde I (GF109203X), calphostin C, PMA, PLC from Bacillus cereus, KT5720, and KT5823 were purchased from Calbio- Determination of ERK activity by in-gel-kinase assay

chem (San Diego, CA). Ionomycin was purchased from ICN Pharmaceu- Downloaded from ticals (Costa Mesa, CA). 8-Bromo-cAMP (8-Br-cAMP) and 8-bromo-cy- This assay was performed as previously described (30). Briefly, 50 ␮gof clic GMP (8-Br-cGMP) were obtained from Fluka Biochemika (Buchs, total protein were separated by 12.5% SDS-PAGE in the presence of 0.1 Switzerland). Sphingomyelinase from B. cereus and wortmannin were ob- mg/ml of myelin basic protein (Sigma) copolymerized in the gel. After tained from Sigma. PD98059 was purchased from New England Biolabs electrophoresis, SDS was removed by washing the gel with two changes of (Beverly, MA). Go¨6976 was a kind gift from Dr. A. Garcıá de Herreros 20% 2-propanol in 50 mM Tris-HCl, pH 8.0, for1hatroom temperature. (Institut Municipal d’Investigacio´Me`dica, Barcelona, Spain). All reagents The gel was then incubated with 50 mM Tris-HCl, pH 8.0, containing 5 were used following the manufacturer’s recommendations. mM 2-ME (buffer A) for1hatroom temperature. The proteins were denatured by incubating the gel with two changes of 6 M guanidine-HCl http://www.jimmunol.org/ Antisense oligonucleotides for1hatroom temperature and then renatured by incubating with five changes of buffer A containing 0.04% Tween 20 for 16 h at 4°C. To Antisense phosphorothioated-oligonucleotides specific for PKC isoforms perform the phosphorylation assay, the gel was first equilibrated in 40 mM ␤ ⑀ ␨ I/II, , and were used to block the expression of specific PKC isoforms. HEPES-NaOH, pH 7.4, containing 2 mM DTT, 0.1 mM EGTA, 15 mM The cells were incubated for 34 h in DMEM with 5% FBS containing 12 MgCl , and 300 ␮M sodium orthovanadate for 30 min at 25°C and then ␮ 2 M thioated-oligonucleotides (Biognostik, Go¨ttingen, Germany). Oligo- incubated for1hinthesame solution containing 50 ␮M ATP and 100 ␮Ci ␤ nucleotides specific for mouse PKC I/II corresponded to the reverse com- [␥-32P]ATP (ICN). The reaction was stopped by washing the gel with 5% plement of a target sequence as described (C. L. Ashendel, unpublished TCA containing 10 mM sodium pyrophosphate to inhibit phosphatase ac- observations) (accession no. X53532). Oligonucleotides against mouse tivity. The gel was dried, exposed to x-ray films (Kodak), and quantified ␨ PKC were designed as described (26). Oligonucleotides specific for with a Bio-Rad molecular analyst. mouse PKC⑀ corresponded to the sequence: 5Ј-GCTCACCGCCTC by guest on October 1, 2021 GCAGATTT-3Ј. Determination of PKC translocation RNA extraction and Northern blot analysis PKC translocation was determined as described previously (31) with some modifications. The cells were lysed by scraping in cold hypotonic buffer The cells were washed twice in cold PBS, and extraction of total RNA was T10 (10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 10 mM NaCl), containing performed as described (27). Total RNA samples (15 ␮g) were separated inhibitors of proteases (1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 86 ␮g/ml on 1.2% agarose gels containing formaldehyde and transferred to nylon iodoacetamide, 1 mM PMSF) and 1 mM sodium orthovanadate. The cell membranes (Genescreen; NEN Life Science Products, Boston, MA). For lysates were centrifuged at 100,000 ϫ g for 30 min at 4°C and the super- MKP-1 mRNA detection, we obtained the full-length cDNA fragment of natants were collected (cytosol fraction). The pellets were resuspended in MKP-1 following purification from a HindIII digestion of the plasmid cold buffer T10 containing 1% Triton X-100 and homogenized on ice pBluescript KS/MKP-1 (kindly provided by Dr. R. Bravo, Bristol-Myers (15–20 strokes with a Dounce homogenizer). To allow PKC extraction Squibb Pharmacology Research Institute, Princeton, NJ). For TNF-␣ from cell membranes, the samples were left for1hat4°Candthen cen- mRNA detection, we used the EcoRI/HindIII fragment of pSP65/TNF-␣ trifuged at 100,000 ϫ g for 30 min at 4°C. The supernatants were collected (kindly supplied by Dr. M. Nabholz, Institut Suisse de Recherches Experi- (plasma membrane fraction) and the pellets were resuspended in cold T10 mentales sur le Cancer, Epalinges, Switzerland). To study the expression of containing 1% SDS, passed through a 19-gauge needle five times, and IL-1␤, we obtained a probe by digesting the construct pGEM1/IL-1␤ boiled at 100°C for 5 min. Insoluble material was removed by centrifuga- (kindly provided by Dr. R. Wilson, Glaxo Research and Development Lim- tion at 13,000 ϫ g for 10 min, and the supernatants were collected (cy- ited, Greenford, U.K.) with EcoRI/PstI. For ␤-actin mRNA detection, we toskeleton fraction). Samples from each fraction (25 ␮g of protein) were obtained the PstI fragment of pSP65/␤-actin (28). To detect the L32 tran- boiled at 95°C in loading buffer and separated by 8% SDS-PAGE. The script, we used the EcoRI/HindIII fragment of pGEM1/L32 as a probe (29). proteins were electrophoretically transferred to Hybond-ECL membranes All probes were labeled with [␣-32P]dCTP (ICN Pharmaceuticals). After (Amersham) and immunoblotted with anti-PKC isoforms Abs in the pres- incubating in hybridization solution (20% formamide, 5ϫ Denhardt’s, 5ϫ ence or absence of competitor peptide. SSC, 10 mM EDTA, 1% SDS, 25 mM Na2HPO4,25mMNaH2PO4, and 0.2 mg/ml salmon sperm DNA) at 65°C, the membranes were washed and Measurement of PKC⑀ activity exposed to Kodak X-AR films (Kodak, Rochester, NY). Besides, bands of This assay was performed as previously described (32) with modifications. interest were quantified with a Molecular Analyst (Bio-Rad, Specific Abs against PKC⑀ (Life Technologies) were used to immunopre- Richmond, CA). cipitate this isoform from subcellular fractions (2 ␮g of Ab per 150 ␮gof Protein extraction and Western blot analysis total protein in a total volume of 300 ␮l). Incubation was conducted for 2 h at 4°C. Immunocomplexes were separated by addition of 75 ␮lof20% The cells were washed twice in cold PBS and lysed on ice with lysis protein A-Sepharose slurry, incubated2hat4°C, and pelleted. The pellets solution (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.5, 150 were washed twice with RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM mM NaCl, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 ␮g/ml iodoacetamide, NaCl, 1% Triton X-100, 1% deoxycholate, 2 mM EDTA, 1 mM EGTA) 1 mM PMSF). When inhibition of the activity of tyrosine phosphatases was supplemented with protease inhibitors and 1 mM sodium orthovanadate, ␤ required, 1 mM sodium orthovanadate was included. For PKC detection once with prereaction buffer (50 mM -glycerophosphate, 10 mM MgCl2, experiments, 1 mM EGTA and 2 mM EDTA were added to the lysis so- 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, protease inhibitors, lution. Insoluble material was removed by centrifugation at 13,000 ϫ g for 1 mM sodium orthovanadate), and then resuspended in reaction buffer The Journal of Immunology 31

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 ␮M) or vehicle (0.1% DMSO) for1hand 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 preincu- Downloaded from bated for 1 h with either PD98059 (50 ␮M) or vehicle (0.1% DMSO) and then stimulated or not with LPS (100 ng/ml) for 45 min. The expression of ␮ FIGURE 1. MKP-1 expression is induced by LPS in bone marrow mac- MKP-1 was analyzed by Northern blotting (15 g of total RNA per sam- ␮ rophages in a time- and dose-dependent manner. A, MKP-1 mRNA ex- ple) (B) and by Western blotting (100 g of total protein per lane) (C). pression was assessed by Northern blotting (15 ␮g of total RNA per lane). These assays were repeated twice with identical results. Total RNA extracts were obtained after treating quiescent bone marrow macrophages with LPS (100 ng/ml) for the indicated periods of time. B, http://www.jimmunol.org/ The expression of the MKP-1 protein was analyzed by Western blotting (100 ␮g of total cell extracts per lane). Cell extracts were obtained after the Because the members of the MKP-1 family play a crutial role in treatment with LPS (100 ng/ml) for the indicated periods of time. The the inactivation of MAPK cascades, we were next interested in expression of ␤-actin mRNA (A) and protein (B) was analyzed to check for determining the signal transduction pathway that leads to the in- differences in sample loading and transfer efficiency. C, Northern blotting duction of this phosphatase after the stimulation of macrophages ␮ was performed with total RNA extracts (15 g per lane) from cells treated with LPS. We first studied whether the induction of MKP-1 by with the indicated concentrations of LPS for 45 min. D, Values of MKP-1 LPS was mediated, as a negative feedback mechanism, by the expression from C were normalized with the L32 control and represented activation of the MAPKs ERK-1/2. Fig. 2 shows that the blockage as percentages of maximal expression. These images are representative of by guest on October 1, 2021 two independent experiments. 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 (prereaction buffer supplemented with 100 ␮M ATP, 33 ␮M1,2-sn-dio- is independent of the activation of the late elements of the ERK 25 leoylglycerol, 40 ␮g/ml L-␣-phosphatidylserine, 1 ␮M Ser-PKC, and 5 cascade (MEK1 and ERK-1/2). ␮Ci [␥-32P]ATP). A 25Ser-substituted peptide obtained from the Maximal induction of MKP-1 was obtained at a concentration of pseudosubstrate region of PKC (Calbiochem) was used as the substrate for 100 ng/ml of LPS (Fig. 1, C and D), a dose that saturates the the phosphorylation assay because it represents an appropiate substrate for binding of LPS to its high-affinity receptor, the molecule CD14 measuring PKC⑀ activity (33). The reaction was conducted for 10 min at 30°C. Each sample was spotted on a phosphocellulose filter (Whatman 3 (34). Although other molecules may also transduce signals at very MM) and subjected to five washes of 30 min each in 5% TCA, 10 mM high doses of LPS (1–10 ␮g/ml), our results suggest that the sig- sodium pyrophosphate. Radioactivity was counted by liquid scintillation naling pathway leading to MKP-1 induction is triggered after the using a Packard Tri-Carb 1400 scintillation counter (Meriden, CT). 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) Results supports this hypothesis. In fact, the recognition of LPS by CD14 The present study was conducted with bone marrow-derived mac- requires the previous association of LPS with the serum protein rophages because they represent an homogeneous population of LBP (LPS-binding protein) (35). Binding of LPS to CD14 results primary macrophages. In these cells, LPS induced the transient in the tyrosine phosphorylation of several cellular proteins (8, 36). expression of the phosphatase MKP-1. The induction of the To determine the involvement of tyrosine kinases in the induction MKP-1 transcript could be detected after 20–30 min of LPS treat- of MKP-1 by LPS, the cells were preincubated with genistein, a ment (Fig. 1A). Maximal expression was observed at 45–60 min general inhibitor of this type of kinases (Fig. 3). A significant and then it gradually decayed to basal levels within 2–3 h. Syn- decrease of the expression of MKP-1 mRNA was observed. As a thesis of the MKP-1 protein tightly correlated with the time course control of the effect of genistein, we also analyzed the LPS-in- of MKP-1 mRNA expression (Fig. 1B). The expression of MKP-1 duced expression of TNF-␣ and IL-1␤. As previously reported was also analyzed after the stimulation of macrophages with dif- (37), the expression of these cytokines was inhibited by this treat- ferent concentrations of LPS for 45 min (Fig. 1, C and D). The ment (Fig. 3). Thus, our results suggest that the LPS-induced ex- induction of MKP-1 by LPS was dose dependent and reached a pression of MKP-1 is dependent on the activation of tyrosine plateau at 100 ng/ml of LPS. Therefore, in additional experiments kinases. we used 100 ng/ml and 1 ng/ml of LPS for saturant and subsaturant cAMP-response elements (CREs) are the binding sites for tran- conditions, respectively. scription factors that belong to the family of CRE binding proteins. 32 PKC⑀ IS REQUIRED FOR MKP-1 INDUCTION BY LPS

activation of PKA has been associated with repression of the LPS- induced expression of TNF-␣ in macrophages (40), the expression of TNF-␣ 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-␣ (Fig. 4A). 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. 4, B and C). The implication of a FIGURE 3. The induction of MKP-1 by LPS depends on the activation cGMP-dependent pathway was also tested. However, 8-Br-cGMP of tyrosine kinases. The cells were untreated or preincubated with either neither induced the expression of MKP-1 nor increased its induc- genistein (100 ␮M) or vehicle (0.1% DMSO) for 1 h and then stimulated tion by a subsaturant dose of LPS (Fig. 4A). Besides, the inhibition with LPS (100 ng/ml) for 45 min. A, Total RNA (15 ␮g) was analyzed by of c-GMP-dependent protein kinase G (PKG) with KT5823 did not Northern blotting. B, Values of gene expression were normalized with the alter the induction of MKP-1 by LPS (Fig. 4, B and C). Our results L32 control. The mean of three independent experiments is represented. 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. Following the elevation of intracellular cAMP levels, these pro- A number of LPS-induced processes have been described to be teins are phosphorylated and thus activated by PKA (38). Because dependent on PKC activation in macrophages (6–8). The MKP-1 LPS is able to activate PKA in macrophages (39) and a CRE el- gene promoter contains a PMA-responsive element at position Downloaded from Ϫ ement has been described at position 137 bp in the promoter of Ϫ450 bp (21). PMA response element sites are recognized by the the MKP-1 gene (21), we analyzed the involvement of cAMP- transcriptional complex AP-1 (41), whose activation may be me- dependent pathways in the induction of MKP-1 by LPS. We first diated by PKC (42). For this reason, we studied whether the acti- studied whether an increase in intracellular cAMP levels alone was vation of PKC was sufficient to induce the expression of MKP-1 in able to induce the expression of MKP-1 in macrophages. The cells bone marrow macrophages. The cells were treated with the phor- were treated with 8-Br-cAMP, which is a membrane-permeable bol ester PMA, a direct activator of PKC. Treatment with PMA http://www.jimmunol.org/ and phosphodiesterase-resistant analogue of cAMP. However, the alone induced the expression of MKP-1 mRNA and the effect of expression of MKP-1 was not detected after treating macrophages PMA could be reversed by preincubating the cells with a specific with this compound (Fig. 4A). Besides, 8-Br-cAMP was unable to PKC inhibitor, GF109203X (43) (Fig. 5A). The cells were also increase the induction of MKP-1 when macrophages were stimu- treated with two natural activators of PKC, PLC and sphyngomy- lated with a subsaturant concentration of LPS. Because sustained elinase. Fig. 5B 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 ionomy- by guest on October 1, 2021 cin, an ionophore that promotes the mobilization of intracellular Ca2ϩ, thus inducing the activation of Ca2ϩ-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 Ca2ϩ mobilization alone does not induce the expression of MKP-1 in macrophages (Fig. 5A). 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 ␮M) before the addition of LPS, we observed a dose-dependent inhibition of the expression of MKP-1 mRNA in response to LPS (Fig. 5, 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), FIGURE 4. The LPS-induced expression of MKP-1 is not mediated by and, as we have shown above, specific blockage of this cascade either PKA or PKG. A, Macrophages were either left untreated or incu- with PD98059 did not inhibit the MKP-1 induction by LPS. In bated for 45 min with 8-Br-cAMP (100 ␮M), 8-Br-cGMP (100 ␮M), or contrast, rapamycin, a selective inhibitor for p70 S6 kinase (46), with LPS (1 ng/ml) in the presence or absence of 8-Br-cAMP (100 ␮M) or did not alter MKP-1 expression in response to LPS (data not 8-Br-cGMP (100 ␮M). Total RNA was extracted and the expression of shown). Besides, calphostin C, another PKC inhibitor not related MKP-1 was analyzed by Northern blotting (15 ␮g per sample). The ex- to GF109203X, also inhibited the LPS-induced expression of pression of TNF-␣ was studied as a control of the effect of 8-Br-cAMP. B, MKP-1 (Fig. 5E), further supporting the involvement of PKC in Cells were pretreated or not with either the PKA inhibitor KT5720 (100 this process. nM) or the PKG inhibitor KT5823 (400 nM) for 1 h and then stimulated We further explored the effect of the inhibition of PKC on the with LPS (100 ng/ml) for 45 min. The expression of MKP-1 was studied by Northern blotting (15 ␮g per sample). C, Macrophages were pretreated pattern of ERK activation. In bone marrow macrophages, LPS in- or not with KT5720 (100 nM) or KT5823 (400 nM) for 1 h and then duced the activation of ERK-1/2 within the first 15 min. ERK stimulated with a subsaturant dose of LPS (1 ng/ml) for 1 h. The expression activity remained high until 30 min of LPS stimulation and de- of MKP-1 protein was analyzed by Western blot. All the experiments cayed drastically thereafter (Fig. 5F). The major part of ERK-1/2 shown were performed twice with identical results. inactivation correlated with the time course of expression of the The Journal of Immunology 33

FIGURE 6. The induction of MKP-1 by LPS is not dependent on PKC␤I. A, Prolonged exposure of macrophages to PMA completely down- regulates PKC␤I. 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 Downloaded from 12 h before the addition of LPS (100 ng/ml). MKP-1 expression was analyzed by Northern blot. C, The PKC␤I inhibitor Go¨6976 does not block the induction of MKP-1 by LPS. The cells were preincubated with Go¨6976 (2 ␮M) 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. http://www.jimmunol.org/ determine the expression of PKC isozymes in bone marrow-derived macrophages. We found that only PKC␤I, ⑀, and ␨ were present in these cells, whereas the rest of the isozymes were not detected (47). Among the three isoforms expressed in bone mar- FIGURE 5. PKC mediates the expression of MKP-1 by LPS. A, Mac- row macrophages, PKC␨ is not effectively inhibited by treating the rophages were treated with PMA (100 ng/ml) in the presence or absence of cells with GF109203X at the doses used in our experiments (48), ␮ GF109203X (GF) (1 M), with ionomycin (Io) (500 nM) or with both thus suggesting that the LPS-induced expression of MKP-1 is not PMA and ionomycin for 45 min. A subset of cells were stimulated with mediated by this isoform. In support of this hypothesis, MKP-1 LPS (1 ng/ml) (positive control) or were not treated (negative control). B, by guest on October 1, 2021 The cells were treated for 1 h with either PLC from B. cereus (5 U/ml), induction was not affected by wortmannin (data not shown), an sphingomyelinase (Smase) from B. cereus (1 U/ml), or LPS (100 ng/ml). inhibitor of phosphatidylinositol 3-kinase. This enzyme catalyzes C, The cells were preincubated during 2 h with the indicated concentrations the production of phosphatidylinositol (3,4,5)-trisphosphate, an ac- of GF109203X and then stimulated with LPS (100 ng/ml) for 45 min. A tivator of PKC␨ (13). subset of cells were stimulated with LPS in the presence of vehicle (0.1% To determine which of the PKC isoforms, ␤Ior⑀, regulated the DMSO) (positive control) or were left untreated (negative control). North- expression of MKP-1, macrophages were treated with PMA for ern blot analysis for MKP-1 was performed with total RNA extracts (15 ␮g 12 h before the addition of LPS. Prolonged exposure to PMA per lane) from A, B, and C. D, The values of MKP-1 mRNA expression completely down-regulated PKC␤I but had only a partial down- from C were normalized with the values of L32 expression. The graphic modulating effect on PKC⑀ (Fig. 6A). Accordingly, this treatment represents the mean of three independent experiments. E, The cells were had only a low inhibitory effect on the induction of MKP-1 mRNA 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 by LPS (Fig. 6B). Furthermore, Go¨6976, a selective inhibitor of ␤ Northern blot. All the experiments were reproduced at least twice. F, The conventional PKC isoforms, including PKC I (48), did not alter pattern of ERK-1/2 activation is extended in GF109203X-treated macro- the LPS-induced expression of this phosphatase (Fig. 6C). Taken phages. The cells were preincubated with GF109203X (5 ␮M) or vehicle together, these results indicate that the activation of PKC␤Iisnot for 2 h and then stimulated with LPS (100 ng/ml) for the indicated periods necessary for the induction of MKP-1 by LPS and thus suggest that of time. ERK activity was measured by an in-gel-kinase assay. PKC⑀ is the main PKC isoform involved in this process. To further confirm this hypothesis, we studied the intracellular localization of PKC␤I, ⑀, and ␨ in response to LPS. Fig. 7 shows MKP-1 protein (Fig. 1). Interestingly, the inhibition of PKC with that PKC␤I and ␨ were mainly present in the cytosol in unstimu- GF109203X lead to a significant prolongation of ERK activity lated cells. Whereas PMA induced the translocation of PKC␤Ito during the macrophage response to LPS (Fig. 5F), correlating with the membrane and cytoskeleton fractions (Fig. 7B), LPS, at the the ability of the drug to inhibit MKP-1 expression (Fig. 5, C and concentrations used in our experiments, did not induce the trans- D). These results reinforce the importance of MKP-1 as a negative location of either PKC␤Ior␨ to any of these compartments (Fig. regulator of ERK activity in LPS-stimulated macrophages. We 7A). In contrast, PKC⑀ was present in the membrane fraction of were also interested in determining which PKC isoform was re- both unstimulated and LPS-treated cells, thus being able to interact sponsible for MKP-1 induction in LPS-stimulated macrophages. with its cofactors once a peak of DAG is generated in response At least 11 PKC isoforms have been described, including ␣, ␤I, to LPS. ␤⌱⌱, ␥, ␦, ⑀, ␩, ␪, ␮, ␫/␭, and ␨ (9, 10). However, only a subset of To study whether LPS was able to efficiently activate mem- PKC isozymes are expressed in a given tissue and at a certain stage brane-bound PKC⑀, we immunoprecipitated PKC⑀ from the mem- of differentiation (10). In previous studies, we used specific Abs to brane fraction of macrophages stimulated or not with LPS and 34 PKC⑀ IS REQUIRED FOR MKP-1 INDUCTION BY LPS

FIGURE 7. PKC⑀ is membrane-bound in unstimulated and LPS-treated

bone marrow macrophages. The cells were either not treated or stimulated Downloaded from 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 ␮g) 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 ⑀

FIGURE 8. PKC is required for the induction of MKP-1 and the neg- http://www.jimmunol.org/ PKC⑀. PKC⑀ was immunoprecipitated from the membrane fractions of ative regulation of ERK-1/2 during the macrophage response to LPS. Mac- macrophages stimulated for different periods of time. PKC⑀ activity was rophages were treated during 34 h with antisense oligonucleotides (12 ␮M) measured as described in Materials and Methods, and the mean of three specific for the indicated PKC isoforms. A, The blocking efficiency of each independent experiments is represented. 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 ex- analyzed its capability to phosphorylate the specific susbtrate pression of the ␤-actin protein was assessed to detect differences in loading 25 ⑀ Ser-substituted PKC peptide in vitro. The PKC activity detected and transfer. C, Normalized values of MKP-1 expression are represented as in the membrane fraction of nonstimulated macrophages was percentage of maximal induction and represent the mean of two indepen- equivalent to the background signal detected in the cytosolic frac- dent assays. D, The state of activation of ERK-1/2 after the treatment with by guest on October 1, 2021 tion of these cells (data not shown). Fig. 7C shows that LPS in- LPS (100 ng/ml) for 70 min was analyzed by an in-gel-kinase assay. duced significant activation of PKC⑀ within the first 15 min of stimulation. These results further correlate PKC⑀ activation with genes, the expression of MKP-1 is regulated mainly at the tran- the induction of MKP-1 in bone marrow macrophages. scriptional level. Both mRNA and protein have a very short half Moreover, the cells were treated with antisense oligonucleotides life, and no mechanisms of posttranslational control have been specific for each of the three PKC isoforms ␤I, ⑀,or␨. Antisense described (19, 21, 22). We have explored the signal transduction oligonucleotides directed against PKC␤Ior␨, although blocking pathway that leads to the expression of this phosphatase in re- the expression of these isoforms (Fig. 8A), did not inhibit the in- sponse to LPS. duction of MKP-1 by LPS (Fig. 8B). In contrast, oligonucleotides In fibroblasts, the activation of ERK-1/2 is sufficient to induce specific for PKC⑀ efficiently blocked the expression of this isoform the expression of MKP-1, thus promoting the attenuation of their and significantly inhibited the induction of MKP-1 in response to own cascade in a direct negative feedback loop (50). However, in LPS. Besides, this treatment lead to an increase in the levels of bone marrow macrophages, the induction of MKP-1 by LPS takes ERK-1/2 activity after 70 min of exposure to LPS in comparison place even when ERK activation is blocked. Thus, LPS seems to to those observed in control cells and in cells treated with specific activate two independent pathways, one leading to ERK-1/2 acti- oligonucleotides against PKC␤Ior␨ (Fig. 8D). These results in- vation and the other one determining the expression of their spe- dicate that PKC⑀ is specifically involved in the induction of the cific phosphatase. Also in contrast to what has been reported in phosphatase MKP-1 and thus in the negative regulation of ERK fibroblasts (21, 50), a cAMP-dependent pathway is not involved in activity in LPS-stimulated macrophages. the induction of MKP-1 in macrophages. Our results suggest that LPS leads to MKP-1 induction through Discussion a pathway that involves the activation of intracellular tyrosine ki- MKP-1 is a tyrosine/threonine-phosphatase that dephosphorylates nases and PKC. Although we have not identified the tyrosine ki- different members of the MAPK superfamily (22–24). In bone nases involved in this response, the members of the src family of marrow macrophages, we found that LPS induces the transient tyrosine kinases are likely candidates for two reasons. First, p53/ expression of both MKP-1 mRNA and protein. This agrees with a 56lyn is associated with CD14 in macrophages (51). Second, p53/ previous report that described the accumulation of MKP-1 mRNA 56lyn, p58/64hck, and p59c-fgr are all transiently activated upon in the macrophagic cell line RAW 264.7 after stimulation with stimulation of macrophages with LPS (15, 51). However, some LPS (49). The study of the mechanisms that induce the expression LPS responses occur in the absence of Hck, Fgr, and Lyn (52), of MKP-1 is critical to understand how the cell machinery controls which suggests that their activity can be replaced either by other the duration of MAPK activity. Like many other immediate early members of the same family or by totally distinct tyrosine kinases. The Journal of Immunology 35

In bone marrow macrophages, PKC activation is sufficient to mental conditions, our results allow us to conclude that PKC⑀ has induce the expression of MKP-1. This has been also described in an important function in the control of the pattern of ERK activity some other cell types (21, 53, 54). By using two unrelated PKC by inducing the expression of MKP-1. inhibitors, we have shown that PKC is also involved in the induc- PKC activation has been frequently associated to its translocation of MKP-1 by LPS. Although PKC has been extensively im- tion to the membrane or the cytoskeleton (9, 10). Surprisingly, we plicated in the control of several LPS-induced events (6–8, 55– have detected PKC⑀ at the membranal fraction of unstimulated 59), it is still unclear which isoform(s) are involved in each of macrophages. This confirms previous reports in which the consti- these effects. In previous studies, we found that bone marrow mac- tutive association of PKC⑀ to the membrane was also described in rophages express PKC isoforms ␤I, ⑀, and ␨ (47). Although LPS other cell types and in a macrophagic cell line (10, 65). However, shows high structural similarities with ceramide (60), a second our results reveal that the presence of PKC⑀ at this compartment messenger that activates PKC␨ (12), and despite the fact that does not mean that PKC⑀ is constitutively active in unstimulated MKP-1 expression is induced in macrophages stimulated with macrophages. In fact, treatment with LPS is required to detect sphyngomyelinase, an upstream activator of PKC␨, it is unlikely significant activation of membrane-bound PKC⑀, thus correlating that this isoform mediates the induction of MKP-1 by LPS for the activation of this isoform with the induction of MKP-1 in LPS- several reasons. First, the PKC inhibitor that blocks MKP-1 ex- stimulated macrophages. pression, GF109203X, was used at doses that inhibit conventional Although we cannot exclude the activation of PKC isoforms and novel PKCs, but not the atypical isoforms, including PKC␨ other than PKC⑀, studies with blocking Abs showed that activation (48). Second, the use of wortmannin to inhibit PI3K, an upstream of PKC⑀ accounts for the majority of PKC activity in murine peri- regulator of PKC␨, did not affect MKP-1 induction by LPS. Third, toneal macrophages treated with LPS (66). Moreover, stimulation we could not detect translocation of PKC␨ in response to LPS. And of human monocytes with LPS results in the specific activation of Downloaded from finally, oligonucleotides against PKC␨ did not block MKP-1 in- aCa2ϩ-independent isoform of PKC (7). These and our observa- duction by LPS. All these observations suggest that PKC␨ is not tions predict a crutial role for PKC⑀ in the biology of macrophages. involved in the LPS signaling pathway that leads to MKP-1 Several reports suggest that generation of DAG by LPS takes place expression. through the action of a phosphatydilcholine-specific form of PLC ⑀ Several observations support the involvement of PKC rather (67). Phosphatydilcholine-PLC activity does not generate IP3,a than PKC␤I in the induction of MKP-1 by LPS. First, GF109203X second messenger that induces the release of Ca2ϩ from intracel- http://www.jimmunol.org/ inhibits conventional PKC isoforms better than novel ones (48). lular storage sites (68). In fact, in accordance with previously pub- Concentrations of up to 1 ␮M of GF109203X completely inhibit lished work (69–71), we have not detected mobilization of intra- the activation of conventional PKCs, including ␤I, whereas con- cellular Ca2ϩ in bone marrow macrophages treated with 1–100 centrations of up to 5 ␮M are needed to completely block novel ng/ml of LPS (A.F.V. et al., manuscript in preparation). Because PKC isoforms, including ⑀. In our hands, maximal inhibition of conventional PKCs require the presence of Ca2ϩ for full activation MKP-1 was reached at concentrations of 3–5 ␮M of GF109203X. (9, 10), the absence of Ca2ϩ mobilization would explain the lack Second, prolonged treatment of macrophages with PMA causes of translocation of PKC␤I from the cytosolic to particulate frac- only a low inhibition of MKP-1 induction by LPS. In bone marrow tions in response to the doses of LPS used in our experiments. by guest on October 1, 2021 macrophages, this treatment leads to a complete depletion of Although we can assume that PKC⑀ is required for the induction PKC␤I, but not of PKC⑀. Similarly, in three macrophagic cell lines of MKP-1 by LPS, we cannot discard the involvement of other and in several other cell systems, PKC⑀ has been also shown to be signaling molecules that have not been analyzed in this report. In resistant to prolonged PMA treatment (6, 61–64). Third, Go¨6976, fact, we did not get a total inhibition of MKP-1 expression in a selective inhibitor of conventional PKCs, does not block MKP-1 macrophages treated with either specific inhibitors or with oligo- induction by LPS. And finally, treatment of these cells with anti- nucleotides against PKC⑀. Besides, the induction of this phospha- sense oligonucleotides specific for PKC⑀, and not with those spe- tase by PMA was substantially lower than that mediated by LPS. cific for PKC␤I, significantly inhibits the induction of MKP-1 by These observations suggest that PKC⑀ is required for this process LPS. All these results suggest that, in bone marrow macrophages, but some other mechanism may participate in the induction of PKC⑀ is specifically involved in the induction of the phosphatase MKP-1 by LPS. We are currently analyzing the involvement of MKP-1 by LPS. In previous studies, we had observed that MKP-1 other pathways that regulate MKP-1 expression. expression was also induced in macrophages stimulated with M- In this report, we have studied the mechanisms that control the CSF (47). A strong parallelism has been observed between the expression of MKP-1 in bone marrow macrophages stimulated mechanism used by M-CSF and LPS to induce MKP-1 expression, with LPS. It should be emphasized that LPS induces the expression although both agents trigger totally opposed effects in macrophage of MKP-1 in an ERK-independent manner. Therefore, in macro- biology (proliferation vs activation). During the macrophage re- phages stimulated with LPS, ERK-1/2 do not attenuate their own sponse to M-CSF, MKP-1 induction was similarly mediated activation by inducing MKP-1. LPS ensures this negative control through a PKC-dependent pathway, and, of all the PKC isoforms by triggering an alternative pathway that is activated via the CD14 detected in bone marrow macrophages, PKC⑀ was the main can- molecule and specifically requires the action of tyrosine kinases didate to mediate MKP-1 expression. These, together with the data and PKC⑀. The results of this report demonstrate the key role of shown in the present report, suggest that, in bone marrow macro- PKC⑀ in the control of the duration of MAPK activity during the phages, PKC⑀ plays a major role in the control of MKP-1 macrophage response to LPS. expression. We have also shown a significant prolongation of the time Acknowledgments course of ERK activity in macrophages treated with the PKC in- We thank Dr. Antonio Garcıá de Herreros (Institut Municipal hibitor GF109203X or with antisense oligonucleotides against d’Investigacio´Me´dica, Barcelona, Spain) for discussing the experiments ⑀ PKC , correlating with the capability of these compounds to in- regarding PKC and for some reagents. We also thank Dr. Rich A. Maki hibit MKP-1 expression. Although we cannot discard the involve- (The Burnham Institute, La Jolla, CA), Dr. Jorge Moscat and Dr. Teresa ment of other phosphatases different to MKP-1, specially because Dıáz-Meco (CBM, Madrid, Spain) for some reagents and for helpful advice partial inactivation of ERK-1/2 still took place in those experi- about signal transduction, Dr. Peter Parker (Imperial Cancer Research 36 PKC⑀ IS REQUIRED FOR MKP-1 INDUCTION BY LPS

Fund, London, U.K.) for the anti-PKC Abs, Dr. Michael Weber (University unique substrate specificities and reduced activity in vivo toward the ERK2 sev- of Virginia, Charlottesville, VA) for the anti-ERK1/2 Abs, Dr. Rodrigo enmaker mutation. J. Biol. Chem. 271:6497. Bravo (Bristol-Myers, Princeton, NJ) for the plasmid containing the full- 25. Celada, A., P. W. Gray, E. Rinderknecht, and R. D. Schreiber. 1984. Evidence for a ␥-interferon receptor that regulates macrophage tumoricidal activity. J. Exp. length MKP-1 cDNA, Dr. Markus Nabholz (Institut Suisse de Recherches Med. 160:55. Experimentales sur le Cancer, Epalinges, Switzerland) for the plasmid con- 26. Goodnight, J., M. G. Kazanietz, P. M. Blumberg, J. F. Mushinski, and taining TNF-␣ cDNA, Dr. Rose Wilson (Glaxo Research and Development H. Mischak. 1992. The cDNA sequence, expression pattern and protein charac- ␨ Limited, Greenford, U.K.) for the IL-1␤-containing plasmid, and Dr. Jose´ teristics of mouse protein kinase C . 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