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Mitogen-activated protein (MAP) kinases mediate PMMA-induction of osteoclasts

Posted on: Wednesday, 19 November 2003, 06:00 CST

Abstract

Inflammatory osleolysis induced by implant-derived wear debris is associated with infiltration of various cell-types to the implant- bone interface leading to abundant secretion of pro-inflammatory cytokines and activation of proteinases that together lead to propagation of the localized inflammatory response and periprosthetic bone erosion. Tumor necrosis factor family members are considered to be direct mediators of inflammation and osteolysis. These cytokines exert their osteoclastic effects via activation of the transcription factor NF-[kappa]B and certain MAP kinases, including c-Jun, Erks and p38, all known to be essential for the development of osteoclasts. We have recently documented that the osteoclastogenic cytokines TNF and RANKL play a pivotal role in the development of inflammatory osteolysis. We have also found that PMMA particles stimulate osteoclastogenesis, at least in part, by induction of RANKL, TNF, and by activation of the transcription factor NF-[kappa]B. More importantly, our data indicate that inhibitors of the osteoclastogenic factors, TNF and RANKL abrogate particle-induced osteoclastogenesis. In the current study, we investigated if PMMA particles activate MAP kinases, and the potential role of these kinases as mediators of osteolysis. Using kinase assays, we show that in osteoclast precursors, PMMA particles markedly and rapidly activate p38 and ERK MAP kinases. This activation was specific, evident by complete blockade with specific inhibitory compounds. Similarly, we show that PMMA particles activate the JNK pathway, which is known to be involved in inflammatory and osteoclastogenic events. We also show that p38 MAP kinase regulates PMMA-activation of NF-[kappa]B, thus providing a possible mechanism for particle action in osteoclast precursors. Finally, we provide evidence that specific inhibitors of MAP kinases are capable of inhibiting PMMA-stimulated osteoclastogenesis. These data provide evidence that MAP kinases are potent mediators of particle-induced osteoclastogenesis.

(C) 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: MAP kinases; p38; ERKs; JNK; Osteoclasts; PMMA

Introduction

Aseptic loosening secondary to wear particle-induced osteolysis is the lead cause of failure of hip and knee arthroplasties, and revision surgery is associated with significant cost and potential morbidity [14,15,29]. The pathogenesis of this disease includes at least two major components, inflammatory and osteolytic processes. Wear particulates, composed of polymethylmethacrylate (PMMA), polyethylene (PE) and metals initiate and sustain a chronic inflammatory response at the prosthetic-bone interface [12]. While there is limited information describing the intracellular signaling pathways activated by PMMA particles, the common hypothesis is that at the cellular level the inflammatory response is manifested by recruitment of macrophages/phagocytes. These cells ingest the debris as an initial host defense mechanism, and in response secrete a host of proinflammatory cytokines, which in turn lead to recruitment and activation of ostcoclasts [29]. Furthermore, the inflammatory response phase includes formation of a fibrous tissue (membrane) at the implant-bone interface rich with macrophages and fibroblasts, the latter cells are rich source of osteoclast differentiation factors [15]. This process is supported by ample evidence indicating that more than one billion particles per g of tissue can be recovered from the fibrous membrane adjacent to the failing prosthesis. In addition high levels of cytokines are produced by macrophages/phagocytes in response to particulate exposure in vitro, and by cells recovered from the fibrous tissue following revision of failed implants. Finally, the importance of cytokines in wear debris- induced osteolysis has been established in animal models [14,15].

The osteolytic component of the disease is mediated directly by osteoclast resorbing activity. In this regard, the pro-inflammatory response provides a mixture of cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6, all are well established as required or potent inducers of osteoclast differentiation, recruitment and activation [38], Recent advances have unveiled receptor activator of NF-[kappa]B ligand (RANKL), also known as ODF and OPGL, as a key cytokine of the TNF superfamily secreted by stromal cells, osteoblasts and activated T lymphocytes that control osteoclast differentiation, survival and activation [20,21,25]. In fact, the presence of the cytokine is documented in healthy and in inflamed tissues. In this regard, RANKL was detected in sites of bone erosion, indicating that recruitment of osteoclasts and their precursor cells to inflamed sites is imminent [11,36]. RANKL is also expressed in T cells and fibroblasts isolated from synovial tissues [20,33]. Thus, RANKL plays a key role in recruitment and differentiation of osteoclasts in sites of inflammatory osteolysis. This cytokine exerts its action by mechanisms that appear to be typical to the TNF family [39]. Namely, in a manner similar to TNF, RANKL binding to its receptor RANK on monocyte/macrophage precursor cells and activates key pathways required for osteoclast differentiation and function. In this regard, we have shown recently that these include activation of the transcription factors NF- [kappa]B, c-Jun/AP-1 and other mitogen-activated protein kinase (MAPK) pathways [3,39].

Our recent studies utilizing a murine in vivo calvaria model and in vitro studies have established that PMMA is a potent recruiter of osteoclasts and inducer of osteolysis. In this regard, we have shown that PMMA activates NF- and osteoclastogenesis. These processes are dependent upon TNF and RANKL signaling, as deletion of TNF or inhibition of RANKL signaling with the decoy molecule OPG, dramatically inhibit PMMA-mediated effects [8,9]. Activation of NF- [kappa]B by PMMA particles (whether secondary to RANKLTNF or not) is significant since the transcription factor is involved in the pathogenesis of most inflammatory diseases including osteolysis [5,35]. Another major set of TNF family member-induced transcriptional activities in macrophages and osteoclasts are transmitted by MAP kinases.

MAP kinases are proline-directed serine/threonine kinases that are important in cell growth, differentiation and apoptosis [13,17]. They become activated by phosphorylation on threonine and tyrosine residues in response to external stimuli, such as cellular stresses, mitogens, reactive oxygen species, and cytokines [32]. Three major subfamilies of MAP kinases have been identified in mammalian cells; extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinase (JNKs), and p38 MAP kinases [13,32], A wide range of stimuli leads to activation of up-stream MAP kinases (MKKKs), such as Raf, MEKK1 and ASK1, which in turn activate MAPKKs (MKKs). MKKs activate in turn MAP kinases such as ERKs, P38s and JNK leading to activation of transcription factors. We and others have shown that all three MAP kinase families are regulated by osteoclastogenic factors of the TNF family (TNF and RANKL), and are essential for osteoclast differentiation and activation [28,31,39], On the mechanistic level, evidence indicates that oligomerization or overexpression of TNF receptor associated factor (TRAF)2 and TRAF6, adapter proteins required for RANK and TNF receptor signaling, is sufficient for MEKK1, JNK and p38 activation [6].

Importantly, the activation of both ERK and JNK/ SAPK subfamilies results in the increased expression of c-fos and trans-activation of c-Jun DNA-binding activity, respectively [34]. On the other hand, p38 and ERK MAP kinases have been established as significant participants of cytokine-mediated inflammatory responses. These kinases have been proven as integral part of both acute and chronic inflammation [23]. Furthermore, a key role for p38 MAP kinase in osteoclastogenesis has been established [28]. Taken together with its key role in RANKL-mediated osteoclast formation, our novel observations that PMMA particles rapidly activate c-Jun, p38 and ERK kinase activity indicate that these kinases may play crucial roles in osteolysis. Thus, investigating the molecular steps of PMMA induction of MAPK may provide new insights in the management of osteolysis.

Materials and methods

Reagents

All cytokines were purchased from R&D industries (Minneapolis, MN). MAPK inhibitors are from Calbiochem (San Diego, CA). E-toxate kit was purchased from Sigma (St. Louis, MO). Other chemicals are from Sigma (St. Louis, MO) unless otherwise indicated.

Polymethylmethacrylate particles

Commercially available, gamma radiation-sterilized PMMA particles (Howmedica, Simplex-P) were used for all experiments. The powder component (mixture of PMMA, methyl methacrylate-styrenecopolymer and barium sulfate) of the two component (methyl methacrylate mixture and liquid monomer) bone cement preparation was used as provided in the commercial product without modification. Stock solutions were prepared with [alpha]-MEM at a concentration of 100 nig/ml and treatment concentrations are indicated for each experiment. Scanning electron microscopy revealed a heterogeneous array of particle sizes (range, 220 nm to 60 [mu]m in diameter) and shapes. Less than 5\1% of the particles consisted of relatively large smooth-surfaced microspheres in the 20-60 [mu]m range. The majority (at least 50%) of the particles were smaller in the 220 nm to 1 [mu]m range and had slightly irregular shapes and smooth surfaces. The majority of these submicron particles were in aggregates and are in the biologically active size range when compared to participates isolated from periprosthetic membrane tissue [27]. To test for particle contamination with LPS, a commercially available technique (E- toxate Kit) was employed. No LPS was detected by this assay in this PMMA particle preparation.

Animals

Approval was obtained from Institutional Animal Care and Use Committee prior to performing this study. C3H/HeN 34 week old male mice were purchased from Marian Industries (Indianapolis, IN) and housed at the Washington University School of Medicine barrier facility.

Cell isolation und purification

Bone marrow macrophages (osteoclast precursors) were isolated from whole bone marrow of 4-6 week old mice and incubated in tissue culture plates, at 37 [degrees]C in 5% CO2, in the presence of 10 ng/ ml M-CSF [10]. After 24 h in culture, the non-adherent cells were collected and layered on a Ficoll-Hypaque gradient. Cells at the gradient interface were collected and plated in [alpha]-MEM, supplemented with 10% heatinactivated fetal bovine scrum, at 37 [degrees]C in 5% CO2 in the presence of 10 ng/ml M-CSF, and plated according to each experimental conditions.

Osteoclast generation

Ostcoclasts were generated by culturing purified precursor cells in the presence of M-CSF and soluble RANKL (20 ng/ml each) for four days. Bona fide osteoclasts develop on days 3-4 of culture at a point cells are fixed and TRAP-stained or subjected to further treatments such as exposure to MAP kinase inhibitors and stimulation with PMMA. TRAP-positive (purple color) multinucleated cells (>3 nuclei/ cell) are bona fide osteoclast-like cells capable of resorbing bone wavers [1]. These cells are counted per surface area under light microscope.

MAP kinase assays

Kinase assay kits for the various MAP kinases were purchased from Cell Signaling (Beverly, MA). MAP kinase activity assays were performed using standard kits and according to manufacturer protocols. In short, following activation, cell lysates were prepared and incubated with relevant specific substrate proteins fused to beads. "Pull Down" reactions were carried overnight followed by washing pellets and incubation in kinase buffer in the presence of 100 [mu]M ATP. Reactions were terminated after 30 min incubation at 30 [degrees]C using 3X SDS sample buffer. Boiled samples were analyzed using SDS-PAGE electrophoresis and immunoblots with relevant antibodies.

Immunoblotting

Total cell lysates were boiled in the presence of 2 x SDS-sample buffer (0.5 M Tris-HCl [pH 6.8], 10% (w/v) SDS, 10% glycerol, 0.05% (w/v) bromophenol blue, distilled water) for 5 min and subjected to electrophoresis on 8-12% SDS-PAGE [22]. Proteins were transferred to nitrocellulose membranes using a semi-dry blotter (Bio-Rad, Richmond, CA) and incubated in blocking solution (10% skim milk prepared in PBS containing 0.05% Tween-20), to reduce non-specific binding. Membranes were washed with PBS/Tween buffer and exposed to primary antibodies (1 h at room temperature), washed again four times and incubated with the respective secondary HRP-conjtigated antibodies (l h at room temperature). Membranes were washed extensively (5 x 15 min), and an ECL detection assay was performed following manufacturer's directions.

Electrophoretic mobility shift assay (EMSA)

Nuclear fractions were prepared as previously described [2,4]. In brief, cells were washed twice with ice-cold phosphate-buffered saline. Cells were then lifted from the dish by treating with 5 mM EDTA and 5 mM EGTA in PBS, resuspended in hypotonic lysis buffer A (10 mM HEPES [pH 7.8] 10 nM KCl, 1.5 mM MgCl, 0.5 mM dithiothreitol, 0.5 mM AEBSF, 5 ng/ml Leupeptin) and incubated on ice for 15 min. NP- 40 was added to a final concentration of 0.64% and samples were vortexed. Nuclei were pelleted and the cytosolic fraction was carefully removed. The nuclei were then re-suspended in nuclear extraction buffer B (20 mM HEPES [pH 7.8] 420 mM NaCl, 1.2 mM MgCl, 0.2 mM EDTA 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM AEBSF, 5 [mu]g/ml Pcpstatin A, 5 [mu]g/ml Leupeptin), vortexed for 30 s and rotated for 30 min in 4 [degrees]C. The samples were then centrifuged and the nuclear proteins in the supernatant were transferred to fresh tubes and protein content was measured using standard BCA kit (Pierce, Rockford, IL). Nuclear extracts (10 [mu]g) were incubated with an end-labeled double stranded oligonucleotide probe commercially available from Santa Cruz (Santa Cruz, CA). The reaction was performed in a total of 20 [mu]l of binding buffer (20 mM HEPES [pH 7.8], 100 mM NaCl, 0.5 mM dithiothreitol, 1 [mu]g poly dI-dC, and 10% glycerol) for 15-20 min at room temperature. After incubation with the labeled probe for 30 min, samples were fractionated on a 4% polyacrylamide gel and visualized by exposing dried gel to film. Results are representative of at least three independent experiments.

Results

PMMA particles activate MAP kinases in osteoclast progenitors

PMMA and other inducers/mediators of osteolysis utilize complex signaling cascades. Their action is often superimposed over preexisting pathways such as those transmitted by the prime osteoelast cytokines, RANKL and TNF. The major pathways activated by these osteoclastogenic cytokines are NF-[kappa]B and MAP kinascs. We have established that PMMA particles activate NF-[kappa]B, most likely by superimposed action over the RANKL and TNF pathways [8,9]. Next, we asked if PMMA activates other TNF down-stream signals, especially MAP kinases. Using kinase assays we show that, in osteoclast precursors, similar to TNF (not shown) and RANKL, PMMA particles markedly activate p38 and ERK MAP kinases within 30 min of treatment (Fig. 1) evident by marked increase in kinase activity (KA). These data further show that ERK1 basal activity in osteoclast precursors is significantly lower than that of ERK2. PMMA particles induce ERK1 and ERK2 activities by 9 and 4-folds, respectively. This activity is more profound in the presence of RANKL alone (a respective 15 and 8-fold increase). Further findings indicate that activation of p38 and ERKs that was observed in Fig. 1 appears specific, evident by complete blockade with selective inhibitory compounds (Figs. 2, 3). Specifically, administration of the p38 MAP kinase inhibitor SB203580 (at 1 [mu]M) dampened PMMA, RANKL and TNF activation of the kinase, without significantly affecting p38 protein expression (Fig. 2). This latter observation further substantiates the specific nature of p38 activation by the various inducers. Likewise, we examined the nature of ERK activation by PMMA and other osteoclastogenic agents utilizing the ERK specific inhibitor U0126. Once again, the data shown in Fig. 3 indicate that PMMA (0.2 mg/ml), RANKL (20 ng/ml), and TNF (20 ng/ml) induce kinase activity of both ERK isoforms with substantially more activation of ERK1. Administration of the ERK inhibitor U0126 (at 1 [mu]M) obliterates activation of both ERK isoforms without affecting total protein expression noted by western blot (WB). Notably, however, U0126 while potently inhibiting basal activation of ERK2 (lane 2) and inhibiting RANKL and TNF activation of ERK2, the compound failed to inhibit ERK2 activation by PMMA (lane 4). This observation suggests that PMMA particle stimulation of ERK2 is unique and differs from RANKL and TNF activation of this kinase.

Fig. 1. PMMA particles activate MAP kinases in osteoclast progenitors. Subconfluent (~75%) monolayers of osleoclast precursors (marrow macrophages) were achieved in culture after three days and were treated with PMMA (0.2 mg/ml), or RANKL (20 ng/ml), for 30 min. Cleared cell lysates were then immunoprecipitated with specific anti- bodies for p38 and ERKs and subjected for kinase assays (KA) with specific substrates for the various MAP kinases indicated.

Fig. 2. The p38 MAP kinase selective inhibitor, SB203580, inhibits particle and cytokine-induced activation of the kinase. Osteoclast progenitors were cultured for three days in media and M- CSF. On day four, cells were treated with PMMA, RANKL, or TNF (as indicated in Fig. 1) in the presence of DMSO (control) or 1 [mu]M SB203580 (30 rain). Cultured were then lysed and subjected to p38 kinase assay (KA) and western blot (WB).

Fig. 3. The selective inhibitor U0126 blocks PMMA, RANKL and TNF activation of ERK MAP kinases. Osteoclast progenitors were cultured to subconfluence (three days) and treated with PMMA (0.2 mg/ml), TNF (20 ng/ml), or RANKL (20 ng/ml) for 30 min in the presence of DMSO (control) or U0126 (1 [mu]M). Cells were then lysed and cleared lysates were subjected to kinase assays (KA). A small fraction of the cell lysates was used for total ERK2 protein western blots (WB) to insure equal protein expression under the various treatment conditions.

Blockade of p38 MAPK inhibits NF-[kappa]B activation

We have reported recently that PMMA particles induce osteoclastogenesis at least in part via activation of NF-[kappa]B. Furthermore, we report in this study that MAP kinases including p38 are potent mediators of PMMA-induced osteoclastogenesis. Given that recent reports indicated that p38 MAP kinase may be involved in mediating NF-[kappa]B activation [19,26] and that both p38 and NF- [kappa]B play a major role in osteoclast development [16,28], we reasoned that NF-[kappa]B activation and subsequent osteoclastogenesis may be dependent upon p38 activation. To test this proposition, osteoclast precursors were stimulated with TNF or PMMA particles in the absence or presence of 1 [mu]M SB203580. EMSA of nuclear extracts revealed that inhibition of p38 activity indeed signific\antly decreased NF-[kappa] activation (Fig. 4). Thus, our findings indicate that PMMA particles specifically activate MAP kinases and that p38 may be mediating NF-[kappa]B activation in osteoclast precursor cells.

PMMA particles strongly induce c-Jun/AP-1 DNA-binding activity

Having established that PMMA particles specifically activate p38 and ERK kinases, we turned to investigate whether these particles induce another MAP kinase pathway that regulates osteoclasts and inflammatory processes, namely, c-Jun/AP-1 pathway. First we examined activation of the upstream c-Jun N-terminal kinase (JNK) in response to PMMA particles. The findings clearly indicate that PMMA particles strongly activate JNK. The data presented in Fig. 5 show that while total protein level of JNK is largely unchanged, activity of the kinase is increased as measured by GST-c-Jun substrate phosphorylation. Specifically, we find that PMMA particles induce JNK activity by 8-fold compared to control as early as 30 min postexposure.

Fig. 4. Inhibition of PMMA or TNF-induced p38 MAP kinase inhibits NF-[kappa]B activation. Osteoclast progenitors were cultured as indicated for Fig. 1 and were treated with the p38 inhibitor SB203580 (1 [mu]M/30 min) followed by PMMA particles (0.2 mg/ml/30 min) or TNF (20 ng/ ml/30 min) as shown. Nuclear extracts were subjected to EMSA using ^sup 32^P-labelled [kappa]B3 oligonucleotide. NF-[kappa]B band shift and free probe are indicated.

Fig. 5. PMMA particles induce kinase activity of c-Jun N- terminal kinase (JNK). Osteoclast progenitors (marrow macrophages) were cultured for three days to reach subconfluence and were then treated with PMMA (0.2 mg/ml), TNF (20 ng/ml) or RANKL (20 ng/ml) for the time points indicated. Cells were lysed and cleared lysates were subjected to JNK kinase assay using GST-cJun as a substrate. Upper panel noted with c-Jun indicates activity of JNK. Lower bands noted with "JNK" represent total JNK 1/2 protein expressed/present in the cells under various treatment conditions.

This activation persists up to 2 h and parallels activation of the kinase with TNF and RANKL. We next examined the end point of the JNK activation pathway, namely DNA-binding activity of c-Jun. Electrophoretic mobility shift assay indicates that similar to TNF and RANKL, PMMA particles induce DNA-binding activity of c-Jun in nuclei of osteoclast progenitors (Fig. 6). Activation with PMMA particles was maximal at 60 min postexposure.

Fig. 6. PMMA particles, TNF and RANKL induce DNA-hinding activity of c-Jun/AP-1. Osteoclast progenitors were cultured in the presence of M-CSF (10 ng/ml) for three days to reach subconfluence. Cells were then treated as described for Fig. 5 for the time points shown. Nuclei were isolated and extracts were prepared and subjected to gel shift assay using radiolabeled c-Jun/AP-1 consensus oligonucleotide.

MAP kinases mediate PMMA-indiiced osteoclastogenesis

Next, we asked if selective MAP kinase inhibitors impact PMMA- induced osteoclastogenesis. To this end, ostcoclasts were generated in vitro with RANKL (four days) and then treated for an additional 24 h with TNF or PMMA in the absence or presence of 1 [mu]M of SB203580 (p38 inhibitor) or U0126 (ERK inhibitor) (Fig. 7a). The data indicate that while TNF or PMMA mounts a strong osteoclastogenic response (3.8, 2.9-folds, respectively) as measured by number of TRAPpositive osteoclasts, inclusion of MAP kinase inhibitors significantly reduced this response (Fig. 7b). Specifically, SB203580 and U0126, while exerting a modest inhibitory effect on basal osteoclast formation in the last 24 h of culture (panels D, G, respectively), these compounds reduced TNF-induced osteoclastogenesis by 85% and 78%, respectively (panels E, H). In a similar manner, SB203580 and U0126 reduced PMMA induction of osteoclastogenesis by 72% and 68% (panels F, I). These data provide evidence that MAP kinases are potent mediators of particle-induced osteoclastogenesis.

Discussion

Despite extensive investigations, the precise mechanism of action of PMMA particles in inflammation and osteolysis remains elusive. Over the past few years, evidence gathered indicates that PMMA particles exert pathological effect via intermediate pro- inflammatory molecules. Our previous work suggests that multiple pro- inflammatory and pro-osteoclastogenic cytokines facilitate particle- stimulated osteoclastogenesis and osteolysis. In this regard, our studies provide compelling evidence that TNF and RANKL are considered as essential mediators of particle-stimulated osteoclastogenesis and osteolysis [8,9,29]. In those studies we show that specific interference with either TNF or RANKL signaling results in blockade of particle induction of osteoclast recruitment. We further provide evidence that activation of the transcription factor NF-[kappa]B appears to be central to particle induction of both pathways, i.e. TNF and RANKL-mediated osteoclastogenesis.

Fig. 7. ERK and p38 MAP kinase inhibitors block osteoclastogenesis. Osteoclast progenitors were placed in culture with M-CSF (10 ng/ml) and RANKL (20 ng/ml) for four days to form osteoclasts. Cultures were then incubated for an additional 24 h with TNF (20 ng/ml) (panels B, E, H) or PMMA (0.2 mg/ml) (panels C, F, I) in the presence of DMSO (panels A, B, C), SB203580 (1 [mu]M) (panels D, E, F), or U0126 (1 [mu]M) (panels G, H, I). Cultures were then fixed and stained with TRAP to detect osteoclasts (arrows) (Fig. 7a). Cells with three or more nuclei were then counted in three different wells and three different experiments. The mean average of the various treatment conditions is presented as percent of control (Fig. 7b). Statistical significance for TNF and PMMA- treated conditions compared to control was **p < 0.001. Conditions of TNF+inhibitors and PMMA + inhibitors were also statistically significant (*p < 0.005) compared to TNF and PMMA-treated conditions, respectively.

In other studies investigating signal transduction by osteoclasts, we documented that TNF family members strongly induce MAP kinase-related pathways including c-Jun/JNK and ERKs [39]. In fact, these pathways are directly regulated by TNF and appear essential for normal osteoclast development. These observations are supported by findings indicating that expression levels and activity of MAP kinases were significantly lower in TNF receptor type 1 null osteoclasts. Osteoclasts from TN Fr1-null mice are developmentally retarded and exhibit lesser activity compared to their wild type counterparts [3]. These observations prompted us to further examine a possible mediator role of MAP kinases in particle-stimulated osteolysis. Indeed, our initial studies indicate that PMMA particles activate all three major MAP kinasc pathways. First, we show that osteoclast progenitors express low level activity of ERK2 and nearly undetectable activity of ERKl. Exposure of these cells to particles, TNF or RANKL rapidly induces the activity of both isoforms of ERK. Interestingly, we noted that U0126, a potent inhibitor of ERK kinases, abolishes basal as well as TNF and RANKL-induced activation of ERK1 and ERK2. Conversely, however, while potently blocking particle-induced ERK1 activation, U0126 only partially blocks ERK2 activation (compare lanes 3, 4; Fig. 1). Although the meaning of these differences is unclear at this time, it is possible that particles utilize additional mechanisms distinct from TNF and RANKL.

Similar to NF-[kappa]B, the JNK/c-Jun pathway is also considered as an integral participant in inflammatory responses [17]. Our findings point out that particles are also potent inducer of the JNK/ c-Jun pathway. These events are not isolated or incidental since previous observation described a signaling cross talk between the two pathways, namely NF-[kappa]B and JNK [30,37]. In this regard, it has been shown that JNKl and its activator MEKK1 synergistically activate transcription by [kappa]B-driven promoter. Moreover, JNK1 associates with NF-[kappa]B member c-Rel by immunoprecipitation studies and binds to it directly as shown by yeast two-hybrid system [37]. All together, these observations suggest that JNK/c-Jun pathway may regulate NF-[kappa]B and down stream target genes relevant to inflammation and osteolysis. Thus, our observations suggest that JNK/e-Jun signaling may contribute to development of certain aspects of inflammatory osteolysis. This is also supported by previous reports attributing a role for JNK pathway in the secretion of the inflammatory cytokine TNF [18]. More importantly, JNK actions related to inflammation were proposed to act in concert with other MAP kinases, primarily p38 MAP kinase.

We also document in this study that particles activate p38 MAP kinase in osteoclast progenitors. This finding may not be surprising in light of ample reports describing the prominent role of this kinase in inflammatory responses and osteoclastogenesis [13,24,28,32]. Nevertheless, we provide direct evidence that p38 MAP kinase is directly and specifically involved in PMMA-induced activation of osteoclast precursors and osteoclastogenesis. More importantly, however, we provide evidence that p38 might be involved in regulating NF-[kappa]B activation, which we have shown in earlier studies as an essential mediator of particle-induced osteoclastogenesis [9]. Just like in the case of JNK pathway, a likely regulation of NF-[kappa]B by p38 provides yet another regulatory mechanism by which inflammatory responses arc orchestrated. Support for this notion comes from other studies in which pharmacological inhibitors of p38 blocked RANKL-mediated osteoclastogenesis [28] and Akt-mediated activation of NF-[kappa]B [26].

Perhaps the most compelling evidence for functional involvement of MAP kinases in particle signaling in ostcoclasts is the fact that induction of osteoclastogenesis by particles is significantly reduced in the presence of selective MAP kinase inhibitors\. Our data clearly show that PMMA-induced osteoclastogenesis is reduced by p38 and ERK inhibitors. Data regarding JNK inhibitory studies are not available at this time due to lack of commercial inhibitors. However, a recent study using a synthetic inhibitor (not available commercially) of the JNK pathway demonstrated that selective inhibition of JNK inhibited phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-[gamma], TNF, and blocked lipopolysaccharide-induced expression of TNF [7].

In summary our data demonstrate that in osteoclast precursors, PMMA particles activate p38, JNK, and ERK MAP kinase pathways, enhance cJun/AP-1 DNA binding activity, and that particles and TNF- mediated-activation of NF-[kappa]B is at least in part regulated by p38 MAP kinase. More importantly, selective MAP kinase inhibitors significantly inhibit the osteoclastogenic effect of PMMA particles and TNF. Therefore, these observations underscore the complex signaling mechanisms activated in particle-stimulated osteoclast precursor cells. In light of these findings, MAP kinases present themselves as potential mediators of particle-induced osteoclastogenesis and as such targets for inhibiting this phenomenon.

Acknowledgements

This study was supported by NIH grants DE 13754, AR 47443 (YAA), AR 47096 (JCC), and by grants from the Shriners Hospital and the Arthritis Foundation (YAA).

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S. Abbas(a), J.C. Clohisy(a), Y. Abu-Amer(a,b,*)

a Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA

b Department of Cell Biology & Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA

Received 6 December 2002; accepted 24 March 2003

* Corresponding author. Address: Department of Orthopaedics Surgery, Washington University School of Medicine, One Barnes Hospital Plaza, 1130 West Pavillion, Campus 8283, St. Louis, MO 63110, USA. Tel.: +1-314-362-0335; fax: +1-314-362-0334.

E-mail address: abuamery@msnotes.wustl.edu (Y. Abu-Amer).

Copyright Journal of Bone and Joint Surgery, Inc. Nov 2003

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