Macrophage-Colony-Stimulating Factor-Induced Proliferation and Lipopolysaccharide-Dependent Activation of Macrophages Requires Raf-1 This information is current as Phosphorylation to Induce Kinase of September 26, 2021. -1 Expression Ester Sánchez-Tilló, Mónica Comalada, Consol Farrera, Annabel F. Valledor, Jorge Lloberas and Antonio Celada Downloaded from J Immunol 2006; 176:6594-6602; ; doi: 10.4049/jimmunol.176.11.6594 http://www.jimmunol.org/content/176/11/6594 http://www.jimmunol.org/ References This article cites 78 articles, 41 of which you can access for free at: http://www.jimmunol.org/content/176/11/6594.full#ref-list-1

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Macrophage-Colony-Stimulating Factor-Induced Proliferation and Lipopolysaccharide-Dependent Activation of Macrophages Requires Raf-1 Phosphorylation to Induce Mitogen Kinase Phosphatase-1 Expression1

Ester Sa´nchez-Tillo´, Mo´nica Comalada, Consol Farrera, Annabel F. Valledor,2 Jorge Lloberas, and Antonio Celada3

Macrophages are key regulators of immune responses. In the absence of an activating signal, murine bone marrow-derived macrophages undergo proliferation in response to their specific growth factor, namely M-CSF. The addition of bacterial LPS results in macrophage growth arrest and their engagement in a proinflammatory response. Although participation of ERKs is Downloaded from required for both macrophage proliferation and activation, ERK phosphorylation follows a more delayed pattern in response to activating agents. In primary macrophages, mitogen kinase phosphatase-1 (MKP-1) is a key regulator of the time course of MAPK activity. Here we showed that MKP-1 expression is dependent on Raf-1 activation. The time course of Raf-1 activation correlated with that of ERK-1/2. However, whereas ERK phosphorylation in response to M-CSF is Raf-1 dependent, in response to LPS, an alternative pathway directs the activation of these kinases. Inhibition of Raf-1 activity increased the expression of -dependent kinase inhibitors and growth arrest. In contrast, no effect was observed in the expression of proinflammatory cytokines and http://www.jimmunol.org/ inducible NO synthase following LPS stimulation. The data reported here reveal new insights into how signaling determines opposing macrophage functions. The Journal of Immunology, 2006, 176: 6594–6602.

acrophages serve essential roles as regulators of im- changes in the activity of these kinases have been associated with munity. A number of stimuli regulate macrophage specific cell fates in a number of cell types (8). In our cellular M gene expression and determine their final biological model, an early peak of ERK activation (5 min) correlated with function. In response to growth factors, such as M-CSF, macro- cellular proliferation whereas a later peak (15 min) was associated

phages differentiate and proliferate (1). In contrast, activation by with the activation program (5). Among the regulatory mecha- by guest on September 26, 2021 LPS, a component of Gram-negative bacteria, makes these phago- nisms that affect ERK phosphorylation, dual specificity mitogen cytic cells stop their proliferating program and acquire effector kinase (MKP4 or DUSP) dephosphorylate tyrosine functions such as the production of NO and proinflammatory cy- and residues, which results in ERK inactivation (9, 10). tokines, including TNF-␣, IL-1, and IL-6 (2–4). In vitro studies and transfection experiments showed that MKP-1 Although macrophages either proliferate or become activated, in expression in response to dephosphorylates mainly ERK- both cases activation of ERK-1 and -2 is required (5). These are 1/2 and has lower activity toward other MAP kinases such as JNK evolutionarily conserved proline-directed serine/threonine protein and p38 (9). In bone marrow-derived macrophages, MKP-1 is a kinases, also known as p44- and p42-MAPKs, responsible for key regulator of the time course of ERK activity and inactivation phosphorylating several transcription factors such as Elk-1, Fos, of ERK-1/2 tightly follows the induction of this phosphatase (5). Jun, and c-Myc family members (6, 7). Interestingly, subtle dif- However, in contrast to what has been reported in other cell types ferences in the time course and strength of ERK activation as well (11), MKP-1 induction by M-CSF and LPS in these cells is inde- as its subcellular localization appear to specify differential down- pendent of ERK activation (12, 13). Interestingly, a number of stream signaling events. In particular, the duration of ERK signal- conditions that inhibit MKP-1 expression in macrophages, for ex- ing is under tight control by positive and negative regulators and ample extracellular matrix proteins such as decorin or fibrinogen, or treatment with cyclosporine A or FK506, elongate ERK activity and reduce proliferation (14, 15). Macrophage Biology Group, Institute of Research in Biomedicine-University of Bar- One of the early players in intracellular signaling is the serine/ celona, Barcelona Science Park, Barcelona, Spain threonine kinase Raf, which regulates a broad range of functions Received for publication November 14, 2005. Accepted for publication March including cellular growth, proliferation, activation, differentiation 9, 2006. and (11, 16). In many cell types, activated Raf kinases The costs of publication of this article were defrayed in part by the payment of page phosphorylate and stimulate the mitogen-activated ERK kinase charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (MEK), the immediate upstream regulator of the ERK module (17). Several Raf isoforms in mammalian cells have been identi- 1 This work was supported by Ministerio de Ciencia y Tecnologı´a Grant BFU2004- 05725/BMC (to A.C.). fied; c-Raf/Raf-1, A-Raf, and B-Raf (18). A and B isoforms are 2 A.F.V. is an investigator from the Programa Ramo´n y Cajal of the Spanish Ministry of Science and Technology. 3 Address correspondence and reprint requests to Antonio Celada, Institute of Re- 4 Abbreviations used in this paper: MKP-1, mitogen-activated phos- search in Biomedicine, Barcelona Science Park, c/ Josep Samitier 1-5, E-08028 Bar- phatase-1; cdk, cyclin-dependent kinase; SA, sodium arsenite; iNOS, inducible NO celona, Spain. E-mail address: [email protected] synthase; siRNA, small-interfering RNA.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 6595 predominantly expressed in neuronal and urogenital tissues, PKC assay whereas Raf-1 is expressed ubiquitously (19). Raf activity can be PKC assay was performed as previously described (29). Briefly, total cel- regulated by direct phosphorylation by other kinases, including lular lysates were obtained and immunoprecipitated for PKC␧. After wash- members of the serine-threonine protein kinase C (PKC) family, ing, kinase assay was performed using 1 ␮g of histone H1 (Santa Cruz Src and AKT/PKB (20–22) and by complex formation with 14-3-3 Biotechnology) as exogenous substrate, 20 ␮M ATP, and 1 ␮Ci of ␥ 32 proteins (23) and Hsp90 chaperone (24). [ - P]ATP. The exact effects of Raf-1 activation on macrophages are con- RNA extraction and Northern blot analysis troversial. Here we studied the role of Raf-1 in MKP-1 expression Total RNA was extracted with the RNA Kit EZ-RNA (Biological Indus- and ERK activation in primary macrophages. We show that Raf-1 tries), separated in agarose gel and transferred to nitrocellulose membrane activation is required for MKP-1 induction in macrophages stim- (Amersham Biosciences). Probes were labeled with [␣-32P]dCTP (Amer- ulated with M-CSF or LPS. In both conditions, we observed that sham Biosciences) using the random prime labeling system (Amersham Raf-1 binds to and regulates PKC␧ activity. Distinct kinetics of Biosciences). 18S was used as a loading and transfer control. Results are representative of three independent experiments. Raf-1 activation were detected in response to the two stimuli. Al- though these time courses correlated with those of activated ERK- Real Time-PCR 1/2, Raf-1 was involved in ERK activation in response to M-CSF cDNA was obtained using M-MLV Reverse Transcriptase (Promega) as only. During LPS signaling, an alternative pathway directed ERK described (30). Primer Express software (Applied Biosystems) was used to activation in the absence of active Raf-1. In correlation with these design primer sequences. Real Time-PCR was conducted with 1ϫ SYBR effects, inhibition of Raf-1 activity compromised the progression Green PCR master mix using the ABI Prism 7900 detection system (Ap- plied Biosystems). Each sample was analyzed in triplicate. Expression lev- of macrophages through the whereas no effect was ob- Downloaded from els were normalized to ␤-actin. Relative values from a representative ex- served in the expression of proinflammatory cytokines or inducible periment out of three independent experiments are represented in each NO synthase (iNOS) upon LPS stimulation. graphic. Cell cycle analysis Materials and Methods Cell cycle was analyzed as described (27). Briefly, 106 treated cells were Reagents fixed and incubated in PBS, 0.2% Triton X-100, 10 ␮g/ml RNase A, 5 http://www.jimmunol.org/ LPS, actinomycin D, propidium iodide, and Abs anti-ERKP Thr183/Tyr185, mg/ml propidium iodide, and analyzed by FACS. Cell cycle distributions and anti-␤-actin were purchased from Sigma-Aldrich. Recombinant M- were analyzed with the Multicycle program (Phoenix Flow Systems). Ex- CSF (1200 U/ml Х 10 ng/ml) and IFN-␥ were obtained from R&D Sys- periments were performed in triplicate. tems. Sodium arsenite (SA) was obtained from Wako Pure Chemicals. The Raf activity assay p38P (Thr180/Tyr182) MAP kinase Ab was purchased from Technology. Monoclonal anti-Raf-1P Ser338 and polyclonal anti-Raf-1 Abs Raf activity assay was performed as previously described (1). The assay is were obtained from Upstate Biotechnology and Biosource, respectively. based on measurement of Raf-1-dependent phosphotransferase activity in a Anti-PKC␧ and anti-phosphotyrosine Abs were from BD Pharmingen. Abs kinase reaction using recombinant MEK1, inactive as a Raf-1 substrate against ERK-1/2 or MKP-1 were purchased from Santa Cruz Biotechnol- (Upstate Biotechnology). Briefly, cell lysates were immunoprecipitated ogy. ZM 336372, geldanamycin, and Abs anti-NOS2 and anti-MEKP with total Raf-1 Ab (Biosource). After several washes, the reaction was Ser218/Ser222 (17) were obtained from Calbiochem. conducted with 1 ␮g of inactive MEK, 2 mCi of [␥ 32-P]ATP, and 500 ␮M by guest on September 26, 2021 cold ATP at 30°C for 30 min. Cell culture “In gel” kinase assay Bone marrow-derived macrophages were obtained from BALB/c mice (Harlan Ibe´rica) and cultured as previously described (25). Animal use was ERK activity was analyzed as previously described (5) using myelin basic approved by the Animal Research Committee of the University of Barce- protein (Sigma-Aldrich) as a substrate, copolymerized in the gel. Results lona (2523). To render cells quiescent, they were deprived of M-CSF for were representative of three independent assays. 18 h before stimulation (25). Transfection of small-interfering RNA (siRNA) Apoptosis assay siRNA were obtained from Dharmacon and transfected by electroporation ϫ 6 ␮ ␮ FITC (31). Then 4 10 cells and 1.5 M siRNA were resuspended in 400 l Cell death was assessed by Annexin V (Bender MedSystems) using and pulsed once at 350 V, 2300 ␮F with a BTX ECM 600 electroporator FACS analysis (Coulter Multisizer II) according to the manufacturer’s in- (BTX). The siRNA sequences were AGUCAAAGAAGAGAGACCU for structions. These studies were confirmed by trypan blue exclusion. Each siRNA1 and UUCCAGAUGUUCCAGCUAA for siRNA2. siRNAu was point was performed in triplicate and the results were expressed as the Ϯ directed against and the sequence was AACCUCGUGUG mean SD. GAUAUCUU (15). Proliferation assay Results The proliferative capacity of macrophages was assessed as previously de- Raf-1 activation by M-CSF or LPS is required for the scribed (26). A 6-h pulse of [3H]thymidine (1 ␮Ci/ml) (Amersham Bio- expression of MKP-1 sciences) was applied to each sample. Each point was performed in trip- licate, and the results were expressed as the mean Ϯ SD. Although resulting in two opposing cellular functions, the macro- phage growth factor M-CSF and the activating bacterial compound Western blot analysis LPS induce the expression of the phosphatase MKP-1 in macro- Total cytoplasmic extracts were obtained and Western blotting was per- phages. This phosphatase has gained interest as a key regulator of formed as previously described (27). Detection was conducted using macrophage function by virtue of its capacity to control the dura- EZ-ECL kit (Biological Industries). ␤-Actin expression was measured as a tion of MAPK activity (5, 12, 13). The involvement of Raf-1 in control for differences in loading and transfer. Figures are representative of macrophage pathways is unclear. There is ev- at least three independent experiments. idence that proliferating and activating stimuli also result in dif- Functional analysis of JNK ferent Raf-1 activation requirements in macrophagic cell lines (32– 34). Here we studied whether Raf-1 activity is required for the JNK activity was measured as previously described (28). Briefly, cell ly- sates were immunoprecipitated for JNK1. The reaction was performed with expression of MKP-1. Since primary cultures of bone marrow- 1 ␮g of GST-c-jun (MBL) as JNK substrate, 20 ␮M ATP, and 1 ␮Ci derived macrophages are scarcely transfectable with plasmids (31), [␥-32P]ATP. we were unable to use mutants or activated constructs of Raf-1 6596 Raf-1 DIRECTS MKP-1 EXPRESSION IN MACROPHAGES proteins. We therefore used chemical inhibitors of Raf-1, assuming 1, C and D). These effects were not caused by toxicity induced by that they are not always completely specific. It has been reported ZM 336372, as shown in Fig. 5B. that fully induced Raf-1 activity requires both Ser338 and Tyr341 To corroborate the results obtained with the Raf-1 inhibitor, we phosphorylation (20, 35, 36), which can be blocked by the syn- selectively abrogated Raf-1 protein expression using siRNA tech- thetic compound ZM 336372 (Fig. 1, A and B). Furthermore, Raf-1 nology. The efficiency of this process was tested using real time- activity was determined by a link MEK kinase assay measuring PCR and Western blot after 48 h of electroporation. Two indepen- MEK phosphorylation after in vitro reaction (Fig. 1A). To study dent siRNAs against Raf-1 were similarly efficient at depleting the effect of Raf-1 activity on MKP-1 expression, we performed Raf-1 (Fig. 1E). As controls, we used a mock transfection and an both Northern and Western blot analyses. We had previously char- siRNA directed to an unrelated gene, calcineurin (siRNAu) (15). acterized the time course of induction of this phosphatase by M- To exclude toxic effects of siRNA transfection, we determined the CSF and LPS; maximal mRNA induction occurred at 30 and 45 levels of apoptosis. No increased cell death was associated with min of treatment, respectively, whereas the protein levels peaked any of the conditions tested compared with actinomycin D used as 15 min later for each case (12, 13). ZM 336372 abolished the a positive control (Fig. 1F). Depletion of Raf-1 resulted in com- induction of MKP-1 mRNA and protein by M-CSF and LPS (Fig. plete inhibition of MKP-1 expression even in the presence of Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. MKP-1 induction by M-CSF and LPS was compromised by Raf-1 inhibition. A, Quiescent cells were pretreated for 1 h with 30 ␮MofZM 336372 and stimulated with 1200 U/ml M-CSF for the times indicated. Western blot of phosphotyrosine was performed on Raf-1 immunoprecipitates. Raf-1 activity was determined using inactive MEK as substrate. B, Western blot of phosphoserine Raf-1 was determined. C, Quiescent macrophages were stimulated with 1200 U/ml M-CSF after1hofpretreatment with 30 ␮M ZM 336372. MKP-1 mRNA induction by M-CSF at 30 min was determined by Northern blot (top panel) and expression of MKP-1 at 45 min was analyzed by Western blot (bottom panel). D, MKP-1 mRNA induction by1hof pretreatment with 30 ␮M ZM 336372 and 10 ng/ml LPS stimulated macrophages at 45 min was determined by Northern blot (top panel) and expression of MKP-1 at 60 min was analyzed by Western blot (bottom panel). E, Raf-1 mRNA was determined by real time-PCR and Western blot in siRNA electroporated macrophages. Mock and siRNAu were used as controls of electroporation and unspecific effects. F, Cytotoxic effects of electroporation were checked by annexin V determination. Act D was used as positive control of cell death. G, Raf-1 disappearance by siRNA inhibits MKP-1 expression in M-CSF and LPS-stimulated macrophages as determined by Western blot at 45 min for M-CSF and 60 min for LPS. Mock was used as control of electroporation. The Journal of Immunology 6597

FIGURE 2. Raf-1 interacts with PKC␧ and regulates its activity. Quiescent cells were stimulated with 1200 U/ml M-CSF or 10 ng/ml LPS in presence or absence of 30 ␮M ZM 336372. A and B, Serine-phosphorylated Raf-1 was determined in PKC␧ immunoprecipitates from macrophages by Western blot. C and D, In vitro PKC␧ activity was determined by kinase assay using histone H1 as exogenous substrate.

M-CSF or LPS (Fig. 1G). Taken together, these results indicate only from 4 to 8 min after the start of M-CSF stimulation, which

that Raf-1 activity is required for MKP-1 expression during M- indicates that the kinetics of this interaction is very tightly con- Downloaded from CSF-induced proliferation and LPS activation. trolled by positive and negative regulators. The phosphorylated Raf-1-PKC␧ complex was also detected during the response to Treatment with M-CSF or LPS promotes the interaction of Raf-1 LPS, although its kinetics was slightly delayed; binding was de- ␧ with PKC tected 8 to 10 min after the start of treatment (Fig. 2B). We previously reported that PKC␧ is a key upstream regulator of To further examine whether Raf-1 regulates PKC␧ activity in

MKP-1 expression in primary macrophages (12, 13). Our next in- macrophages, we performed immune complex kinase assays for http://www.jimmunol.org/ terest was to examine whether cross-talk occurs between Raf-1 and PKC␧. In this case, PKC␧ was immunoprecipitated from total cell PKC␧ during signal transduction to M-CSF or LPS. PKC␧ was lysates and its activity was measured in vitro using histone H1 as immunoprecipitated from equivalent amounts of cellular lysates a substrate. Both M-CSF and LPS stimulation increased PKC␧ and the samples were subsequently immunoblotted with an anti- activity, which was abolished in the presence of the Raf-1 inhibitor phosphoserine Raf-1 Ab, which is a good indicator of the active ZM 336372 (Fig. 2, C and D). These observations indicate that state of Raf-1. Phosphorylated Raf-1 coimmunoprecipitated with Raf-1 activity is required for PKC␧ activation during M-CSF and PKC␧ in response to M-CSF (Fig. 2A). This complex was detected LPS signaling. Moreover, in correlation with the time course of by guest on September 26, 2021

FIGURE 3. M-CSF induces an earlier activation of Raf-1, MEK-1/2, and ERK-1/2 pathway than LPS. Quiescent macrophages were stimulated for the times indicated with 1200 U/ml M-CSF or 10 ng/ml LPS. A, Raf-1 immunoprecipitates of total cell lysates of quiescent cells stimulated with M-CSF or LPS were blotted with anti-phosphotyrosine Ab. B, Total M-CSF or LPS-stimulated cell lysates were blotted with anti-Raf-1 phosphoserine peptide Ab. C, MEK-1/2 phosphorylation by M-CSF or LPS was determined by Western blot. D, ERK-1/2 activation by M-CSF or LPS measured by “in-gel” kinase assay. 6598 Raf-1 DIRECTS MKP-1 EXPRESSION IN MACROPHAGES

Raf-1 phosphorylation (Fig. 2, A and B), activation of PKC␧ by We next examined whether Raf-1 activity was indeed required M-CSF and LPS followed distinct kinetics. Maximal levels of ac- for ERK-1/2 activation. Total cell lysates from M-CSF-stimulated tive PKC␧ were detected at 5–10 min of M-CSF stimulation and at macrophages were immunoblotted with an Ab that recognizes 15–30 min of LPS treatment. Thr183 and Tyr185 phosphorylation of ERK-1/2. Activation of Interestingly, although Raf-1 phosphorylation was necessary for ERK-1/2 by M-CSF was blocked by the Raf-1 inhibitor ZM PKC␧ activation as assessed by ZM 336372, time course experi- 336372 (Fig. 4A). Gel kinase assays using myelin basic protein as ments indicate that Raf-1 was required only for triggering the ini- a substrate confirmed the complete inhibition of ERK-1/2 activity tial activation of PKC␧, since it remained active after Raf-1 by ZM 336372 (Fig. 4B). Two nonrelated inhibitors of Raf-1 func- . tion, namely SA and geldanamycin, a disruptor of Raf-1 interac- tion with hsp90 chaperone (37), had similar effects on ERK-1/2 Raf-1 activation is required for ERK-1/2 phosphorylation phosphorylation by M-CSF (Fig. 4, C and D). Likewise, the use of induced by M-CSF but not by LPS two independent Raf-1 siRNAs inhibited the phosphorylation of Our results in Fig. 2 indicated that M-CSF and LPS stimulation ERK-1/2 in response to M-CSF (Fig. 4G). Taken together, these resulted in differential patterns of Raf-1 activation. We already data indicate that Raf-1 activity is required for ERK-1/2 activation reported subtle changes in the activation pattern of ERK-1/2 fol- by M-CSF. In contrast, inhibition of Raf-1 with ZM 336372 did lowing stimulation with M-CSF or LPS (5). Here we further char- not reduce ERK phosphorylation or activation (Fig. 4, E and F) acterized overall changes in the Raf-1-MEK-ERK cascade in re- after stimulation with LPS. Identical results were obtained using sponse to the proliferative or the activation signal (Fig. 3). Raf-1 SA (data not shown) and siRNA technology (Fig. 4H). These re- was phosphorylated in both serine and tyrosine residues within 3–5 sults indicate that LPS signaling includes alternative pathways that Downloaded from min of M-CSF stimulation (Fig. 3, A and B), which was followed can direct ERK phosphorylation and activation in the absence of by fast and transient phosphorylation of MEK-1/2 (Fig. 3C), and active Raf-1. Interestingly, Raf-1 inhibition in LPS-treated mac- subsequent activation of ERK-1/2 (Fig. 3D and Ref. 5). As de- rophages appeared to extend the period of ERK-1/2 activation, scribed previously, the time course of ERK-1/2 activation by LPS which can be explained by the lack of expression of MKP-1, re- was more delayed than that induced by M-CSF (Fig. 3D and Ref. sponsible for dephosphorylating several members of the MAPK

5), which correlated with the delayed kinetics of Raf-1 and MEK- family (Fig. 1D). Elongation of JNK and p38 MAPK activation http://www.jimmunol.org/ 1/2 phosphorylation (Fig. 3, A–C). was also observed under these conditions (data not shown). by guest on September 26, 2021

FIGURE 4. Raf-1 is required for ERK-1/2 activation by M-CSF but not for LPS. A, Raf-1 inhibition affects M-CSF-dependent phosphorylation of ERK-1/2. ERK-1/2 phosphorylation of 1200 U/ml M-CSF-treated cells in the presence or absence of 30 ␮M ZM 336372 was determined by Western blot. B, Raf-1 is required for ERK-1/2 activity induced by M-CSF. IGK analysis was performed. C, Western blot of phosphorylated ERK-1/2 of macrophages pretreated with 10 ␮M SA and then M-CSF-stimulated. D, Geldanamycin inhibits ERK-1/2 activity induced by M-CSF. IGK analysis was performed on M-CSF-stimulated macrophages in the presence or absence of 3 ␮M geldanamycin. E, Quiescent macrophages were preincubated with 30 ␮M ZM 336372 and then stimulated with 10 ng/ml LPS for the times indicated. ERK-1/2 phosphorylation was determined by Western blot. F, IGK analysis was performed. G, Western blot of ERK-1/2 phosphorylation of Raf-1 siRNA electroporated cells after 15 min of M-CSF stimulation. H, siRNA inhibition of Raf-1 does not affect ERK-1/2 activation by LPS at 30 min as assessed by Western blot. Mock-electroporated cells were used as control of electroporation. The Journal of Immunology 6599

Raf-1 activation by M-CSF is required for macrophage In addition, SA also inhibited M-CSF-dependent proliferation proliferation and progression through the cell cycle (Fig. 5C) without causing toxic side effects (data not shown). We next determined the role of Raf-1 in macrophage proliferation These results were corroborated using siRNA technology. Macro- by measuring [3H]thymidine incorporation as an indicator of DNA phages transfected with two independent siRNAs directed against synthesis (Fig. 5A). ZM 336372 blocked macrophage proliferation Raf-1 showed decreased DNA synthesis in response to M-CSF, in a dose-dependent manner, with maximal inhibition at 30 ␮M. compared with cells transfected with a control siRNA (Fig. 5D). To exclude toxic effects of this drug, we measured the induction of To further characterize the role of Raf-1 in the control of mac- apoptosis by annexin V staining. Anti-proliferative doses of ZM rophage cell cycle, we measured the cellular DNA content by pro- 336372 did not increase macrophage cell death in the presence pidium iodide staining. Gated viable cells were analyzed for their (Fig. 5B) or absence of M-CSF (data not shown). The lack of DNA content and the percentage of cells in the S phase of the cycle cellular toxicity was corroborated by trypan blue exclusion (data is shown in Fig. 5E. In the absence of growth factors, the cells not shown). were arrested at G0-G1 phase and very few cells progressed through the S phase (Fig. 5E and Ref. 27). Upon M-CSF stimu- lation, 30% of the cells re-entered the cell cycle and were pro- gressing through the S phase 30 h after the start of the stimulation. ␥ As a positive control of G1-S blockage, we used IFN- , as previ- ously described (27). Inhibition of Raf-1 activity with ZM 336372 decreased the numbers of cells entering the S phase compared with

cells stimulated with the growth factor. No cycling activity was Downloaded from observed when the inhibitor was used alone. In conclusion, inhi- bition of Raf-1 decreased the entry of cells into the S phase of the cell cycle, which correlates with reduced [3H]thymidine incorpo- ration (Fig. 5A). We also studied the effect of ZM 336372 on several regulators

of the cell cycle. Progression throughout the G1 and S phases of the http://www.jimmunol.org/ cell cycle is controlled by the sequential activation of a number of cyclin D/cdk complexes (38). We thus performed real time-PCR assays to monitor the expression of various . M-CSF-stim- ulated cells enter the cell cycle by increasing the expression of cyclin D1, D2, and D3 (38). However, inhibition of Raf-1 by ZM 336372 did not modify the mRNA levels of any of these cyclins (Fig. 5F; data not shown). Because cyclin-Cdk activity is also regulated by Cdk inhibitory proteins such as p21Waf-1 and p27Kip-1 (39, 40), we measured their mRNA levels after ZM 336372 treat- by guest on September 26, 2021 ment. Raf-1 inhibition increased the expression of p21Waf-1 and p27Kip-1 (Fig. 5, G and H) at different time points after M-CSF stimulation, indicating that these two regulators may mediate the blockage in S phase entry caused by ZM 336372.

Raf-1 activation by LPS is not required for cytokine production Northern blot assays were used to measure mRNA expression of proinflammatory cytokines in response to LPS. Raf-1 inhibition did not compromise the induction of IL-1␤, TNF-␣, or IL-6 in macrophages treated with subsaturant concentrations of LPS. In- terestingly, this inhibition enhanced their induction (Fig. 6A), FIGURE 5. Raf-1 inhibition causes a macrophage growth arrest at the S which could be related to extended MAPK activation in response phase of the cell cycle and up-regulates p21Waf-1 and p27Kip-1 cdk inhib- itors. A, Raf-1 inhibition affects M-CSF-dependent thymidine incorpora- tion in macrophages. Quiescent macrophages were pretreated for 1 h with the concentrations of ZM 336372 indicated, before incubation with 1200 U/ml M-CSF for 24 h. B, ZM 336372 has a minor effect on cell viability. Quantification of annexin V-positive-gated macrophages incubated with M-CSF in the presence of 30 ␮M ZM 336372. Act D was used as positive control of cell death. C, SA inhibits M-CSF-dependent proliferation in macrophages. Quiescent macrophages were pretreated for 1 h with the concentrations of SA indicated and then stimulated with 1200 U/ml M-CSF for 24 h. D, Proliferation by M-CSF was assessed by thymidine incorpo- ration in Raf-1 siRNA electroporated macrophages. E, Raf-1 inhibition blocks M-CSF-induced S phase; 106 quiescent macrophages were prein- cubated for 1 h with 30 ␮M ZM 336372 or 300 U/ml IFN-␥. M-CSF (1200 U/ml) was then added for 30 h. DNA content was measured and cell cycle FIGURE 6. Raf-1 is not required in cytokine production and NOS2 in- distribution was analyzed. F, Raf-1 inhibition does not modify cyclin D1 duction by LPS. A, TNF-␣, IL-1␤, and IL-6 mRNA expression was deter- mRNA. The induction of cyclin D1 was analyzed by real time-PCR. G, mined by Northern blot. Expression of 18S was used as control. B, Real Raf-1 inhibition causes an up-regulation of p21Waf-1 induction as deter- time-PCR of NOS2 mRNA was measured using ␤-actin expression to nor- mined by real time-PCR. H, Raf-1 inhibition increases p27Kip-1 mRNA as malize the values. C, NOS2 expression was determined by Western blot. analyzed by real time-PCR. ␤-actin was used as control. 6600 Raf-1 DIRECTS MKP-1 EXPRESSION IN MACROPHAGES to LPS (Fig. 4F). Raf-1 had no effect on LPS-induced NOS2 ex- work, we established PKC␧ as a key regulator of MKP-1 expres- pression in bone marrow-derived macrophages (Fig. 6, B and C). sion in macrophages (12, 70). Indeed, the promoter of the MKP-1 Taken together, these results indicate that LPS activates signaling gene contains AP-1 sites that respond to PKC-derived signals (12), events that are capable of bypassing Raf-1 activation and still in- and inhibition of PKC activity elongates ERK activation possibly ducing ERK-1/2 activity and proinflammatory cytokine due to inhibited induction of MKP-1. However, our observations production. are in agreement with previous studies that demonstrated that Ras recruits Raf-1 to the plasma membrane where it can interact with Discussion PKC␧ and act as a coregulator and cooperator of PKC-derived Here we report on differential requirements of Raf-1 for macro- signals (36). Indeed, full Raf-1 phosphorylation is dependent upon phage function. During macrophage signal transduction to M-CSF, its association with the plasma membrane (12) and PKC␧ is con- Raf-1 plays two crucial roles that result in tight regulation of the stitutively located in the plasma membrane in macrophages (5). pattern of ERK activation. First, Raf-1 activity accounts for pos- Although M-CSF and LPS activate a common target, the ERK itive regulation of the MEK-ERK module, which is later required pathway, distinct kinetics of ERK-1/2 activation and deactivation for macrophage proliferation. Our data confirm previous reports correlate with a range of macrophagic functions (5, 71). An early that demonstrated that Raf-1 is critical for activation of ERK-1/2 peak of ERK-1/2 activity is associated with proliferative signals, during M-CSF stimulation (41, 42). Second, Raf-1 is involved in whereas macrophage-activating signals trigger a more delayed and expression of the phosphatase MKP-1, a key negative regulator of lasting peak. Here we have demonstrated that distinct kinetics are MAPK activity (43). also displayed by the upstream regulators MEK-1/2 and Raf-1 in

Therefore, the overall duration and strength of M-CSF-induced response to M-CSF or LPS. This observation indicates that specific Downloaded from ERK activity falls under the control of Raf-1. During the activation events that occur upstream of Raf-1 signaling may be responsible of bone marrow-derived macrophages by LPS, Raf-1 activity is for these differences (Fig. 7). For example, Ras has been shown to dispensable for ERK phosphorylation, because activation of the be involved in M-CSF, but not in LPS, signaling toward Raf-1 ERK module can proceed in the absence of fully active Raf-1. activation, whereas phosphatidylcholine- appears Other macrophagic cells, including the RAW 264.7 cell line and to mediate Raf-1 activation in LPS-stimulated cells (71). Also,

primary alveolar macrophages, phosphorylate ERK during LPS differences in receptor assembly and activation could account for http://www.jimmunol.org/ stimulation without activation of Raf-1 (33, 44), which indicates the differential time courses; c-fms receptor requires noncovalent that LPS generally bypasses Raf-1 activity to activate the ERK dimerization and autophosphorylation of tyrosine kinase domains cascade in macrophages. Although MEK-1/2 are the major sub- (72), whereas recognition of LPS requires the assembly of TLR4 strates of Raf proteins, the presence of an alternative upstream with CD14, LPS-binding protein, and MD-2 (73, 74). pathway that activates MEK-1/2 has been proposed and includes The involvement of Raf-1 in mediating proliferation has also the serine/threonine kinase Cot/Tpl2 (45, 46), the proto-oncogene been described in other cellular types. In fibroblasts, Raf-1 activity

Mos kinase (47), and a 73-kDa MEKK in EGF-treated PC-12 cells is essential for the induced G1-S phase transition and mitogenic (48). In correlation with these observations, our data indicate that effects of growth factors, as demonstrated by antisense constructs proinflammatory cytokine production and expression of iNOS in and dominant inhibitory mutants (75). Furthermore, oncogenically by guest on September 26, 2021 response to LPS do not require Raf-1 activity either. Moreover, activated Raf-1 initiates DNA synthesis (76–78) and induces the increased cytokine production was observed in the absence of transcription of several genes required for proliferation (77). In this MKP-1 and Raf-1 inhibition. This may be because the inhibition of context, the Raf-1 knockout has confirmed that Raf-1 is required MKP-1 extended MAPK activation. In fact, it has been reported for the viability of mice embryos and for ERK activation by mi- that an increase of MKP-1 limits cytokine production (2, 49). togens in fibroblasts (41, 42). However, although oncogenic Raf-1 In contrast, MKP-1 induction by LPS is a Raf-1-dependent increases cyclin D1 protein in fibroblasts (77), we did not observe event, which indicates that Raf-1 is required for the control of the any modifications in the expression levels of any of the members duration of MAPK activity during signal transduction to LPS. Pre- of the cyclin D family. Interestingly, the Cdk inhibitors p21Waf-1 vious reports showed that ectopic expression of v-raf in macro- and p27Kip-1 were negatively regulated by Raf-1 in macrophages, phages also induces MKP-1 (34). The contribution of MKP-1 which may explain the occurrence of growth arrest in the absence phosphatase to deactivation of ERK, JNK, and p38 kinases has of active Raf-1. In agreement with our data, oncogenic activation been widely reported (43, 52). MKP-1 was the first phosphatase of Raf-1 suppresses the expression of p27Kip-1 in NIH3T3 cells identified as an in vitro ERK-specific phosphatase (53–55). Further (75). The data presented here provide new insights into the mech- studies reveal that MKP-1 was ubiquitously induced by a wide anisms used by macrophages to direct their response toward pro- variety of stimuli such as mitogens, hormones, oxidative-DNA liferation or activation. damage cellular stresses, LPS, proinflammatory cytokines, and anti- inflammatory agents (53, 55–58). In addition, depending on the circumstances, MKP-1 dephosphorylates JNK and p38 (2, 49, 59). Discrepancies in the role of MKP-1 are related to the stimuli, the cell type or the kinetics of induction. In our model, we observed that MKP-1 inhibition was correlated with elongated MAPK acti- vation, similar to that described (62). Therefore, in several cells MKP-1 is a crucial regulator of many functions, including prolif- eration, differentiation, activation or apoptosis (2, 63–66). Further- more, MKP-1 has been described as a novel target since it is in- volved in human tumors (67–69). The mechanism used by Raf-1 to induce MKP-1 expression in macrophages probably involves the activation of PKC␧. In this FIGURE 7. Schematic representation of Raf-1 involvement in M-CSF report, we demonstrate that Raf-1 interacts and mediates the acti- and LPS induction of ERK-1/2 kinase phosphorylation and MKP-1 vation of PKC␧ in response to M-CSF and to LPS. In our previous expression. The Journal of Immunology 6601

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