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Endocrinology, doi:10.1210/en.2003-0166
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Endocrinology Vol. 144, No. 11 4999-5005
Copyright © 2003 by The Endocrine Society

p38 Mitogen-Activated Protein Kinase Is Crucially Involved in Osteoclast Differentiation But Not in Cytokine Production, Phagocytosis, or Dendritic Cell Differentiation of Bone Marrow Macrophages

Xiaotong Li, Nobuyuki Udagawa, Masamichi Takami, Nobuaki Sato, Yasuhiro Kobayashi and Naoyuki Takahashi

Institute for Oral Science (X.L., Y.K., N.T.), Department of Biochemistry (N.U., N.S.), Matsumoto Dental University, Nagano 399-0781, Japan; Department of Biochemistry (M.T.), School of Dentistry, Showa University, Tokyo 142-8555, Japan; and Department of Periodontology (N.S.), School of Dentistry, Aichi Gakuin University, Aichi 464-8651, Japan

Address all correspondence and requests for reprints to: Naoyuki Takahashi, Ph.D., Institute for Oral Science, Matsumoto Dental University, 1780 Gobara, Hiro-oka, Shiojiri, Nagano 399-0781, Japan. E-mail: takahashinao{at}po.mdu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that p38 MAPK signaling is required for osteoclast differentiation but not osteoclast function. Here we further investigated the role of p38 MAPK in the function and differentiation of mouse bone marrow macrophages (BMM{phi}), common precursors of osteoclasts and dendritic cells. Lipopolysaccharide (LPS) activated the p38 MAPK signaling pathway in BMM{phi} by sequential phosphorylation of MAPK kinase 3/6, p38 MAPK, and activating transcription factor-2. Treatment of BMM{phi} with SB203580, a p38 MAPK inhibitor, suppressed LPS-induced phosphorylation of activating transcription factor-2. LPS stimulated production of IL-1ß, TNF{alpha}, and IL-6 in BMM{phi}, and SB203580 failed to inhibit the LPS-induced cytokine production. BMM{phi} incorporated latex beads via phagocytosis, and SB203580 had no effect on this phagocytosis. BMM{phi} differentiated into dendritic cells when treated with granulocyte macrophage colony-stimulating factor together with CD40 ligand, TNF{alpha}, or LPS, and SB203580 failed to inhibit this differentiation. Thus, p38 MAPK-mediated signals are not involved in either BMM{phi} function or BMM{phi} differentiation into dendritic cells. The differentiation of BMM{phi} into osteoclasts in response to receptor activator of nuclear factor-{kappa}B ligand or TNF{alpha} was strongly inhibited by SB203580. These findings emphasize the crucial roles of p38 MAPK-mediated signaling in osteoclast differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HEMOPOIETIC CELLS OF the monocyte-macrophage lineage differentiate into bone-resorbing osteoclasts under the control of osteoblasts or bone marrow stromal cells (1, 2, 3, 4). Osteoblasts/stromal cells express two factors essential for osteoclastogenesis, RANKL [receptor activator of nuclear factor-{kappa}B (RANK) ligand] and macrophage colony-stimulating factor (M-CSF) (1, 2, 3, 4). RANKL together with M-CSF induces osteoclast formation from monocytes/macrophages in the absence of osteoblasts/stromal cells. Mature osteoclasts as well as osteoclast precursors express RANK, a receptor of RANKL, and RANKL stimulates the survival, fusion and bone-resorbing activity of osteoclasts. TNF{alpha} has been shown to stimulate osteoclast differentiation from monocytes/macrophages through a mechanism independent of RANKL-RANK interaction (5, 6).

Monocytes/macrophages play critical roles in innate defenses against viral and bacterial infections. They produce proinflammatory cytokines such as IL-1ß, TNF{alpha}, and IL-6 in response to viral and bacterial constituents (7, 8, 9). These cytokines activate the immune system to defend the host from infections. Another important function by which macrophages fight infectious diseases is phagocytosis (10). Macrophages show strong phagocytic activity against foreign substances. In addition, monocyte/macrophage-lineage cells can give rise not only to osteoclasts but also to dendritic cells, antigen-presenting immune cells (11, 12, 13). Monocyte/macrophage lineage cells differentiate into immature dendritic cells in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) (14, 15). CD40 ligand (CD40L), TNF{alpha}, or lipopolysaccharide (LPS) then stimulates the maturation of GM-CSF-induced immature dendritic cells (16).

The p38 MAPK family is a group of 38-kDa intracellular signal transduction proteins (17, 18, 19). c-Jun N-terminal kinase and ERK, together with p38 MAPK, form the MAPK family (19). p38 MAPK is predominantly activated through its phosphorylation by upstream MAPK kinase 3 (MKK3) and MKK6 (17, 18). Activated p38 MAPK then phosphorylates downstream targets, including activating transcription factor-2 (ATF-2). SB203580, a specific inhibitor of p38 MAPK, has been widely used to investigate the roles of p38 MAPK in the differentiation and function of cells (17, 18, 19). SB203580 binds to the ATP pocket of the activated p38 MAPK and inhibits phosphorylation of the downstream targets (19). Recent studies have shown that signals mediated by p38 MAPK are involved in the differentiation of chondrocytes and epithelial cells (20, 21). Kumar et al. (22) also reported that SB203580 inhibited IL-6 production induced by IL-1 and TNF{alpha} in osteoblasts and chondrocytes. These results suggest that p38 MAPK-mediated signals are involved in cell differentiation and function.

We and others (23, 24) have reported that SB203580 inhibits RANKL-induced differentiation of precursor cells into osteoclasts, Interestingly, it was previously found that SB203580 fails to inhibit the survival and bone-resorption activity of osteoclasts induced by RANKL. Moreover, p38 MAPK is phosphorylated in response to RANKL, IL-1, TNF{alpha}, and LPS in bone marrow macrophages (BMM{phi}), which are the osteoclast precursors, but not in mature osteoclasts (23). We therefore concluded that p38 MAPK-mediated signals are required for osteoclast differentiation but not osteoclast function. It was also shown that RANKL is a survival factor of dendritic cells (25).

In the present study, we further investigated the role of p38 MAPK-mediated signaling in the function and differentiation of BMM{phi}, common precursors of osteoclasts and dendritic cells. We showed here that inhibition of p38 MAPK activity by SB203580 in BMM{phi} did not suppress the cytokine production or phagocytic activity of BMM{phi}. SB203580 failed to inhibit dendritic cell differentiation of BMM{phi}, whereas it strongly inhibited the induction of BMM{phi} differentiation into osteoclasts by RANKL and TNF{alpha}. These findings suggest that p38 MAPK-mediated signaling is crucially involved in osteoclast differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and chemicals
Five- to 8-wk-old male ddY mice were obtained from Shizuoka Laboratories Animal Center (Shizuoka, Japan). Three- to 4-wk-old C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All procedures for animal care were approved by the Animal Management Committee of Matsumoto Dental University. Recombinant human M-CSF (Leukoprol) was obtained from Kyowa Hakko Kogyo Co. (Tokyo, Japan). Recombinant murine GM-CSF and soluble RANKL were purchased from PeproTech EC Ltd. (London, UK). Mouse TNF{alpha} was obtained from Genzyme TECHNE (Minneapolis, MN). Mouse CD8-conjugated CD40L in insect cell culture supernatant was provided by Dr. Yongwon Choi (University of Pennsylvania, Philadelphia, PA). LPS was purified from Escherichia coli strain K235 as described (26). SB203580 was purchased from Calbiochem Co. (La Jolla, CA). Latex beads (0.75-µm microspheres, 2.68%) were from Polysciences, Inc. (Warrington, PA). Rabbit polyclonal antibodies against phospho-p38 MAPK, p38 MAPK, phospho-MKK3/6, MKK3, phospho-ATF-2, ATF-2, phospho-ERK, and ERK were purchased from Cell Signaling Technology, Inc. (Beverly, MA). ELISA kits for mouse IL-1ß and TNF{alpha} were obtained from Genzyme TECHNE, and that for mouse IL-6 was obtained from ENDOGEN (Woburn, MA). Specific PCR primers for mouse IL-1ß, IL-6, TNF{alpha}, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by Life Technologies, Inc. (Tokyo, Japan). Other chemicals and reagents were of analytical grade.

Preparation of mouse bone marrow macrophages
Bone marrow cells obtained from tibiae of 5- to 8-wk-old adult mice were suspended in {alpha}MEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS) in 60-mm diameter dishes for 16 h in the presence of M-CSF (100 ng/ml) (5). Then, nonadherent cells were harvested and further cultured for 2 d with M-CSF (100 ng/ml). Nonadherent cells were completely removed from the cultures by pipetting. The adherent cells, almost all of which expressed macrophage-specific antigens such as Mac-1, Moma-2, and F4/80, were used as BMM{phi} (5). Usually, bone marrow cells from three animals were pooled and used for the BMM{phi} preparation in each experiment.

Osteoclast differentiation
BMM{phi} were further cultured for 4 d with M-CSF (100 ng/ml) together with RANKL (100 ng/ml) or TNF{alpha} (20 ng/ml) in 48-well plates in the presence or absence of SB203580 at 10-7 M or 10-6 M. Cells were then fixed and stained for tartrate-resistant acid phosphatase (TRAP; a marker enzyme of osteoclasts) as described (27). TRAP-positive multinucleated cells judged by microscopic examination contain three or more nuclei were counted as osteoclasts. The number of osteoclasts formed in the culture varied greatly in each experiment (28). Therefore, the results obtained from one experiment typical of three independent experiments were expressed as the means ± SD of three cultures. The significance of the differences was determined using Student’s t test.

Cytokine production and phagocytosis assay
BMM{phi} were further cultured for 48 h with vehicle (control) or LPS (1 µg/ml). Some cultures were pretreated with SB203580 (10-6 M) overnight in the presence of M-CSF (100 ng/ml). The concentrations of IL-1ß, TNF{alpha}, and IL-6 in the conditioned medium were determined using the respective ELISA kits. BMM{phi} were cultured on 18-mm coverslips in 12-well plates at 105 cells/1 ml/well with M-CSF (100 ng/ml) in the presence or absence of SB203580 at 10-6 M. BMM{phi} were further maintained in serum-free {alpha}MEM for 4 h, and then latex beads (1:500 dilution) were added to each well for 10 min, 20 min, 40 min, or 1 h. The cells on the coverslips were then rinsed twice with cold PBS, fixed with methanol and stained with Giemsa’s solution. Cells containing latex beads were counted as bead-positive cells. Phagocytic activity of macrophages was measured as the percentage of bead-positive cells among the total cells. The results obtained from one typical experiment of three independent experiments were expressed as the means ± SD of three cultures.

Dendritic cell differentiation
Bone marrow cells prepared from C57BL/6J mice were suspended in {alpha}MEM supplemented with 10% FBS in 100-mm diameter dishes (107 cells/10 ml/dish) in the presence of M-CSF (30 ng/ml). After the cells were cultured for 2 d, the adherent cells were harvested by treatment with 0.05% trypsin and EDTA (Life Technologies, Inc.) for 5 min. The harvested cells were resuspended in RPMI 1640 medium (Sigma) supplemented with 5% FBS, because dendritic cell differentiation from BMM{phi} is generally examined in this culture condition (29). At d 0, cells were seeded in 24-well plates (2 x 105 cells/0.5 ml/well) with GM-CSF (10 ng/ml) in the presence of increasing concentrations of SB203580. On d 2, two thirds of the culture supernatant was replaced with fresh medium containing the same concentrations of GM-CSF and SB203580. On d 4, another milliliter of fresh medium containing CD40L (1:500 dilution of the original solution), TNF{alpha} (20 ng/ml final concentration) or LPS (1 µg/ml final concentration) was added to some cultures to stimulate the maturation of dendritic cells. After further culturing for 2 d, the cells were stained with biotin-conjugated anti-CD11c antibody followed by streptavidin-fluorescein isothiocyanate and phycoerythrin-conjugated CD86 antibody (all from BD PharMingen, San Diego, CA). The stained cells were analyzed by fluorescence-activated cell sorting as described (29).

PCR amplification of reverse-transcribed mRNA
BMM{phi} were cultured for 24 h in {alpha}MEM containing 10% FBS with vehicle (control) or LPS (1 µg/ml) in the presence or absence of SB203580 (10-6 M) on 60-mm diameter dishes. Some cultures of BMM{phi} were pretreated with SB203580 (10-6 M) for overnight in the presence of M-CSF (100 ng/ml). Total cellular RNA was then extracted using TRIzol solution (Life Technologies, Inc.). First-strand cDNA was synthesized from total RNA with random primers and subjected to PCR amplification with Ex Taq polymerase (Takara Biochemicals, Shiga, Japan) using the following specific PCR primers: mouse IL-1ß, 5'-AAGCTCTCCACCTCAATGGA-3' (forward, nucleotides 431–450) and 5'-TGCTTGAGAGGTGCTGATGT-3' (reverse, nucleotides 713–732); mouse TNF{alpha}, 5'-ACGTGGAACTGGCAGAAGAG-3' (forward, nucleotides 26–45) and 5'-TGGAAGACTCCTCCCAGGTA-3' (reverse, nucleotides 589–608); mouse IL-6, 5'-TTCCATCCAGTTGCCTTCTT-3' (forward, nucleotides 26–45) and 5'-TCTTGGTCCTTAGCCACTCC-3' (reverse, nucleotides 546–565); and mouse GAPDH, 5'-ACCACAGTCCATGCCATCAC-3' (forward, nucleotides 566–585) and 5'-TCCACCACCCTGTTGCTGTA-3' (reverse, nucleotides 998–1017). The PCR products were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining with UV light illumination. The sizes of the PCR products for mouse IL-1ß, TNF{alpha}, IL-6, and GAPDH were 302, 583, 541, and 452 bp, respectively.

Western blot analysis
BMM{phi} prepared in 60-mm diameter dishes were further incubated with vehicle (control), or LPS (1 µg/ml) in the presence or absence of SB203580 (10-6 M) for 30 min, and then washed twice with PBS and lysed in cell lysate buffer [62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10.2% 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue]. Some cultures of BMM{phi} were pretreated with SB203580 (10-6 M) overnight in the presence of M-CSF (100 ng/ml). Whole cell extracts were electrophoresed on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Millipore, Bedford, MA). After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), the anti-phospho-MKK3/6 antibody, anti-MKK3 antibody, anti-phospho-p38 MAPK antibody, anti-p38 MAPK antibody, anti-phospho-ATF2 antibody, anti-ATF2 antibody, anti-phospho-ERK antibody or anti-ERK antibody (1:1000 dilution) was added in TBS-T containing 5% BSA, and the bound antibodies were visualized by using the enhanced chemiluminescence (ECL) assay with reagents from Amersham Co. (Arlington Heights, IL) followed by exposure to x-ray film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BMM{phi} were prepared from mouse bone marrow cultures treated with M-CSF for 3 d. When BMM{phi} were treated with RANKL and M-CSF, more than 70% of BMM{phi} differentiated into TRAP-positive mononuclear cells and multinucleated cells within 4 d. SB203580, a specific inhibitor of p38 MAPK, strongly inhibited the induction of osteoclast differentiation by RANKL (see Table 1Go). We first examined whether SB203580 specifically blocked p38 MAPK signaling in BMM{phi} (Fig. 1Go). SB203580 is known to bind to the ATP binding site of the activated p38 MAPK and to inhibit the kinase activity (19). We found that MKK3/6, p38 MAPK, ATF2, and ERK were markedly phosphorylated in BMM{phi} treated with LPS for 30 min. We previously showed that RANKL and TNF{alpha} as well as LPS stimulate phosphorylation of p38 MAPK in BMM{phi} (24). SB203580 did not inhibit LPS-induced phosphorylation of p38 MAPK, ERK, or MKK3/6, which is the upstream activator of p38 MAPK, but strongly suppressed the phosphorylation of ATF2, which is one of the downstream targets of p38 MAPK (Fig. 1Go). The total amounts of the MKK3, p38 MAPK, ERK, and ATF2 proteins in BMM{phi} remained unchanged in the presence and absence of LPS and/or SB203580 (Fig. 1Go). Altogether, these results demonstrate that SB203580 inhibited p38 MAPK-mediated signaling in BMM{phi}.


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  		TABLE 1. Effect of SB203580 on CD40L-induced dendritic cell maturation1

 


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  		FIG. 1. Effect of SB203580 on the phosphorylation of MKK3/6, p38 MAPK, ATF2, and ERK induced by LPS in BMM{phi}. Bone marrow cells were cultured for 16 h with M-CSF (100 ng/ml) as described in Materials and Methods. Then nonadherent cells were harvested and further cultured with M-CSF (100 ng/ml). After culturing for 2 d, nonadherent cells were carefully removed, and adherent cells were used as BMM{phi}. Some cultures of BMM{phi} were pretreated with SB203580 (10-6 M) overnight in the presence of M-CSF (100 ng/ml). BMM{phi} were then incubated with LPS (1 µg/ml) for 30 min in the presence or absence of SB203580 (10-6 M), and total-cell lysates were prepared. The lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with the indicated antibodies (1:1000 dilution).

 
The role of p38 MAPK-mediated signaling in BMM{phi} function was then examined using SB203580. Treatment of BMM{phi} with LPS for 48 h markedly stimulated the production of proinflammatory cytokines such as IL-1ß, TNF{alpha}, and IL-6 in BMM{phi} (Fig. 2Go, A–C). BMM{phi} were preincubated with SB203580 overnight and further treated with LPS together with SB203580. The SB compound showed no inhibitory effect on the LPS-induced production of IL-1ß and TNF{alpha} in BMM{phi} (Fig. 2Go, A and B). RT-PCR analysis confirmed that the expression of IL-1ß and TNF{alpha} mRNAs in BMM{phi} was up-regulated by LPS, and SB203580 showed no inhibitory effects on the mRNA expression (Fig. 2DGo). There was a downward trend in IL-6 production in the presence of SB203580 at both the mRNA and protein levels, but the inhibitory effect of SB203580 was not marked (Fig. 2Go, C and D). These results suggest that p38 MAPK activity is not crucially involved in the production of these cytokines in BMM{phi}.



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  		FIG. 2. Effect of SB203580 on the production of IL-1ß, TNF{alpha}, and IL-6 induced by LPS in mouse BMM{phi}. BMM{phi} were prepared in 48-well plates. Some cultures of BMM{phi} were pretreated with SB203580 (10-6 M) overnight. The BMM{phi} were then incubated with or without LPS (1 µg/ml) for 48 h in the presence or absence of SB203580 (10-6 M). The conditioned medium of each well was collected and the concentrations of IL-1ß (A), TNF{alpha} (B), and IL-6 (C) in the conditioned medium were measured using the respective ELISA kits. The results were expressed as the means ± SD of three cultures. D, BMM{phi} were prepared in 60 mm diameter dishes. Some cultures of BMM{phi} were also pretreated with SB203580 (10-6 M) for overnight. The BMM{phi} were then incubated for 24 h with LPS (1 µg/ml) in the presence or absence of SB203580 (10-6 M). Total RNA was then extracted from BMM{phi}, and the expression of IL-1ß, TNF{alpha}, IL-6, and GAPDH mRNAs was analyzed by RT-PCR. The PCR products for IL-1ß, TNF{alpha}, IL-6, and GAPDH were 302, 583, 541, and 452 bp, respectively. a, Significantly different from the culture treated with LPS; P < 0.05.

 
Macrophages can ingest great quantities of latex beads by phagocytosis. We next examined whether phagocytosis of BMM{phi} is affected by the treatment of BMM{phi} with SB203580. BMM{phi} were incubated with latex beads for 10–60 min, and the BMM{phi} that incorporated latex beads were counted as phagocytic cells (Fig. 3AGo). The number of BMM{phi} incorporating latex leads increased as the incubation period increased. Almost all BMM{phi} showed phagocytic activity within 60 min (Fig. 3AGo). The time course of changes in the phagocytosis of BMM{phi} was not affected by the addition of SB203580. The phagocytic activity of BMM{phi} was also evaluated after incubation for 40 min. The proportions of BMM{phi} incorporating more than 100 latex beads, 50–100 beads, and less than 50 beads were about 10%, 25%, and 50% of total BMM{phi}, respectively (Fig. 3Go, B and C). Again, SB203580 failed to inhibit the phagocytic activity of BMM{phi}.



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  		FIG. 3. Effect of SB203580 on the phagocytosis of BMM{phi}. BMM{phi} were prepared on 18-mm diameter glass coverslips in 12-well plates. BMM{phi} were then maintained in serum-free medium for 4 h. Some cultures of BMM{phi} were preincubated with SB203580 (10-6 M) for overnight, and maintained in serum-free medium in the presence of SB203580. BMM{phi} were incubated with latex beads for 10 min, 20 min, 40 min, or 1 h in the presence or absence of SB203580 (10-6 M). BMM{phi} on coverslips were then fixed and stained with Giemsa’s solution, and observed by microscopic examination. A, Time course of changes of phagocytic activity in BMM{phi} treated or not treated with SB203580. Cells incorporating beads were counted as bead-positive cells. The percentage of bead-positive cells was calculated at the indicated time points. The SD was small enough to be included at each mean point. B, Effect of SB203580 on phagocytic activity of BMM{phi}. BMM{phi} were incubated with latex beads for 40 min. Cells incorporating beads were counted as bead-positive cells, and divided into three groups according to the number of beads incorporated (>100 beads, 50–100 beads, <50 beads) in each cell. The percentage of bead-positive cells was calculated. C, Dark- and bright-field images of BMM{phi} treated or not treated with SB203580. BMM{phi} were incubated with latex beads for 40 min. Latex beads incorporated in BMM{phi} appeared as bright green dots in the dark field with UV light illumination (upper panels). Nuclei of BMM{phi} appeared in purple in the bright field (lower panels). Bar, 50 µm.

 
Finally, we examined the possible involvement of p38 MAPK-mediated signaling in dendritic cell differentiation, because dendritic cells differentiate from the same precursors as osteoclasts. BMM{phi} were cultured for 4 d in the presence of GM-CSF to induce dendritic cell differentiation. The cells were further cultured for 2 d in the presence of GM-CSF with or without CD40L to induce their maturation (Fig. 4AGo). Without CD40L stimulation, the majority of the cells were CD11c (a marker of dendritic cells) positive and CD86 (a marker of mature dendritic cells) negative (Fig. 4AGo, upper panels). With CD40L stimulation, the majority of the cells were CD11c and CD86 double positive (Fig. 4AGo, lower panels). SB203580 at 10-7 M to 3 x 10-6 M had no effect on the expression of either CD11c or CD86 (Fig. 4AGo, upper panels). Treatment with TNF{alpha} or LPS similarly stimulated the maturation of dendritic cells in the presence of GM-CSF (Fig. 4BGo). The induction of dendritic cell maturation by TNF{alpha} and LPS was not affected by the addition of SB203580 (Fig. 4BGo).



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  		FIG. 4. Effect of SB203580 on the differentiation and maturation of dendritic cells. BMM{phi} were cultured for 4 d in 24-well plates (2 x 105 cells/1 ml/well) with GM-CSF (10 ng/ml) to induce the dendritic cell differentiation in the presence of SB203580 at the indicated doses. Then, the culture was further treated for 2 d with or without CD40L (1:1000 dilution of the original solution), TNF{alpha} (20 ng/ml) or LPS (1 µg/ml) to stimulate the maturation of dendritic cells in the presence or absence of SB203580. Cells were stained with antibodies for CD11c and CD86, and analyzed by fluorescence-activated cell sorting. Dot plots show the expression of CD11c and CD86 on the cells (upper panels in A). Histograms show the distribution of CD86-positive cells in the CD11c-positive cell population (lower panels in A and B).

 
The effect of SB203580 on CD40L-induced dendritic cell differentiation was compared with that on RANKL- and TNF{alpha}-induced osteoclast differentiation. CD40L markedly increased the number of CD86-positive mature dendritic cells (Table 1Go). The induction of dendritic cell maturation by CD40L was not affected by SB203580 even at 10-6 M. Treatment of BMM{phi} with RANKL or TNF{alpha} stimulated osteoclast formation (Table 2Go). SB203580, even at 10-7 M, inhibited the induction of osteoclastic differentiation of BMM{phi} by RANKL (33.4 ± 15.4% of the control, the mean ± SD of experiments 1–3) and TNF{alpha} (45.6 ± 11.7% of the control, the mean ± SD of experiments 4–6). SB203580 at 10-7 M showed no inhibitory effect on the dendritic cell differentiation induced by CD40L, TNF{alpha} or LPS (Fig. 4Go). Neither cytokine production nor phagocytic activity of BMM{phi} was significantly inhibited by 10-7 M SB2035807 (data not shown).


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  		TABLE 2. Effect of SB203580 on RANKL- and TNF{alpha}-induced osteoclast differentiation1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using SB203580, a specific inhibitor of p38 MAPK, we investigated the roles of p38 MAPK in the function and differentiation of BMM{phi}, the common precursors of osteoclasts and dendritic cells. Pretreatment of BMM{phi} with SB203580 inhibited the induction of ATF2 phosphorylation by LPS, suggesting that SB203580 shuts off the p38 MAPK-mediated signaling in BMM{phi}. However, SB203580 failed to inhibit LPS-induced production of IL-1ß and TNF{alpha} by BMM{phi}. LPS-induced IL-6 production in BMM{phi} was only slightly inhibited by SB203580. Moreover, SB203580 had no inhibitory effect on the phagocytic activity of BMM{phi}. BMM{phi} differentiated into dendritic cells in the presence of GM-CSF, and matured in response to the stimulation by CD40L, TNF{alpha} or LPS. SB203580 again failed to inhibit the differentiation and maturation of dendritic cells. In the present study, dendritic cell differentiation was examined in cultures of BMM{phi} derived from C57BL/6J mice, whereas osteoclast differentiation was examined in cultures of BMM{phi} from ddY mice. We have confirmed that BMM{phi} derived from C57BL/6J mice differentiated similarly into osteoclasts in response to RANKL or TNF{alpha}, and the osteoclast differentiation was strongly inhibited by SB203580. These results suggest that p38 MAPK-mediated signaling is not involved in either BMM{phi} functions such as cytokine production and phagocytosis or dendritic cell differentiation from the common precursors of osteoclasts and dendritic cells.

The role of p38 MAPK in cytokine production and cell differentiation is highly controversial. Baldassare et al. (30) reported that SB203580 inhibited the induction of IL-1ß production by LPS in RAW264.7 cells and J774 cells, but SB203580 did not alter IL-1ß mRNA levels in another ANA-1 macrophage cell line. Rawadi et al. (31) showed that Raw 264.7 cells produced IL-1ß, TNF{alpha}, and IL-6 in response to Mycoplasma fermentans membrane lipoproteins, and this production was strongly inhibited by SB203580. In contrast, peritoneal macrophages obtained from MKK3-deficient mice, in which p38 MAPK activation was diminished, produced TNF{alpha} normally in response to LPS (32). Yosimichi et al. (20) reported that connective tissue growth factor stimulated both differentiation and proliferation of chondrocytes, and the differentiation of chondrocytes induced by connective tissue growth factor was selectively inhibited by SB203580. Bhowick et al. (21) also reported that TGFß induced epithelial to mesenchymal transdifferentiation in mammary epithelial cells, and inhibition of p38 MAPK activity by the expression of a dominant-negative p38 MAPK blocked epithelial to mesenchymal transdifferentiation induced by TGFß. In contrast, SB203580 has been shown to enhance monocytic differentiation of HL60 cells induced by 1,25-dihydroxyvitamin D3 (33). These results suggest that the role of p38 MAPK differs in different types of cells and/or different physiological states. The divergence in the effects of SB203580 on several cell types may be due to differences in isoforms of p38 MAPK involved in the differentiation of each cell type. SB203580 has been shown to inhibit p38 MAPK, p38{alpha}, and p38ß2, but not p38{gamma} (34, 35). It is also possible that the role of p38 MAPK may differ in cells of different origin and/or different states. In some cells, the target of p38 MAPK will function at the translational level (36, 37), whereas in others, its target will function at the transcriptional level (19, 30). Further studies will be necessary to elucidate the different roles of p38 MAPK in cytokine production and cell differentiation.

CD40-mediated signals have been shown to be quite similar to those of RANK (38). Nevertheless, CD40L-induced maturation of dendritic cells was not suppressed by the addition of SB203580. Like RANKL, TNF{alpha} has been shown to stimulate osteoclast differentiation from monocyte/macrophage lineage cells, including BMM{phi} (5, 6). TNF{alpha}-induced osteoclast formation in BMM{phi} was strongly inhibited by SB203580 even at 10-7 M. Matsumoto et al. (23) also reported that treatment of RAW264.7 cells with SB203580 inhibited TNF{alpha}-induced osteoclast differentiation. Thus, the p38 MAPK pathway plays a crucial role in not only RANKL-mediated but also TNF{alpha}-mediated osteoclast differentiation. In contrast to its effect on osteoclast differentiation, SB203580 failed to inhibit the induction of dendritic cell differentiation and maturation by TNF{alpha} as well as CD40L. The induction of human osteoclast formation by RANKL plus M-CSF from CD14-positive cells in peripheral blood was also inhibited by the addition of SB203580 (data not shown). These results emphasize the importance of p38 MAPK-mediated signaling in the differentiation of osteoclasts but not dendritic cells from their common precursors. Recently, a transcription factor, NFAT2 (NFATc1), has been shown to play a critical role in the differentiation of osteoclasts induced by RANKL (39, 40). Therefore, it is proposed that the p38 MAPK signaling is involved in the induction of NFAT2 activation.

We previously reported that osteoclasts expressed a certain amount of p38 MAPK but failed to phosphorylate p38 MAPK in response to any of the stimuli examined (24). LPS did not induce the phosphorylation of MKK3/6 or ATF2 in osteoclasts, suggesting that the p38 MAPK signaling pathway is not entirely functional in osteoclasts. These findings raise novel questions concerning the role of p38 MAPK in osteoclast differentiation and function: why is p38 MAPK-mediated signaling important for osteoclast but not dendritic cell differentiation? How is the p38 MAPK signaling pathway selectively shut down in osteoclasts after the completion of their differentiation? Further studies on p38 MAPK-mediated events in osteoclast precursors will provide new insights into the regulatory mechanisms of osteoclast differentiation and function.


   Footnotes
 
This work was supported in part by Grants-in-Aid 12137209, 13557155, and 13470394 and a Grant for Asian Researchers awarded by the Tokyo Biochemical Research Foundation, Japan.

Abbreviations: ATF2, Activating transcription factor-2; BMM{phi}, bone marrow macrophages; CD40L, CD40 ligand; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte macrophage colony-stimulating factor; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MKK, MAPK kinase; RANK, receptor activator of nuclear factor-{kappa}B ligand; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase.

Received February 3, 2003.

Accepted for publication July 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ 1999 Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357[Abstract/Free Full Text]
  2. Roodman GD 1999 Cell biology of the osteoclast. Exp Hematol 27:1229–1241[CrossRef][Medline]
  3. Teitelbaum SL 2000 Bone resorption by osteoclasts. Science 289:1504–1508[Abstract/Free Full Text]
  4. Chambers TJ 2000 Regulation of the differentiation and function of osteoclasts. J Pathol 192:4–13[CrossRef][Medline]
  5. Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S, Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Morinaga T, Higashio K, Martin TJ, Suda T 2000 Tumor necrosis factor {alpha} stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 191:275–286[Abstract/Free Full Text]
  6. Azuma Y, Kaji K, Katogi R, Takeshita S, Kudo A 2000 Tumor necrosis factor-{alpha} induces differentiation of and bone resorption by osteoclasts. J Biol Chem 275:4858–4864[Abstract/Free Full Text]
  7. Lynn WA, Golenbock DT 1992 Lipopolysaccharide antagonists. Immunol Today 13:271–276[CrossRef][Medline]
  8. Ulevitch RJ, Tobias PS 1995 Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 13:437–457[CrossRef][Medline]
  9. Hla T, Lee MJ, Ancellin N, Paik JH, Kluk MJ 2001 Lysophospholipids-receptor revelations. Science 294:1875–1878[Abstract/Free Full Text]
  10. Djaldetti M, Salman H, Bergman M, Djaldetti R, Bessler H 2002 Phagocytosis—the mighty weapon of the silent warriors. Microsc Res Tech 57:421–431[CrossRef][Medline]
  11. Heath WR, Carbone FR 2001 Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 19:47–64[CrossRef][Medline]
  12. Banchereau J, Steinman RM 1998 Dendritic cells and the control of immunity. Nature 392:245–252[CrossRef][Medline]
  13. Flores-Romo L, Bjorck P, Duvert V, van Kooten C, Saeland S, Banchereau J 1997 CD40 ligation on human cord blood CD34+ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells. J Exp Med 185:341–349[Abstract/Free Full Text]
  14. Caux C, Vanbervliet B, Massacrier C, Dezutter-Dambuyant C, de Saint-Vis B, Jacquet C, Yoneda K, Imamura S, Schmitt D, Banchereau J 1996 CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF{alpha}. J Exp Med 184:695–706[Abstract/Free Full Text]
  15. Young JW, Szabolcs P, Moore MA 1995 Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor {alpha}. J Exp Med 182:1111–1119[Abstract/Free Full Text]
  16. Bleharski JR, Niazi KR, Sieling PA, Cheng G, Modlin RL 2001 Signaling lymphocytic activation molecule is expressed on CD40 ligand-activated dendritic cells and directly augments production of inflammatory cytokines. J Immunol 167:3174–3181[Abstract/Free Full Text]
  17. Cobb MH, Goldsmith EJ 1995 How MAP kinases are regulated. J Biol Chem 270:14843–14846[Free Full Text]
  18. Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ 1996 MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol 16:1247–1255[Abstract]
  19. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW 1994 A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739–746[CrossRef][Medline]
  20. Yosimichi G, Nakanishi T, Nishida T, Hattori T, Takano-Yamamoto T, Takigawa M 2001 CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK). Eur J Biochem 268:6058–6065[Medline]
  21. Bhowmick NA, Zent R, Ghiassi M, McDonnell M, Moses HL 2001 Integrin ß1 signaling is necessary for transforming growth factor-ß activation of p38MAPK and epithelial plasticity. J Biol Chem 276:46707–46713[Abstract/Free Full Text]
  22. Kumar S, Votta BJ, Rieman DJ, Badger AM, Gowen M, Lee JC 2001 IL-1- and TNF-induced bone resorption is mediated by p38 mitogen activated protein kinase. J Cell Physiol 187:294–303[CrossRef][Medline]
  23. Matsumoto M, Sudo T, Saito T, Osada H, Tsujimoto M 2000 Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-{kappa}B ligand (RANKL). J Biol Chem 275:31155–31161[Abstract/Free Full Text]
  24. Li X, Udagawa N, Itoh K, Suda K, Murase Y, Nishihara T, Suda T, Takahashi N 2002 p38 MAPK-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function. Endocrinology. 143:3105–3113
  25. Wong BR, Josien R, Choi Y 1999 TRANCE is a TNF family member that regulates dendritic cell and osteoclast function. J Leukoc Biol 65:715–724[Abstract]
  26. Koga T, Nishihara T, Fujiwara T, Nisizawa T, Okahashi N, Noguchi T, Hamada S 1985 Biochemical and immunobiological properties of lipopolysaccharide (LPS) from Bacteroides gingivalis and comparison with LPS from Escherichia coli. Infect Immun 47:638–647[Abstract/Free Full Text]
  27. Suda T, Jimi E, Nakamura I, Takahashi N 1997 Role of 1{alpha}, 25-dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol 282:223–235[Medline]
  28. Takahashi N, Yamana H, Yoshiki S, Roodman GD, Mundy GR, Jones SJ, Boyde A, Suda T 1988 Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122:1373–1382[Abstract/Free Full Text]
  29. Inaba K, Turley S, Iyoda T, Yamaide F, Shimoyama S, Reis e Sousa C, Germain RN, Mellman I, Steinman RM 2000 The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J Exp Med 191:927–936[Abstract/Free Full Text]
  30. Baldassare JJ, Bi Y, Bellone CJ 1999 The role of p38 mitogen-activated protein kinase in IL-1ß transcription. J Immunol 162:5367–5373[Abstract/Free Full Text]
  31. Rawadi G, Ramez V, Lemercier B, Roman-Roman S 1998 Activation of mitogen-activated protein kinase pathways by Mycoplasma fermentans membrane lipoproteins in murine macrophages: involvement in cytokine synthesis. J Immunol 160:1330–1339[Abstract/Free Full Text]
  32. Lu HT, Yang DD, Wysk M, Gatti E, Mellman I, Davis RJ, Flavell RA 1999 Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO J 18:1845–1857[CrossRef][Medline]
  33. Wang X, Studzinski GP 2001 Inhibition of p38MAP kinase potentiates the JNK/SAPK pathway and AP-1 activity in monocytic but not in macrophage or granulocytic differentiation of HL60 cells. J Cell Biochem 82:68–77[CrossRef][Medline]
  34. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC 1995 SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364:229–3334[CrossRef][Medline]
  35. Enslen H, Raingeaud J, Davis RJ 1998 Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J Biol Chem 273:1741–1748[Abstract/Free Full Text]
  36. Young P, McDonnell P, Dunnington D, Hand A, Laydon J, Lee J 1993 Pyridinyl imidazoles inhibit IL-1 and TNF production at the protein level. Agents Actions 39:C67–C69
  37. Prichett W, Hand A, Sheilds J, Dunnington D 1995 Mechanism of action of bicyclic imidazoles defines a translational regulatory pathway for tumor necrosis factor {alpha}. J Inflamm 45:97–105[Medline]
  38. Bachmann MF, Wong BR, Josien R, Steinman RM, Oxenius A, Choi Y 1999 TRANCE, a tumor necrosis factor family member critical for CD40 ligand-independent T helper cell activation. J Exp Med 189:1025–1031[Abstract/Free Full Text]
  39. Ishida N, Hayashi K, Hoshijima M, Ogawa T, Koga S, Miyatake Y, Kumegawa M, Kimura T, Takeya T 2002 Large scale gene expression analysis of osteoclastogenesis in vitro and elucidation of NFAT2 as a key regulator. J Biol Chem 277:41147–41156[Abstract/Free Full Text]
  40. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T 2002 Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3:889–901[CrossRef][Medline]



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