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Originally published In Press as doi:10.1074/jbc.M411189200 on January 12, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11395-11403, March 25, 2005
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Prostaglandin E2 Enhances Osteoclastic Differentiation of Precursor Cells through Protein Kinase A-dependent Phosphorylation of TAK1*

Yasuhiro Kobayashi{ddagger}, Toshihide Mizoguchi{ddagger}, Ikuko Take{ddagger}§, Saburo Kurihara§, Nobuyuki Udagawa¶, and Naoyuki Takahashi{ddagger}||

From the {ddagger}Institute for Oral Science, the §Department of Orthodontics and the Department of Biochemistry, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781, Japan

Received for publication, September 29, 2004 , and in revised form, December 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-{kappa}B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor {beta}-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409–412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 -> Ala mutant TAK1. Ser412 -> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I{kappa}B{alpha}, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor {alpha}-induced I{kappa}B{alpha} degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 -> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor {alpha}. Ser412 -> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte-macrophage lineage (1–3). The differentiation and activation of osteoclasts are tightly regulated by osteoblasts or bone marrow-derived stromal cells (4–6). Osteoblasts express two cytokines essential for osteoclast differentiation: receptor activator of NF-{kappa}B ligand (RANKL)1 and macrophage colony-stimulating factor (M-CSF) (7, 8). The expression of RANKL in osteoblasts is up-regulated by several osteotropic factors such as interleukin 11 (IL-11), parathyroid hormone, 1{alpha},25-dihydroxyvitamin D3, IL-1, and lipopolysaccharide (LPS) (7, 9). Osteoclast precursors differentiate into mature osteoclasts in the presence of RANKL and M-CSF (10, 11). Recent studies have shown that mouse macrophage-like RAW264.7 cells can differentiate into osteoclasts in response to RANKL even in the absence of M-CSF (12). We and others (13–15) have reported that tumor necrosis factor {alpha} (TNF{alpha}) stimulates osteoclastic differentiation from bone marrow macrophages through a mechanism independent of the RANKL-RANK interaction. RAW264.7 cells also differentiate into osteoclasts in response to TNF{alpha} even in the absence of RANKL (16). Thus, two cytokines, RANKL and TNF{alpha}, induce the differentiation of osteoclasts from the precursor cells of the monocyte-macrophage lineage.

PGE2 has been proposed to be a potent stimulator of osteoclastic bone resorption involved in inflammatory diseases such as rheumatoid arthritis and osteomyelitis (17–20). Like other osteotropic factors, PGE2 stimulates RANKL expression in osteoblasts (21, 22). The functions of PGE2 in the target cells are mediated by four different G protein-coupled receptor subtypes, EP1, EP2, EP3, and EP4 (23, 24). The signal of EP1 increases intracellular Ca2+ and activates protein kinase C. EP2 and EP4 activate Gs, which stimulates cAMP generation, followed by the activation of cAMP-dependent protein kinase A (PKA), in the target cells. Conversely, EP3 acts via Gi to inhibit cAMP generation. Among these PGE2 receptor subtypes, EP4 mainly mediates PGE2-induced RANKL expression in osteoblasts (21, 25). In addition, PGE2 synergistically stimulates the differentiation of bone marrow macrophages into osteoclasts induced by RANKL and M-CSF (26, 27). Thus, PGE2 stimulates osteoclastic bone resorption through the following two different pathways: the induction of RANKL expression in osteoblasts, and the direct enhancement of RANKL-induced differentiation of osteoclast precursor cells into osteoclasts. The mechanism of the synergistic effect of PGE2 on the RANKL-induced osteoclastic differentiation of precursor cells has not yet been explained.

When RANKL binds to RANK, the receptor for RANKL, TNF receptor-associated factor 1 (TRAF1), TRAF2, TRAF3, TRAF5, and TRAF6 interact with the cytoplasmic tail of RANK (28, 29). The ligand-dependent interaction of TRAFs with RANK induces activation of nuclear factor-{kappa}B (NF-{kappa}B), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK) (30). Activation of NF-{kappa}B and MAPKs in osteoclast precursors is believed to be involved in osteoclast differentiation. Recent studies (31) have revealed that TRAF6-mediated signals are particularly important for RANKL-induced osteoclast differentiation and function. TRAF6 knock-out mice exhibit severe osteopetrosis with defects in bone resorption due to the impaired osteoclast differentiation and function (32, 33). In contrast to RANK, TNF receptors interact with TRAF2 but not with TRAF6 in their signaling pathway (34).

Transforming growth factor {beta} (TGF-{beta})-activated kinase 1 (TAK1) was first identified as a MAPK kinase kinase (MAP-KKK) activated by TGF-{beta} family ligands (35). Recent studies (36–38) have shown that TAK1, which forms a complex with the TAK1-binding protein 1 and 2 (TAB1/2), functions as an adaptor molecule in the interaction between TRAF6 and downstream molecules such as NF-{kappa}B, JNK, and p38 MAPK in signaling cascades induced by IL-1, LPS, and RANKL. Endogenous TAK1 is activated in response to RANKL stimulation, and a dominant-negative form of TAK1 inhibits the RANK-induced activation of NF-{kappa}B in RAW264.7 cells (38). It has been shown that TAK1 is also involved in TRAF2-mediated signaling (39). These results suggest that TAK1 is involved in the RANK- and TNF receptor-induced signaling pathways and may regulate the MAPK and NF-{kappa}B pathways activated by the interaction of RANKL-RANK or TNF{alpha}-TNF receptors.

In the present study, we explored the mechanism of PGE2 action on the osteoclastic differentiation of precursor cells. The stimulatory effect of PGE2 on the RANKL-induced osteoclast differentiation of the precursor cells was mediated through EP2 and EP4. TAK1 acted as an adaptor molecule linking PKA-induced signals, and RANKL and TNF{alpha} induced such signals in osteoclast precursors. Furthermore, TAK1 is involved in the synergistic effect of cAMP/PKA signals on TNF receptor- and Toll-like receptor 4 (TLR4)-induced signaling pathways. The cAMP/PKA signal may enhance bone resorption induced by RANKL and TNF{alpha} through TAK1 in osteoclast precursors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Chemicals—Human recombinant RANKL was purchased from PeproTech EC Ltd. (London, UK), and mouse TNF{alpha} was from R & D Systems (Minneapolis, MN). Human M-CSF (Leukoprol) was obtained from Kyowa Hakko Kogyo Co. (Tokyo, Japan). PGE2 were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Purified LPS (Escherichia coli 055:B5) was from Sigma. Elcatonin, a synthetic analogue of eel calcitonin (CT), was kindly provided by Asahi Kasei (Tokyo, Japan). Forskolin and 3-isobutyl-1-methylxanthine (IBMX) were from Biomol (Plymouth Meeting, PA). Polyclonal antibodies against p38 MAPK, phosphorylated p38 MAPK, ERK, phosphorylated ERK, JNK, phosphorylated JNK, phospho-(Ser/Thr) PKA substrates, and I{kappa}B{alpha} were purchased from Cell Signaling Technology Inc. (Beverly, MA). Mouse monoclonal antibody against TAK1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Fluo-4 AM, Fura Red AM, and Pluronic F127 were from Molecular Probes Inc. (Eugene OR). All other chemicals were of analytical grade.

Plasmids and cDNA Cloning—Mouse TAK1 cDNA (GenBankTM accession number D76446 [GenBank] ) was amplified by RT-PCR from cDNA of RAW264.7 cells using high fidelity Taq polymerase (Pyrobest, Takara Biochemicals, Tokyo, Japan) and TAK1-specific primers (forward primer 5'-gATATCCTGTCGACAGCCTCCGC and reverse primer 5'-AACGTAACGGGCCCAGAGAA). The PCR product was verified to be TAK1 cDNA by DNA sequencing. The TAK1 cDNA fragment was inserted into the BamHI-EcoRI site of pc DNA3.1/His, a mammalian expression vector (Invitrogen). The mutant TAK1, Ser412 -> Ala TAK1, was generated by PCR-directed site-specific mutagenesis. Coding regions of all plasmids were sequenced in both directions prior to the transfection.

Cell Culture and Transfection—RAW264.7 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan) (RCB0535). RAW 264.7 cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (JRH Biosciences, Lenexa, KS) in 100-mm dishes. The RAW264.7 cells were transfected with the indicated expression plasmids (10 µg) by TransFast transfection reagents (Promega Corp., Madison, WI) according to the manufacturer's instructions. After 24 h of cultivation, 1 mg/ml of G418 was added to the medium, and the medium was replaced 2–3 times during a 2-week period. Clonal lines were prepared from the drug-resistant cultures. To evaluate clones expressing the TAK1 transgenes, Western blotting was performed on several cell lines. We obtained three different lines in each transfectant.

Cultures of Bone Marrow-derived Macrophages and RAW264.7 Cells—Bone marrow-derived macrophages (BMM{varphi}) were prepared as osteoclast precursors from 5- to 8-week-old male ddY mice (Shizuoka Laboratories Animal Center, Shizuoka, Japan). All procedures for animal care were approved by the Animal Management Committee of Matsumoto Dental University. Bone marrow cells obtained from mouse tibia were suspended in {alpha}-MEM (Sigma) supplemented with 10% FBS in 60-mm diameter dishes for 16 h in the presence of M-CSF (50 ng/ml). Then nonadherent cells were harvested and further cultured for 2 days with M-CSF (50 ng/ml). The adherent cells, most of which expressed macrophage-specific antigens such as Mac-1, Moma-2, and F4/80, were used as BMM{varphi}. RAW264.7 cells were cultured in {alpha}-MEM in the presence of RANKL (50 ng/ml) to induce their differentiation into osteoclasts. After the cells were cultured for 5 days, they were fixed and stained for tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts). TRAP-positive multinucleated cells containing more than three nuclei were observed under a microscope and counted as osteoclasts. The results were expressed as the mean ± S.D. of quadruplicate cultures. All experiments were performed at least three times, and similar results were obtained. Statistical analysis of the results was performed by Student's t test.

RT-PCR for PGE2 Receptor mRNAs—Total RNA was extracted from cultured mouse bone marrow macrophages and RAW264.7 cells using the acid guanidinium-phenol-chloroform method. cDNA was synthesized from 10 µg of the total RNA by using reverse transcriptase (Revatra Ace, Toyobo Co. Ltd., Tokyo) and amplified using PCR. Sequences of primers used in RT-PCR for EP subtypes, CT receptor, and mouse glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were described in previous reports (21, 40). The PCR conditions for EP subtypes were as follows: denaturation 94 °C, 30 s, annealing 65 °C, 30 s, and primer extension 75 °C, 60 s. The conditions for the CT receptor and G3PDH were as follows: denaturation 94 °C, 30 s, annealing 60 °C, 30 s, and primer extension 72 °C, 60 s. Preliminary experiments were performed to ensure that the number of PCR cycles was within the exponential phase of the amplification curve. PCR products were subjected to electrophoresis in a 2% agarose gel followed by staining with ethidium bromide.

Immunoprecipitation and Western Blotting—Cells were washed once with phosphate-buffered saline and lysed in 200 µl of 0.1% Nonidet P-40 lysis buffer (20 mM Tris (pH 7.5), 50 mM {beta}-glycerophosphate, 150 mM NaCl, 1 mM EDTA, 25 mM NaF, 1 mM sodium orthovanadate, 1x protease inhibitors mixture (Sigma)). After removal of the cellular debris, the lysates (1 mg of protein) were incubated with 1 µg of various antibodies and 20 µl of protein G-Sepharose (Amersham Biosciences). The immune complexes were washed three times with Nonidet P-40 lysis buffer. The Sepharose beads were suspended in 30 µl of Laemmli sample buffer and boiled for 2 min. The cell lysates and immunoprecipitates were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (Clear blot P membrane, Atto Instruments, Tokyo, Japan). The membrane was blotted with antibodies to specific proteins and visualized using the enhanced chemiluminescence system (Amersham Biosciences).

Assay of cAMP Production and IL-6—To measure the amount of cAMP produced, cells were preincubated for 5 min at 37 °C in {alpha}-MEM containing 1 mM IBMX, and then incubated for 5 min at 37 °C with CT (Elcatonin, 10-9 M) or PGE2 (10-6 M). Cells were washed with ice-cold phosphate-buffered saline containing 1 mM IBMX and then were dissolved. The amounts of intracellular cAMP were determined using a cAMP enzyme immunoassay kit (Amersham Biosciences). To determine the effect of forskolin on LPS-induced IL-6 production, RAW264.7 cells transfected with mock, wild-type TAK1, or Ser412 -> Ala TAK1 (0.8 x 105 cells/well, 48-well plate) were cultured with or without LPS (100 ng/ml) in the presence or absence of forskolin (100 µM) for 24 h. The culture medium was collected, and the concentration of IL-6 in the culture medium was measured by ELISA (R & D Systems).

Measurements of Intracellular Ca2+—The effects of PGE2 and CT on intracellular Ca2+ in RAW264.7 cells was measured using a confocal microscope (LSM510, Carl Zeiss, Jena, Germany) according to the methods described previously (41). RAW264.7 cells were incubated in glass-bottom dishes (Asahi Techno Glass Corp., Tokyo) for 6 h. Cells were then incubated with 5 µM fluo-4 AM, 5 µM Fura Red AM, and 0.05% Pluronic F127 for 30 min in Dulbecco's modified Eagle's medium. Cells loaded with these dyes were washed twice with {alpha}-MEM and post-incubated in {alpha}-MEM containing 10% FBS. Cells were further washed three times with Hanks' balanced salt solution and then excited at 488 nm. Emission at 505–530 nm for fluo-4 and at 600–680 nm for Fura Red was acquired simultaneously at 2-s intervals. The ratio of the fluorescence intensity of fluo-4 to Fura Red was calculated to estimate intracellular Ca2+ influx in single cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of PGE2 Receptors in Osteoclast Precursors—We first analyzed the expression of PGE2 receptors in bone marrow macrophages and RAW264.7 cells using RT-PCR. Bone marrow macrophages expressed EP1, EP2, EP3{beta}, and EP4 mRNAs, whereas RAW264.7 cells expressed EP1, EP2, and EP4 mRNAs (Fig. 1A). Bone marrow macrophages differentiated into TRAP-positive osteoclasts in response to RANKL together with M-CSF (Fig. 1B, upper panel). PGE2 (10-6 M) alone did not induce osteoclast formation in cultures of bone marrow macrophages but did enhance the osteoclast formation induced by RANKL plus M-CSF (Fig. 1B, upper panel). RAW264.7 cells differentiated into osteoclasts in the presence of RANKL (Fig. 1B, lower panel). PGE2 similarly enhanced the osteoclast differentiation induced by RANKL in cultures of RAW264.7 cells (Fig. 1B, lower panel). EP1 activates Ca2+ signals, whereas EP2 and EP4 couple to Gs protein, which stimulates adenylate cyclase activity. We then examined calcium signaling in RAW264.7 cells treated with PGE2 and eel CT (Fig. 1C). An increase in intracellular calcium was induced by PGE2 (10-5 M) but not CT (10-9 M) in RAW264.7 cells. We next examined the effects of PGE2 and CT on cAMP production in RAW264.7 cells (Fig. 1D). PGE2 (10-6 M) but not CT (10-9 M) stimulated cAMP production in RAW264.7 cells. We also analyzed the expression of EPs in osteoclasts purified from co-cultures of mouse calvarial osteoblasts and bone marrow cells treated with 1{alpha},25-dihydroxyvitamin D3. Osteoclasts expressed only EP1 mRNA but not EP2, EP3, or EP4 mRNA (data not shown). These results indicate that expression of EP2 and EP4 mRNAs in osteoclast precursors was down-regulated during their differentiation into osteoclasts. CT (10-9 M) enhanced intracellular calcium concentrations and cAMP production in osteoclasts formed from RAW264.7 cells treated with RANKL (data not shown). These results suggest that functional EP1, EP2, and EP4 are expressed in RAW264.7 cells.



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  		FIG. 1.
Expression of EP subtypes in mouse bone marrow macrophages and RAW264.7 cells. A, RT-PCR analysis of EP subtypes in mouse bone marrow macrophages and RAW264.7 cells. Total RNA was extracted from mouse BMM{varphi} and RAW264.7 cells, and cDNA was synthesized from the total RNA by using reverse transcriptase. The expression of mRNA of EP1, EP2, EP3{alpha}, EP3{beta}, EP3{gamma}, EP4, CT receptor (CTR), and G3PDH was detected by PCR in the presence (+) or absence (-) of RT. B, effects of PGE2 on osteoclast differentiation induced by RANKL. BMM{varphi} cells were cultured with or without PGE2 (10-6 M) or RANKL (50 ng/ml) or RANKL plus PGE2 in the presence of M-CSF (50 ng/ml) (upper panel). RAW264.7 cells were cultured with or without PGE2 (10-6 M), RANKL (50 ng/ml), or RANKL plus PGE2 (lower panel). After the cells were cultured for 5 days, they were fixed and stained for TRAP, and TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts. Results are expressed as the mean ± S.D. of quadruplicate cultures. *, significantly different between the culture treated with RANKL + M-CSF and that treated with RANKL + M-CSF + PGE2 (upper panel), or between that with RANKL and that with RANKL+ PGE2 (lower panel); p < 0.01. C, effects of PGE2 and CT on calcium signaling in RAW264.7 cells. RAW264.7 cells loaded with fluo-4 AM, Fura Red AM, and Pluronic F127 were subjected to calcium measurement. Cells were excited at 488 nm, and emission at 505–530 nm for fluo-4 and at 600–680 nm for Fura Red was acquired simultaneously at 2-s intervals. Cells were stimulated by the addition (an arrow) of PGE2 (10-5 M) or CT (10 -9 M). The ratio of the fluorescence intensity of fluo-4 to Fura Red was calculated to estimate intracellular Ca2+ influx in single cells. The results represent calcium signals in six single cells treated with PGE2 or CT. D, effects of PGE2 and CT on cAMP production in RAW264.7 cells. RAW264.7 cells were preincubated for 5 min with IBMX (1 mM) and then incubated for 5 min with PGE2 (10-6 M) or CT (10 -9 M). The amounts of intracellular cAMP were determined by ELISA. *, significantly different from the control culture; p < 0.01.

 
Enhancement of RANKL-induced Osteoclast Differentiation by EP2/EP4-mediated Signals—PGE2 has been shown to enhance synergistically RANKL-induced osteoclast differentiation from the precursors (26, 27). RAW264.7 cells expressed functional EP1, EP2, and EP4 receptors (Fig. 1). We then examined which type of PGE2 receptors is involved in the stimulatory effect of PGE2 on osteoclastic differentiation of RAW264.7 cells (Fig. 2). RANKL-induced osteoclast formation was enhanced by PGE2 in a dose-dependent manner (Fig. 2A). The synergistic effect of PGE2 on RANKL-induced osteoclast differentiation was dose-dependently inhibited by H-89, a specific inhibitor of PKA (Fig. 2, B and C). Dibutyryl cAMP (100 µM), a cell-permeable analogue of cAMP, enhanced the osteoclast differentiation induced by RANKL (Fig. 2B). PGE2 and dibutyryl cAMP also enhanced osteoclastic differentiation of bone marrow macrophages treated with M-CSF and RANKL (data not shown). These results suggest that PGE2 enhances RANKL-induced osteoclast differentiation through the signals mediated by EP2 and EP4 in the precursor cells.



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  		FIG. 2.
Enhancement of RANKL-induced osteoclast differentiation by EP2/EP4-mediated signals in RAW-264.7 cells. A, dose-dependent effects of PGE2 on RANKL-induced osteoclast formation in RAW 264.7 cells. RAW264.7 cells were cultured with or without various concentrations of PGE2 in the presence or absence of RANKL (50 ng/ml). After the cells were cultured for 5 days, they were fixed and stained for TRAP, and TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts. Results are expressed as the mean ± S.D. of quadruplicate cultures. *, significantly different between the culture treated with RANKL and that with RANKL + PGE2; p < 0.01. B, effects of PGE2, H-89, and Bt2 cAMP (db-cAMP) on RANKL-induced osteoclast formation in RAW264.7 cell cultures. RAW264.7 cells were cultured with or without RANKL (50 ng/ml), PGE2 (10-6 M), H-89 (1 µM, 10 µM), and/or Bt2 cAMP (100 µM). After the cells were cultured for 5 days, they were fixed and stained for TRAP, and TRAP-positive multinucleated cells were counted as osteoclasts. Results are expressed as the mean ± S.D. of quadruplicate cultures. *, significantly different between the culture treated with RANKL and that with RANKL together with H89 or Bt2 cAMP; p < 0.01. C, TRAP staining of RAW264.7 cells treated with or without RANKL, RANKL + PGE2, or RANKL + PGE2 + H-89 (10 µM). Bar = 25 µm.

 
Enhancement of RANK-induced Signals by EP2/EP4-mediated Signals—Binding of RANKL to RANK activates various signaling pathways, including those involving NF-{kappa}B, p38 MAPK, ERK, and JNK, in the target cells. We and others (42, 43) have demonstrated previously that p38 MAPK activity is essentially involved in RANKL-induced osteoclastic differentiation. We first examined the effects PGE2 (10-6 M) on the phosphorylation of p38 MAPK in RAW264.7 cells in the presence or absence of RANKL (50 ng/ml) (Fig. 3A). PGE2 alone failed to induce the phosphorylation of p38 MAPK in RAW264.7 cells but synergistically enhanced RANKL-induced phosphorylation of p38 MAPK within 15 min. Pretreatment of RAW264.7 cells with H-89 (10 µM) completely inhibited the synergistic effect of PGE2 on RANKL-induced phosphorylation of p38 MAPK (Fig. 3B). This suggests that the stimulatory effect of PGE2 on RANKL-induced phosphorylation of p38 MAPK is mediated by PKA. We then examined the effects of PGE2 on the activation of NF-{kappa}B, p38 MAPK, ERK, and JNK in RAW264.7 cells treated with RANKL at different time points (Fig. 3C). PGE2 synergistically enhanced RANKL-induced degradation of I{kappa}B{alpha} (NF-{kappa}B activation) and phosphorylation of p38 MAPK in RAW264.7 cells for 5–15 min. RANKL-induced phosphorylation of JNK was also enhanced by PGE2 for 15–30 min (Fig. 3C). The synergistic effect of PGE2 on RANKL-induced phosphorylation of ERK was much weaker than on RANKL-induced phosphorylation of p38 MAPK in RAW264.7 cells throughout the experimental period. These results suggest that PKA-induced signals mainly cross-talk with an upstream effector(s) of p38 MAPK, NF-{kappa}B, and JNK.



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  		FIG. 3.
Effects of PGE2 on RANKL-induced activation of MAPKs and NF-{kappa}B in RAW264.7 cells. A, effects of PGE2 on RANKL-induced phosphorylation of p38 MAPK in RAW264.7 cells. RAW264.7 cells were incubated for 15 min with or without PGE2 (10-6 M), RANKL (50 ng/ml), or RANKL plus PGE2. Cell lysates were prepared, immunoblotted with anti-phosphorylated-p38 MAPK antibody (P-p38 MAPK), and re-blotted with anti-p38 MAPK antibody (p38 MAPK). B, effect of H-89 on PGE2-induced enhancement of phosphorylation of p38 MAPK in RAW264.7 cells. RAW264.7 cells were pre-cultured for 1 h in the presence or absence of H-89 (10 µM) and then incubated for 15 min with or without RANKL (50 ng/ml) in the presence or absence of PGE2 (10-6 M). Cell lysates were prepared, immunoblotted with anti-phosphorylated p38 MAPK antibody, and re-blotted with anti-p38 MAPK antibody. C, time course of changes in degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK, JNK, and ERK in RAW264.7 cells. RAW264.7 cells were incubated with RANKL (50 ng/ml) in the presence or absence of PGE2 (10-6 M) for the indicated times. Cell lysates were prepared and immunoblotted with the antibodies indicated in the panel. Amounts of p38 MAPK, JNK, and ERK in the lysates were determined by re-blotting the membrane with the indicated antibodies.

 
Involvement of TAK1 in EP2/EP4-mediated Enhancement of RANKL-induced Signals and Osteoclast Differentiation—PKA recognizes a consensus sequence (RRX(S/T) motif) of the target proteins and phosphorylates the Ser/Thr residue in the sequence (44, 45). We searched for effectors having the RRX(S/T) motif in RANK-induced signaling molecules, including TRAF6, TAK1, TAB1, TAB2, and other MAPKKs and MAPKKKs. We found that murine TAK1 contains a consensus PKA recognition sequence (Arg409-Arg-Arg-Ser-Ilu-Gln414) at the C-terminal region (Fig. 4A). Human TAK1 also possesses this consensus motif. Recent studies showed that in IL-1 and RANKL-induced signaling cascades, TAK1 functioned as an adaptor molecule in the interaction between TRAF6 and the downstream molecules such as NF-{kappa}B and MAPKs (36, 38). We then examined whether the endogenous TAK1 was phosphorylated in RAW264.7 cells in response to PGE2 (10-6 M) (Fig. 4B). Phosphorylation of TAK1 detected by antibody against phosphorylated Ser/Thr of PKA substrates was markedly induced in RAW264.7 cells treated with PGE2 (Fig. 4B). The phosphorylation of TAK1 induced by PGE2 was strongly suppressed by pretreatment of RAW264.7 cells with H-89 (10 µM). RANKL (50 ng/ml) failed to induce phosphorylation of Ser/Thr residues in TAK1 and showed no effect on the PGE2-induced phosphorylation of TAK1. These results suggest that PGE2-induced phosphorylation of TAK1 is PKA-dependent in RAW264.7 cells.



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  		FIG. 4.
Effects of PGE2 and H-89 on phosphorylation of TAK1 in RAW264.7 cells. A, structure of TAK1. TAK1 possesses the kinase domain in the N-terminal region, the TAB1-binding domain in the kinase domain, and the TAB2-binding domain in the C-terminal region. A consensus motif (RRXS motif) recognized by PKA is located in the C-terminal region and consists of Arg409-Arg-Arg-Ser-Ilu-Gln414 in mouse and human TAK1. B, phosphorylation of TAK1 in RAW264.7 cells treated with PGE2. RAW264.7 cells were pre-cultured for 1 h in the presence or absence of H-89 (10 µM) and then stimulated with or without RANKL (50 ng/ml) in the presence or absence of PGE2 (10-6 M) for 15 min. Cell lysates were subjected to immunoprecipitation with anti-TAK1 antibody. The immunoprecipitates were separated, immunoblotted (IB) with antibody against phosphorylated Ser/Thr in PKA substrates, and re-blotted with anti-TAK1 antibody.

 
We further analyzed whether the Ser412 residue in TAK1 was phosphorylated in RAW264.7 cells in response to PGE2 by using a Ser412 -> Ala mutant form of TAK1 (Ser412 -> Ala TAK1). RAW264.7 cells were stably transfected with empty vector (mock) or expression vector for wild-type TAK1 or Ser412 -> Ala TAK1 (Fig. 5A). Endogenous and transfected wild-type TAK1 were phosphorylated in response to PGE2 (10-6 M) but not to RANKL (50 ng/ml) in RAW264.7 cells (Fig. 5B). In contrast, when RAW264.7 cells were transfected with Ser412 -> Ala TAK1, the phosphorylation of both endogenous and transfected TAK1 was strongly suppressed even in the presence of PGE2 (Fig. 5B). The other two RAW264.7 cell lines established using each transfectant showed similar responsiveness to RANKL and PGE2 (data not shown). This suggests that Ser412 -> Ala TAK1 acts as a dominant-negative mutant in RAW264.7 cells. We then examined whether PKA-induced phosphorylation of TAK1 was involved in the synergistic effect on the RANK-induced signals (Fig. 5C). RANKL-induced degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK were enhanced by forskolin (100 µM), an activator of PKA, in RAW264.7 cells transfected with mock or wild-type TAK1 but not in those transfected with Ser412 -> Ala TAK1 (Fig. 5C). RAW264.7 cells transfected with mock or wild-type TAK1 differentiated into osteoclasts in response to RANKL (50 ng/ml), and PGE2 (10-6 M) enhanced RANKL-induced osteoclast differentiation (Fig. 5D). RANKL similarly stimulated osteoclastic differentiation of RAW264.7 cells transfected with Ser412 -> Ala TAK1. However, the stimulatory effect of PGE2 on RANKL-induced osteoclast differentiation was suppressed in RAW264.7 cells expressing Ser412 -> Ala TAK1. These results suggest that EP2/EP4 signals induce the phosphorylation of TAK1 in osteoclast precursors, which in turn enhances RANK-mediated signals that induce osteoclast differentiation.



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  		FIG. 5.
Effect of Ser412 -> Ala TAK1 on PGE2-induced up-regulation of osteoclast differentiation in RAW264.7 cells treated with RANKL. A, transfection of RAW264.7 cells with wild-type TAK1 or Ser412 -> Ala (S412A) TAK1 cDNAs. RAW264.7 cells were stably transfected with mock, wild-type TAK1 (WT TAK1), or S412A TAK1. Cell lysates were prepared, immunoblotted with anti-TAK1 antibody, and re-blotted with anti-{beta}-actin antibody. B, phosphorylation of TAK1 in RAW264.7 cells transfected with mock, WT TAK1, and S412A TAK1. RAW264.7 cells stably expressing mock, WT TAK1, or S412A TAK1 were treated with or without PGE2 (10-6 M) or RANKL (50 ng/ml) for 15 min. Cell lysates were subjected to immunoprecipitation with anti-TAK1 antibody. The immunoprecipitates (IP) were separated and immunoblotted (IB) with antibodies against phosphorylated Ser/Thr in PKA substrates. C, effects of forskolin on the activation of NF-{kappa}B and p38 MAPK induced by RANKL in RAW264.7 cells transfected with mock, WT TAK1, and S412A TAK1. RAW264.7 cells transfected with mock, WT TAK1, or S412A TAK1 were treated with or without RANKL (50 ng/ml) in the presence or absence of forskolin (100 µM) for 15 min. Cell lysates were separated and immunoblotted with anti-I{kappa}B{alpha} antibody (I{kappa}B{alpha}) and with anti-phosphorylated-p38 MAPK antibody (P-p38 MAPK) followed by re-blotting with anti-p38 MAPK antibody (p38 MAPK). D, effect of PGE2 on RANKL-induced osteoclast formation in RAW264.7 cells transfected with mock, WT TAK1, and S412A TAK1. RAW264.7 cells transfected with mock, WT TAK1, or S412A TAK1 were cultured with or without RANKL (50 ng/ml) in the presence or absence of PGE2 (10-6 M). After the cells were cultured for 5 days, cells were fixed and stained for TRAP. TRAP-positive multinuclear cells containing three or more nuclei were counted as osteoclasts. Results are expressed as the mean ± S.D. of quadruplicate cultures. *, significantly different between cultures treated with RANKL and those with RANKL + PGE2 in each cDNA transfectant; p < 0.01. #, significantly different between mock-transfected cultures and wild-type TAK1- or Ser412 -> Ala TAK1-transfected cultures in the same treatment groups; p < 0.01.

 
Involvement of TAK1 in PKA-mediated Enhancement of TNF{alpha}-induced Signals and Osteoclast Differentiation—We and others (13–15) have also reported that TNF{alpha} stimulated osteoclastic differentiation from osteoclast precursors through a mechanism independent of the RANKL-RANK interaction. TAK1 is also implicated in the TNF receptor-mediated signaling (39). We then examined whether PKA-mediated signals enhanced TNF{alpha}-induced osteoclast differentiation through TAK1-mediated signals (Fig. 6). TNF{alpha} (40 ng/ml) stimulated degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK in RAW264.7 cells transfected with mock or wild-type TAK1, both of which were enhanced by forskolin (100 µM) (Fig. 6A). In contrast, the synergistic effects of forskolin on the degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK were completely suppressed by the transfection with Ser412 -> Ala TAK1. TNF{alpha} (40 ng/ml) stimulated osteoclastic differentiation of RAW264.7 cells transfected with mock or wild-type TAK1, and forskolin (100 µM) enhanced TNF{alpha}-induced osteoclastic differentiation in those transfected cells (Fig. 6B). TNF{alpha} similarly stimulated osteoclastic differentiation of RAW264.7 cells transfected with Ser412 -> Ala TAK1, but the stimulatory effect of forskolin on TNF{alpha}-induced osteoclast differentiation was completely suppressed in those cells (Fig. 6B). These results suggest that the phosphorylation of TAK1 by PKA signals synergistically enhances TNF{alpha}-induced activation of p38 MAPK and NF-{kappa}B and osteoclast differentiation in cultures of RAW264.7 cells.



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  		FIG. 6.
Effect of Ser412 -> Ala TAK1 on forskolin-induced up-regulation of osteoclast differentiation in RAW264.7 cells treated with TNF{alpha}. A, effects of forskolin on the activation of NF-{kappa}B and p38 MAPK induced by TNF{alpha} in RAW264.7 cells transfected with mock, WT TAK1, and S412A TAK1. RAW264.7 cells transfected with mock, WT TAK1, or S412A TAK1 were treated with or without TNF{alpha} (40 ng/ml) in the presence or absence of forskolin (100 µM) for 15 min. Cell lysates were immunoblotted with anti-I{kappa}B{alpha} antibody (I{kappa}B{alpha}) or with anti-phosphorylated-p38 MAPK (P-p38 MAPK) antibody followed by re-blotting with anti-p38 MAPK antibody (p38 MAPK). B, effect of forskolin on TNF{alpha}-induced osteoclast formation RAW264.7 cells transfected with mock, WT TAK1, and S412A TAK1. RAW264.7 cells transfected with mock, WT TAK1, or S412A TAK1 were cultured with or without TNF{alpha} (40 ng/ml) in the presence or absence of forskolin (100 µM). After the cells were cultured for 5 days, cells were fixed and stained for TRAP. TRAP-positive multinuclear cells containing three or more nuclei were counted as osteoclasts. Results are expressed as the mean ± S.D. of quadruplicate cultures. *, significantly different between cultures treated with TNF{alpha} and those with TNF{alpha} + forskolin in each cDNA transfectant; p < 0.01. #, significantly different between mock-transfected cultures and WT TAK1- or S412A TAK1-transfected cultures in the same treatment groups; p < 0.01.

 
Involvement of TAK1 in PKA-mediated Enhancement of LPS-induced IL-6 Production—PGE2 has been shown to enhance LPS-induced IL-6 mRNA expression in mouse macrophages (46). LPS activates NF-{kappa}B and MAPKs through TAK1 in TLR4 signaling (37). We finally examined whether the phosphorylation of Ser412 in TAK1 by PKA is involved in PGE2-induced enhancement of IL-6 production (Fig. 7). LPS (100 ng/ml) stimulated degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK in RAW264.7 cells transfected with either mock, wild-type TAK1, or Ser412 -> Ala TAK1. Forskolin (100 µM) enhanced LPS-induced degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK in RAW264.7 cells transfected with mock or wild-type TAK1 (Fig. 7A). The synergistic effects of forskolin on LPS-induced degradation of I{kappa}B{alpha} and phosphorylation of p38 MAPK were strongly suppressed in RAW264.7 cells transfected with Ser412 -> Ala TAK1. Forskolin (100 µM) enhanced LPS-induced IL-6 production in RAW264.7 cells transfected with mock or wild-type TAK1 (Fig. 7B). The stimulatory effect of forskolin on the LPS-induced IL-6 production was significantly suppressed in RAW264.7 cells transfected with Ser412 -> Ala TAK1.



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  		FIG. 7.
Effect of the Ser412 -> Ala TAK1 on forskolin-induced up-regulation of IL-6 production in RAW264.7 cells treated with LPS. A, effects of forskolin on the activation of NF-{kappa}B and p38 MAPK induced by LPS in RAW264.7 cells transfected with mock, WT TAK1, and S412A TAK1. RAW264.7 cells transfected with mock, WT TAK1, or S412A TAK1 were treated with or without LPS (100 ng/ml) in the presence or absence of forskolin (100 µM) for 15 min. Cell lysates were immunoblotted with anti-I{kappa}B{alpha} antibody (I{kappa}B{alpha}), or with anti-phosphorylated-p38 MAPK (P-p38 MAPK) antibody followed by re-blotting with anti-p38 MAPK antibody (p38 MAPK). B, effect of forskolin on LPS-induced IL-6 production in RAW264.7 cells transfected with mock, WT TAK1, and S412A TAK1. RAW264.7 cells transfected with mock, WT TAK1, or S412A TAK1 were cultured with or without LPS (100 ng/ml) in the presence or absence of forskolin (100 µM) for 24 h. The culture medium was then collected, and IL-6 concentrations were measured using an ELISA for IL-6. *, significantly different between cultures treated with LPS and those with LPS + forskolin in each transfectant; p < 0.01, significantly different between mock-transfected cultures and WT TAK1- or S412A TAK1-transfected cultures in the same treatment groups; p < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have shown that PGE2 stimulates osteoclastic bone resorption through two different mechanisms as follows: the induction of RANKL expression in osteoblasts (21, 22), and the direct enhancement of RANKL-induced differentiation of the precursor cells into osteoclasts (27). In the present study, we examined the mechanism of the synergistic effect of PGE2 on RANKL-induced osteoclastic differentiation. We have shown here that PGE2 synergistically enhances osteoclastic differentiation of RAW264.7 cells through EP2 and EP4, and that TAK1 is a key molecule in cAMP/PKA-induced up-regulation of osteoclast differentiation stimulated by not only RANKL but also TNF{alpha}.

Osteoclast precursors of bone marrow macrophages and RAW264.7 cells expressed EP1 as well as EP2 and EP4 (Fig. 1). Treatment of RAW264.7 cells with PGE2 but not CT increased Ca2+ influx, suggesting that osteoclast precursors express functional EP1. The synergistic effect of PGE2 on RANKL-induced osteoclast differentiation was inhibited by H-89, a specific inhibitor of PKA, and dibutyryl cAMP enhanced the osteoclast differentiation induced by RANKL (Fig. 2). We further examined which receptor, EP2 or EP4, was mainly involved in the synergistic effect of PGE2 on RANKL-induced osteoclastic differentiation of RAW264.7 cells, using specific EP1, EP2, and EP4 agonists. The number of osteoclasts formed in RAW264.7 cell cultures treated with 10-6 M ONO-AE1-259 (EP2 agonist) and 10-6 M ONO-AE1-329 (EP4 agonist) increased by 1.4 and 2.4 times, respectively (data not shown). In contrast, ONO-DI-004 (EP-1 agonist) at 10-6 M showed no effect on osteoclast formation in RAW264.7 cell cultures. Thus, the effect of EP agonists on osteoclast formation was comparable with the expression level of EP2 and EP4 mRNAs (Fig. 1A). These results suggest that EP4 mainly mediates the synergistic effect of PGE2 on RANKL-induced osteoclast differentiation in RAW 264.7 cells.

TAK1 is a key MAPKKK in the IL-1 receptor- and TLR4-mediated signaling pathway (36, 37). TAK1 mediates MAPK and NF-{kappa}B activation via interaction with TRAF6, and TAB2 acts as an adaptor linking TAK1 and TRAF6. Mizukami et al. (38) first reported that TAK1 participates in the RANK signaling pathway. Endogenous TAK1 was activated in response to RANKL in RAW264.7 cells, and the kinase-negative form of TAK1 (K63W TAK1) attenuated JNK and NF-{kappa}B activation induced by RANKL. We have confirmed that expression of K63W TAK1 in RAW264.7 cells significantly inhibited the osteoclast formation induced by RANKL and TNF{alpha}.2 By using antibody against phosphorylated Ser/Thr of PKA substrates, we showed that phosphorylated Ser/Thr of TAK1 was induced by PGE2 (Figs. 4 and 5). However, the phosphorylated Ser/Thr of TAK1 was not detected in RAW264.7 cells transfected with the Ser412 -> Ala mutant TAK1 (Fig. 5B), suggesting that Ser412 -> Ala TAK1 acted as a dominant-negative mutant of TAK1. These findings indicate that Ser412 in TAK1 is a major phosphorylation site by PKA.

The expression of Ser412 -> Ala TAK1 in RAW264.7 cells did not inhibit RANKL- or TNF{alpha}-induced osteoclast formation (Figs. 5 and 6). However, the expression of Ser412 -> Ala TAK1 suppressed the PKA signal-mediated up-regulation of the degradation of I{kappa}B{alpha} degradation, phosphorylation of p38 MAPK, and osteoclast differentiation induced by RANKL and TNF{alpha} in RAW264.7 cells (Figs. 5 and 6). It was reported that Ser192 in the kinase domain in TAK1 was phosphorylated by the kinase in response to IL-1 stimulation, and the phosphorylation of Ser192 was important for the IL-1-induced signal transduction (47). These results together with our findings suggest that overexpression of wild-type or the Ser412 -> Ala mutant TAK1 itself enhances osteoclastic differentiation induced by RANKL and TNF-{alpha}. The Ser412 residue appears to be involved in the synergistic action of cAMP/PKA signaling in osteoclast differentiation. Our experiments also suggest that the Ser412 residue regulates the synergistic action of cAMP/PKA signaling in osteoclast differentiation (Fig. 8).



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  		FIG. 8.
A possible role of phosphorylation of Ser412 in TAK1 in osteoclast differentiation induced by RANKL and TNF{alpha}. Cyclic AMP-dependent PKA phosphorylates the Ser412 residue in TAK1 in osteoclast precursors in response to the binding of PGE2 to EP2 or EP4. The phosphorylation of TAK1 itself does not induce osteoclastic differentiation of the precursors but synergistically enhances downstream signals such as MAPKs and NF-{kappa}B in response to other factors including RANKL and TNF{alpha}. Phosphorylation of Ser192 in TAK1 appears to be indispensable for the signal transduction induced by RANKL and TNF{alpha} as well as IL-1 (47). Osteoclastic differentiation induced by RANKL and TNF{alpha} is also enhanced by the phosphorylation of the Ser412 residue of TAK1 in osteoclast precursors.

 
TRAF6 plays essential roles in osteoclast differentiation and function induced by RANK-mediated signals (31–33). In contrast to RANK, TNF receptors selectively interact with TRAF2 in the signaling pathway (34). Forskolin enhanced TNF{alpha}-induced signals and osteoclast differentiation in cultures of RAW264.7 cells (Fig. 6). These results suggest that cAMP/PKA signals cross-talk with TRAF2-mediated signals as well as TRAF6-mediated ones. Recent studies (48) have shown that TAK1 is involved in not only TRAF6-mediated signaling but also TRAF2-mediated signaling. TNF{alpha} induced the binding of TAK1 to TRAF2 in HeLa cells. TAB2 has been shown to activate NF-{kappa}B by linking TAK1 to TRAF6 (49). Recently, it was shown that TAB3, a TAB2-like molecule that associates with TAK1 and activates NF-{kappa}B, interacts with both TRAF2 and TRAF6 (39). RAW264.7 cells have been shown to express TAB3 as well as TAB2 (50). We have confirmed that those two molecules are expressed in bone marrow macrophages as well as RAW264.7 cells (data not shown). These results suggest that the interaction of TAK1 and TAB2 (or TAB3) is involved in osteoclast differentiation induced by RANKL and TNF{alpha}.

TLR and IL-1 receptors use myeloid differentiation factor 88 (MyD88) as a common signaling molecule (51). In response to LPS, MyD88 interacts with TRAF6, which activates downstream signals. Recent studies have shown that Toll-IL-1 receptor domain-containing adaptor inducing interferon-{beta} (TRIF)-mediated signaling is involved in a MyD88-independent pathway induced by LPS (52). Both MyD88-dependent and TRIF-dependent pathways are required for LPS-induced cytokine production in macrophages (52). Forskolin significantly enhanced LPS-induced I{kappa}B{alpha} degradation, p38 MAPK phosphorylation, and IL-6 production in RAW264.7 cells (Fig. 7). In contrast to the effect of S412A TAK1 on PKA signal-enhanced osteoclast differentiation, the mutant TAK1 significantly but not completely suppressed forskolin-induced enhancement of IL-6 production in RAW264.7 cells treated with LPS. These results suggest that TAK1 signals are certainly involved in the PKA signal-induced enhancement of LPS signals, but signaling molecules other than TAK1 are also involved in the cross-talk between PKA-activated signals and TLR4-induced signals in macrophages. The TRIF-dependent pathway may be another target for the PKA signals in osteoclast precursors. McCoy et al. (53) reported that the production of IL-6 significantly decreased in EP4 receptor-deficient mice in collagen antibody-induced arthritis. Moreover, PKA signals have been shown to enhance LPS-induced IL-6 production in mouse macrophages and Swiss 3T3 cells (46, 54, 55). Thus, these previous findings and our study strongly support the idea that PKA-induced phosphorylation of TAK1 enhances LPS-induced IL-6 production, although we cannot completely rule out the possibility that there is an alternative target for PKA in TLR4 signaling.

At present, it is not known how the phosphorylation of TAK1 by PKA enhances TRAF6- and TRAF2-induced signals. However, it should be noted that the site phosphorylated by PKA is located in the TAB2 binding domain of the TAK1 molecule (Fig. 4). TAB3 has also been proposed to bind to the TAB2 binding domain (39). These results suggest that phosphorylation of TAK1 by PKA may influence the signaling complex formation (TAK1-TAB1-TAB2/TAB3) induced by the various ligands studied here. Therefore, we examined whether RANKL-induced formation of the complex of TAK1-TAB2 in RAW264.7 cells was affected by the treatment with PGE2. However, we could not find significant changes in the complex formation in response to PGE2 (data not shown). Further studies will be necessary to elucidate the molecular mechanism of the interaction between PKA-activated signals and TRAF-mediated signals in osteoclast precursors.

In conclusion, we demonstrated that PKA-activated signals enhanced RANKL-, TNF{alpha}-, and LPS-induced signals in osteoclast precursors. PKA selectively phosphorylated the Ser412 residue in TAK1, which was crucially involved in the synergistic action of PGE2 on RANK-, TNF receptor-, and TLR-mediated signaling. The cAMP/PKA signal may enhance RANKL- and inflammatory cytokine-induced bone resorption through TAK1 in osteoclast precursors (Fig. 8). Signaling molecules involved in the TAK1 pathway in osteoclast precursors would be novel targets for drugs to inhibit osteoclast function induced by inflammatory diseases.


   FOOTNOTES
 
* This work was supported in part by Grants-in-Aid 12137209, 13470394, 14207075, 14370599, 15390565, and 15390641 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Institute for Oral Science, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781, Japan. Tel.: 81-263-51-2135; Fax: 81-263-51-2223; E-mail: takahashinao{at}po.mdu.ac.jp.

1 The abbreviations used are: RANKL, receptor activator for NF-{kappa}B ligand; IL, interleukin; PGE2, prostaglandin E2; NF-{kappa}B, nuclear factor-{kappa}B; RANK, receptor activator of NF-{kappa}B; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; MAPKK, MAPK kinase; PKA, protein kinase A; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; M-CSF, macrophage-colony-stimulating factor; TGF-{beta}, transforming growth factor-{beta}; TAK1, TGF-{beta}-activated kinase 1; TAB, TAK1-binding protein; CT, calcitonin; IBMX, 3-isobutyl-1-methylxanthine; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; TLR, toll-like receptor; MyD88, myeloid differentiation factor 88; TRIF, Toll-IL-1 receptor domain-containing adaptor-inducing interferon-{beta}; RT, reverse transcription; {alpha}-MEM, {alpha}-minimum Eagle's medium; FBS, fetal bovine serum; ELISA, enzyme-linked immunosorbent assay; BMM{varphi}, bone marrow-derived macrophages; Bt2 cAMP, dibutyryl cyclic AMP; TRAP, tartrate-resistant acid phosphatase; WT, wild type. Back

2 Y. Kobayashi and N. Takahashi, unpublished observations. Back



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