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J. Biol. Chem., Vol. 280, Issue 25, 24035-24042, June 24, 2005

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Prostaglandin E₂ Receptors EP2 and EP4 Are Down-regulated during Differentiation of Mouse Osteoclasts from Their Precursors^*

Yasuhiro Kobayashi, Ikuko Take, Teruhito Yamashita¶, Toshihide Mizoguchi, Tadashi Ninomiya, Toshimi Hattori||, Saburo Kurihara, Hidehiro Ozawa, Nobuyuki Udagawa¶, and Naoyuki Takahashi**

From the Institute for Oral Science, Department of Orthodontics, ^¶Department of Biochemistry, and ^||Department of Pharmacology, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781, Japan

Received for publication, January 25, 2005 , and in revised form, April 7, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Prostaglandin E₂ (PGE₂) has been proposed to be a potent stimulator of bone resorption. However, PGE₂ itself has been shown to directly inhibit bone-resorbing activity of osteoclasts. We examined the role of PGE₂ in the function of mouse osteoclasts formed in vitro. Bone marrow macrophage osteoclast precursors expressed PGE₂ receptors EP1, EP2, EP3, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by receptor activator of NF-B ligand and macrophage colony-stimulating factor. In contrast, functional EP1 was continuously expressed in mature osteoclasts. PGE₂ as well as calcitonin caused intracellular Ca²⁺ influx in osteoclasts. However, PGE₂ and 17-phenyltrinol-PGE₂ (an EP1 agonist) failed to inhibit actin-ring formation and pit formation by osteoclasts cultured on dentine slices. When EP4 was expressed in osteoclasts using an adenovirus carrying EP4 cDNA, both actin-ring and pit-forming activities of osteoclasts were inhibited in an infectious unit-dependent manner. Treatment of EP4-expressing osteoclasts with PGE₂ further inhibited their actin-ring and pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are similar to those that are calcitonin receptor-mediated. Thus, osteoclast precursors down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts to escape inhibitory effects of PGE₂ on bone resorption.

	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; they are receptor activator of NF-B ligand (RANKL)¹ and macrophage colony stimulating factor (M-CSF) (7, 8). The expression of RANKL in osteoblasts is up-regulated by various osteotropic factors such as parathyroid hormone, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), interleukin 11 (IL-11), IL-1, and lipopolysaccharide (7, 9). Osteoclast precursors differentiate into mature osteoclasts in the presence of RANKL and M-CSF (10, 11). Mature osteoclasts as well as osteoclast precursors express RANK, a receptor of RANKL. RANKL also induces bone-resorbing activity of osteoclasts (4, 7, 8).

Bone-resorbing osteoclasts form unique cellular structures such as clear zones and ruffled borders toward the bone surface (12). The clear zone, also called the "actin ring," consists of a ring-like alignment of filamentous actin (F-actin) dots and surrounds the ruffled border, from which protons and lysosomal enzymes are secreted into the resorption lacunae (13). Calcitonin (CT), a bone resorption-inhibiting hormone, disrupts actin rings and inhibits the pit-forming activity of osteoclasts cultured on bone or dentine slices (14, 15). Osteoclasts express abundant CT receptors, which are coupled to G_s and G_q proteins (16). We have previously shown that signals mediated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) play important roles in CT-induced inhibition of pit formation and actin ring formation by osteoclasts cultured on dentine slices (17).

Prostaglandin E₂ (PGE₂) has diverse biological activities in a variety of tissues (18). The actions of PGE₂ in the target cells are mediated by four different G protein-coupled receptor subtypes, EP1, EP2, EP3, and EP4 (19). The EP subtypes differ in tissue distribution, ligand binding affinity, and coupling to intracellular signaling pathways. The signal of EP1 predominantly increases intracellular Ca²⁺ and activates protein kinase C (PKC) (19). EP2 and EP4 activate G_s followed by increases in adenylate cyclase activity, adenosine cAMP production, and PKA activity in the target cells. In contrast, EP3 acts via G_i to inhibit cAMP generation.

PGE₂ has been proposed to be a potent stimulator of bone resorption involved in inflammatory diseases such as rheumatoid arthritis (20-23). However, the mechanism of PGE₂-induced bone resorption has not yet been clearly explained. Like other osteotropic factors, PGE₂ stimulates expression of RANKL in osteoblasts (21, 24). Among PGE₂ receptor subtypes, EP4 has been shown to mainly mediate PGE₂-induced RANKL expression in osteoblasts (24). In addition, PGE₂ directly and synergistically with RANKL and M-CSF stimulates the differentiation of mouse bone marrow-derived macrophages (BMM), osteoclast precursors, into osteoclasts (25, 26). We have recently shown that the synergistic effect of PGE₂ on RANKL-induced osteoclast differentiation is mediated through EP2 and EP4. Transforming growth factor--activated kinase 1 (TAK1) acts as an adapter molecule linking PKA-induced signals and RANKL-induced signals in osteoclast precursors (27). Thus, PGE₂ stimulates osteoclastic bone resorption through two pathways, the induction of RANKL expression by osteoblasts and direct enhancement of RANKL-induced osteoclast differentiation of the precursors.

Paradoxically, PGE₂ has been shown to inhibit bone-resorbing activity of osteoclasts when added to osteoclasts cultured on bone or dentine slices (28, 29). Lerner et al. (30) also reported that PGE₂ transiently inhibited bone resorption and the release of lysosomal enzymes in mouse calvarial cultures. This inhibitory effect of PGE₂ on bone resorption may be due to a direct activation of adenylate cyclase in mature osteoclasts, thereby mimicking the effect of CT. Thus, the role of PGE₂ in the function of mature osteoclasts is still a matter of controversy.

In the present study we explored the role of PGE₂ in osteoclast function. Osteoclast precursors of BMM expressed EP1, EP2, EP3, and EP4, but mature osteoclasts expressed only EP1. PGE₂ affected neither the actin ring-forming nor the resorption pit-forming activity of osteoclasts cultured on dentine slices. Forced expression of EP4 in osteoclasts suppressed both activities of osteoclasts. The inhibitory effects of the EP4 expression on osteoclast function were quite similar to those of CT. These results suggest that the down-regulation of EP2 and EP4 in osteoclast precursors during osteoclastic differentiation is an important event for escaping the inhibitory effect of PGE₂ on bone resorbing activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemicals—Recombinant human RANKL and M-CSF (Leukoprol) were purchased from PeproTech EC Ltd. (London) and Kyowa Hakko Kogyo Co. (Tokyo), respectively. PGE₂, bacterial collagenase, and Pronase E were obtained from Sigma. 3-Isobutyl-1-methylxanthine (IBMX) and 17-phenyltrinol-PGE₂ were from BIOMOL Research Laboratory Inc. (Plymouth Meeting, PA). 1,25(OH)₂D₃ was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Elcatonin, a synthetic analogue of eel calcitonin, was kindly provided by Asahi Kasei Pharma (Tokyo). Fura Red AM, fluo-4 AM, and Pluronic F127 were from Molecular Probes Inc. (Eugene, OR). All other chemicals were of analytical grade.

Cultures of Bone Marrow-derived Macrophages—BMM were prepared as osteoclast precursors as described previously (31). Briefly, bone marrow cells were obtained from tibiae of 5-8-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 were suspended in -modified minimum essential medium (-MEM, Sigma) supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS) on 60-mm diameter dishes for 16 h in the presence of M-CSF (100 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.

Survival Assay of Purified Osteoclasts—Osteoclasts were generated in co-cultures of mouse primary osteoblasts and bone marrow cells in collagen gel-coated dishes as described previously (32). Primary osteoblasts and bone marrow cells were co-cultured in -MEM supplemented with 10% FBS in 100-mm tissue culture dishes (Corning Inc., Corning, NY) precoated with type I-collagen gel (Nitta Gelatin, Osaka, Japan) in the presence of 1,25(OH)₂D₃ (10^-8 M). After the cells were cultured for 7 days, all cells were recovered from the dishes by treatment with 0.2% collagenase. The purity of osteoclasts in this preparation was about 5%. To purify osteoclasts the crude osteoclast preparation was plated in 100-mm tissue culture dishes. After the cells were cultured for 6 h, osteoblasts were removed by treatment of cells with phosphate-buffered saline (PBS(-)) containing 0.001% Pronase E and 0.02% EDTA for 5 min. The purity of osteoclasts in this preparation was about 95%. For the osteoclast survival assay, purified osteoclasts were further incubated for the indicated periods in the presence or absence of test chemicals and stained for tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts) as described. TRAP-positive multinuclear cells containing more than three nuclei were counted as viable osteoclasts.

Actin Ring and Pit Formation by Osteoclasts Cultured on Dentine Slices—The crude osteoclast preparation recovered from the co-cultures was placed on dentine slices (4 mm-diameter) for 2 h in 96-well plates. Dentine slices were transferred into 48-well plates and further cultured for 46 h in the presence or absence of calcitonin or PGE₂ or 17-phenyltrinol-PGE₂. For F-actin staining, cells cultured on dentine slices were fixed with 3.7% formalin diluted with phosphate-buffered saline and rinsed with phosphate-buffered saline containing 0.1% Triton X-100 (Sigma) for 1 min. F-actin in the cells was labeled with rhodamine-conjugated phalloidin (3 units/ml, Molecular Probes, Inc.) for 2 h and observed using a fluorescence microscope. For the pit formation assay, dentine slices were recovered from the culture. The surface of the dentine slices was rubbed strongly with a cotton bud to remove all cells on the slices. Ten microliters of Mayer's hematoxylin (Wako Pure Chemical Industries) was placed on the surface using surface tension for 35-45 s to visualize resorption pits. The percentage of resorbed area relative to the total area of dentine slices was determined using an image analysis system. The results were expressed as the mean ± S.D. of triplicate cultures.

RT-PCR for PGE₂ Receptor mRNAs—Total RNA was extracted from cultured mouse bone marrow cells and purified mature osteoclasts (>95% purity) using the acid guanidinium-phenol-chloroform method. cDNA was synthesized from 10 µg of the total RNA using reverse transcriptase (Revatra Ace, TOYOBO BIOCHEMICALS, Inc., Osaka) and amplified using PCR. Primers used in PCR for EP receptor isoforms were as follows: EP1, 5'-TTAACCTGAGCCTAGCGGATG-3' (forward) and 5'-CGCTGAGCGTATTGCACACTA-3' (reverse) for an EP1 fragment of 669 bp; EP2, 5'-CCACGATGCTCCTGCTGCTT-3' (forward) and 5'-CAGCCCCTTACACTTCTCCAATGA-3' (reverse) for an EP2 fragment of 554 bp; EP3, 5'-TGACCTTTGCCTGCAACCTG-3' (forward) and 5'-AGCTGGAAGCATAGTTGGTG-3' (reverse) for an EP3 fragment of 359 bp; EP3, 5'-TGACCTTTGCCTGCAACCTG-3' (forward) and 5'-GAGCCAGGGAAACAGGTACT-3' (reverse) for an EP3 fragment of 401 bp; EP3, 5'-TGACCTTTGCCTGCAACCTG-3' (forward) and 5'-AGACAATGAGATGGCCTGCC-3' (reverse) for an EP3 fragment of 410 bp; EP4, 5'-GGTCATCTTACTCATCGCCACCTCTC-3' (forward) and 5'-TCCCACTAACCTCATCCACCAACAG-3' (reverse) for an EP4 fragment of 599 bp; mouse calcitonin receptor, 5'-TTTCAAGAACCTTAGCTGCCACAG-3' (forward) and 5'-ACGCGGACAATGTTGAGAAG-3' (reverse) for a fragment of 562 bp; mouse glyceraldehyde-3-phosphate dehydrogenase, 5'-ACCACAGTCCATGCCATCAC-3' (forward) and 5'-TCCACCACCCTGTTGCTGTA-3' (reverse) for a fragment of 562 bp. The PCR conditions for EP subtypes were denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, and primer extension at 75 °C for 60 s for 34 cycles. The PCR conditions for calcitonin receptors were denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and primer extension at 72 °C for 60 s for 34 cycles. The PCR conditions for glyceraldehyde-3-phosphate dehydrogenase were denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and primer extension at 72 °C for 60 s for 28 cycles. The PCR products were subjected to electrophoresis in a 2% agarose gel followed by staining with ethidium bromide. We confirmed that cDNA prepared from mouse primary osteoblasts was specifically amplified using the primers for each EP isoform. Each amplified cDNA was verified as the published EP isoform (EP1, GenBank^TM accession number D16338 [GenBank] ; EP2, GenBank^TM accession number NM_008964 [GenBank] ; EP3, GenBank^TM accession number D10204 [GenBank] ; EP3, GenBank^TM accession number D13321 [GenBank] ; EP3, GenBank^TM accession number D17406 [GenBank] ; EP4, GenBank^TM accession number BC011193 [GenBank] ) by DNA sequencing (data not shown).

Real-time PCR—cDNA (20 ng) was utilized for 40-cycle 2-step PCR in an DNA Engine Opticon system (MJ Japan Ltd., Tokyo) using a SYBR Green qPCR kit (Finnzymes oy, Espoo, Finland) and 300 nM primers. The amplicon size and reaction specificity was confirmed by agarose gel electrophoresis. Each transcript in the samples was assayed three times, and the fold-change ratios between experimental and control samples for each gene used in the analysis were calculated. The results were expressed as the mean ± S.D. of three assays. The sequences of primers for EP receptor isoforms were as follows: EP2, 5'-ATGCTCCTGCTGCTTATCGT-3' (forward) and 5'-TAATGGCCAGGAGAATGAGG-3' (reverse); EP4, 5'-CCATCGCCACATACATGAAG-3' (forward) and 5'-TGCATAGATGGCGAAGAGTG-3' (reverse).

Assay of cAMP Production—To measure the amount of cAMP produced, cells cultured in 48-well plates (BMM, 8 x 10⁴/well; purified osteoclasts, 2 x 10⁴ cells/well) were preincubated for 5 min at 37 °C in -MEM containing 1 mM IBMX, then incubated for 5 min at 37 °C with elcatonin (10^-10 M) or PGE₂ (10^-6 M). Cells were washed with ice-cold phosphate-buffered saline containing 1 mM IBMX and then lysed. The content of intracellular cAMP was determined using a cAMP enzyme immunoassay kit (Amersham Biosciences) according to the manufacturer's instructions. The results were expressed as the mean ± S.D. of quadruplicate cultures.

Measurement of Intracellular Ca²⁺ Influx—The effects of PGE₂ and calcitonin on intracellular Ca²⁺ influx in mature osteoclasts and in BMM were measured using a confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany) according to the methods described previously (27). Osteoclasts or BMM were incubated in a glass-bottom dish (ASAHI TECHNO GLASS Corp., Tokyo) for 6 h. Cells were incubated in the presence of 5 µM fluo-4 AM, 5 µM Fura Red AM, and 0.05% pluronic F127 for 30 min in serum-free Dulbecco's modified Eagle's medium (Sigma). Cells loaded with these dyes were washed twice with -MEM and postincubated in -MEM containing 10% FBS. Cells were further washed three times with Hanks' balanced salt solution and then excited at 488 nm, and emission at 505-530 nm for fluo-4 and 600-680 nm for Fura Red were acquired simultaneously at 2-s intervals. The ratio of the fluorescence intensity of the fluo-4 to Fura Red was calculated to estimate intracellular Ca²⁺ concentrations in single cells.

Construction of Adenovirus—A pBluescript vector containing the entire open reading frame of EP4 was a kind gift of Dr. S. Narumiya (Kyoto University, Japan). The EP4 cDNA was inserted into the ApaI-XbaI site of a p-shuttle vector (BD Clontech Laboratories, Inc. Palo Alto, CA), and the DNA fragment was inserted into the PI-SceI/I-CeuI site of a pAdenoX vector (BD Clontech Laboratories, Inc.). Two micrograms of the pAdenoX vector containing EP4 cDNA linearized by PacI was transfected into HEK293 cells (ATCC, Manassas, VA) using TransFast transfection reagents (Promega Corp., Madison, WI). Amplified crude viral stocks were purified by CsCl gradient ultracentrifugation and used for the infection. Adenovirus carrying -galactosidase cDNA (Ad-LacZ) was also generated and used for the control infection. The infectious units (ifu) were determined using an Adeno-X rapid titer kit (BD Clontech Laboratories, Inc.).

Infection of Osteoclasts with Adenovirus Carrying EP4 cDNA—Infection of osteoclasts with Ad-LacZ or adenovirus carrying EP4 cDNA (Ad-EP4) was carried out according to the methods described previously (33). Primary osteoblasts and bone marrow cells were co-cultured for 4 days in collagen gel-precoated dishes (100-mm diameter). Co-cultures were then incubated for 1 h with 2 ml of -MEM containing the indicated ifu of recombinant adenoviruses and 10% FBS and further incubated with 15 ml of -MEM with 10% FBS in the presence of 1, 25(OH)₂D₃ (10^-8 M) for an additional 48 h.

Statistical Analysis—For the determination of cAMP production, data were expressed as the mean ± S.D. of quadruplicate cultures. For the real-time PCR analysis, osteoclast survival assay, and pit forming assay, representative data were expressed as the mean ± S.D. of three determinations. All experiments were independently repeated three times, and similar results were obtained. Statistical analyses were performed using Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of PGE₂ Receptor Subtypes in Osteoclast Precursors and Mature Osteoclasts—We first analyzed the expression of PGE₂ receptor subtypes in BMM, osteoclast precursors, and mature osteoclasts using RT-PCR. BMM were prepared from bone marrow cultures treated with M-CSF (see Fig. 1C). Purified mature osteoclasts were prepared from collagen-gel co-cultures of calvarial osteoblasts and bone marrow cells treated with 1,25(OH)₂D₃. BMM expressed EP1, EP2, EP3, and EP4 mRNAs, whereas purified osteoclasts expressed only EP1 mRNA (Fig. 1A). CT receptor mRNA was expressed in osteoclasts but not in the precursors. In our experiments co cultures were treated for 7 days with 1,25(OH)₂D₃ to induce formation of osteoclasts. Ikegami et al. (34) showed that treatment of mouse peritoneal macrophages with PGE₂ down-regulated EP4 mRNA expression within 12 h in a cAMP/PKA-dependent manner. These results raise the possibility that 1,25(OH)₂D₃ or endogenously produced PGE₂ may down-regulate EP4 and EP2 expression in osteoclast precursors. Next, we examined the effects of PGE₂ and 1,25(OH)₂D₃ on the expression of EP2 and EP4 mRNAs in BMM in the presence of M-CSF (Fig. 1B). PGE₂ (10^-5 M) and 1,25(OH)₂D₃ (10^-8 M) significantly decreased the expression of EP4 mRNA but not EP2 mRNA in BMM (Fig. 1B). Treatment of BMM with RANKL induced osteoclastic differentiation of BMM in the presence of M-CSF. Expression of both EP2 and EP4 mRNAs was significantly down-regulated in BMM treated with RANKL for 24 h (Fig. 1B). Parallel experiments showed that the purified osteoclast preparation and the BMM culture treated with RANKL for 72 h contained more than 95 and 65% TRAP-positive cells, respectively (Fig. 1C). The BMM culture treated with RANKL for 24 h contained less than 2% TRAP-positive cells (data not shown). These results suggest that EP2 and EP4 are down-regulated in osteoclasts during osteoclastic differentiation.

View larger version (75K): [in this window] [in a new window]   FIG. 1. Expression of PGE2 receptor subtypes in osteoclast precursors and mature osteoclasts. A, RT-PCR analysis of PGE receptor subtypes in BMM and mature osteoclasts. Purified osteoclasts were prepared from co-cultures grown on collagen gel-coated dishes as described under "Experimental Procedures." Total RNA was extracted from BMM and purified osteoclasts, and cDNA was synthesized from the total RNA. The expression of EP1, EP2, EP3, EP3, EP3, EP4, CT receptor, and glyceraldehyde-3-phosphate dehydrogenase mRNAs was detected by PCR in the presence (+) or absence (-) of reverse transcriptase. B, effects of PGE2, 1, 25(OH)2D3, or RANKL on expression of EP2 and EP4 mRNAs in BMM. BMM prepared from bone marrow cells were cultured with or without PGE2 (10-5 M), 1, 25(OH)2D3 (10-8 M), or RANKL (100 ng/ml) in the presence of M-CSF (50 ng/ml) for 24 h or cultured with or without RANKL (100 ng/ml) in the presence of M-CSF for 72 h. Total RNA was extracted from these cultures. The expression levels of EP2 mRNA (upper panel) and of EP4 mRNA (lower panel) were determined by quantitative real-time RT-PCR as described under "Experimental Procedures." The ratio of the expression in the culture treated with PGE2, 1,25(OH)2D3 or RANKL to that in the control culture was calculated. Results are expressed as the mean ± S.D. of triplicate cultures. * and #, Significantly different between the treated and the control culture; *, p < 0.01; #, p < 0.05. C, TRAP staining of BMM, purified osteoclasts, and BMM treated with RANKL and M-CSF for 72 h in parallel experiments to those in A and B. Percentages of TRAP-positive cells in each cell preparation were expressed as the mean ± S.D. of quadruplicate cultures and are described below the panels. Bar, 25 µm.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Effects of PGE2 and CT on cAMP production and calcium signaling in osteoclast precursors and mature osteoclasts. A, effects of PGE2 and CT on cAMP production in BMM (upper panel) and purified osteoclasts (lower panel). Cells were preincubated for 5 min with IBMX (1 mM) and incubated for 5 min with PGE2 (10-6 M) or CT (10-10 M). The amount of intracellular cAMP was determined by enzyme-linked immunosorbent assay. Results are expressed as the mean ± S.D. of quadruplicate cultures. *, Significantly different from the control culture; p < 0.01. B-D, crude osteoclast and BMM preparations were loaded with fluo-4 AM and Fura Red AM and subjected to single-cell calcium measurements. 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 (down arrow) of PGE2 (10-5 M) or CT (10-10 M). B, typical confocal images of an osteoclast before (left panels) and after the addition of PGE2 for 16 s (right panels). The images of fluo-4 and those of Fura Red are merged in the lower panels. The ratio of the fluorescence intensity of the fluo-4 to Fura Red was calculated to estimate intracellular Ca2+ influx in single osteoclasts treated with PGE2 (C) and CT (D) and in single BMM treated with PGE2 (E). The results represent calcium signals in five to six single cells treated with PGE2 or CT.

View larger version (50K): [in this window] [in a new window]   FIG. 3. Effects of PGE2, RANKL, and CT on the survival of osteoclasts. A, time course of the survival of osteoclasts treated with RANKL, CT, or PGE2. The crude osteoclast preparation obtained from the collagen gel culture was plated for 6 h in 24-well plates. The cells were treated with Pronase E/EDTA solution to remove osteoblasts. The purified osteoclasts were further cultured in the presence or absence of RANKL (100 ng/ml), CT (10-9 M), and PGE2 (10-6 M). After culturing for the indicated periods, cells were fixed and stained for TRAP. TRAP-positive multinuclear cells containing more than three nuclei were counted as surviving osteoclasts. The values were expressed as the mean ± S.D. of triplicate cultures. *, Significantly different from the control culture; p < 0.01. B, TRAP staining of purified osteoclasts incubated for 36 h with or without PGE2 (10-6 M), RANKL (100 ng/ml), or CT (10-9 M). Bar, 25 µm. C, the effects of a higher dose of PGE2 and EP1 agonist on the survival of osteoclasts. Purified osteoclasts were cultured for 24 h in the presence or absence of PGE2 (10-5 M), 17-phenyltrinol-PGE2 (an EP1 agonist, 10-5 M), or RANKL (100 ng/ml). Cells were then fixed and stained for TRAP. The number of surviving TRAP-positive multinucleated cells was counted. The values were expressed as the mean ± S.D. of triplicate cultures. *, Significantly different from the control culture; p < 0.01.

View larger version (62K): [in this window] [in a new window]   FIG. 4. Effect of PGE2 on actin ring formation and pit formation by osteoclasts. Crude osteoclast preparations were cultured on dentine slices in the presence or absence of PGE2 (10-7-10-5 M), 17-phenyltrinol-PGE2 (an EP1 agonist, 10-6 and 10-5 M), or CT (10-10 M). After cells were cultured for 46 h the dentine slices were recovered. A, some dentine slices were processed for F-actin staining (upper panels, bar = 25 µm). The other slices were processed for Mayer's hematoxylin staining to visualize resorption pits (lower panels, bar = 100 µm). B, percentages of resorbed area on dentine slices. Percentages of resorbed area relative to the total surface were determined using an image analysis system. The values were expressed as the mean ± S.D. of triplicate cultures. *, Significantly different from the control cultures; p < 0.01.

Inhibition of Osteoclast Function by Forced Expression of EP4 —Our final question was why osteoclast precursors lose EP2 and EP4 during the differentiation into osteoclasts; that is, what are the functional consequences of the disappearance of EP2 and EP4 in osteoclasts. To address this question, EP4 was expressed in osteoclasts using Ad-EP4 (Fig. 5). RT-PCR analysis showed that osteoclasts purified from the co-cultures that had been infected with Ad-EP4 at 10⁹ ifu expressed high levels of EP4 mRNA, but those infected with Ad-LacZ did not (Fig. 5A). To examine whether EP4 expressed in the infected osteoclasts is functional, the concentration of intracellular cAMP was determined in Ad-EP4- and Ad-LacZ-infected osteoclasts in the presence or absence of exogenous PGE₂ (Fig. 5B). Intracellular cAMP levels were significantly higher in osteoclasts infected with Ad-EP4 than in those infected with Ad-LacZ even in the absence of exogenously added PGE₂ (Fig. 5B). PGE₂ (10^-6 M) significantly increased cAMP production in Ad-EP4-infected osteoclasts but not in Ad-LacZ-infected osteoclasts. These results suggest that the EP4 expressed in osteoclasts is functionally active.

We finally examined the effects of the forced expression of EP4 on the function of osteoclasts (Fig. 6). Crude osteoclast preparations from co-cultures infected with Ad-EP4 or Ad-LacZ were placed on dentine slices, and the cells were further cultured for 48 h in the presence or absence of PGE₂ or CT. Similar numbers of TRAP-positive cells were observed on the slices cultured with cells expressing Ad-EP4 and Ad-LacZ (Fig. 6A). However, the number of actin rings formed in EP4-expressing osteoclasts was markedly decreased. When PGE₂ (10^-6 M) was added to the culture, actin rings in osteoclasts expressing Ad-LacZ remained unchanged, but those in osteoclasts expressing Ad-EP4 disappeared completely (Fig. 6A). The pit-forming activity of osteoclasts was not affected by Ad-LacZ but was markedly inhibited by Ad-EP4 infection in an ifu-dependent manner (Fig. 6, B and C). The addition of PGE₂ (10^-6 M) to the cultures of osteoclasts infected with Ad-LacZ had no inhibitory effects on the pit-forming activity (Fig. 6, B and C). However, PGE₂ significantly suppressed the pit-forming activity of osteoclasts infected with Ad-EP4 at 1 x 10⁸ ifu. CT (10^-10 M) completely inhibited the pit-forming activity of osteoclasts infected with either Ad-LacZ or Ad-EP4 (Fig. 6C). Thus, EP4-mediated signals were similar to those mediated by CT receptors in osteoclasts.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effect of forced expression of EP4 in osteoclasts on the cAMP production. Co-cultures grown on collagen gel-coated dishes (100-mm diameter) were infected with either Ad-LacZ or Ad-EP4 on day 4 at a dose of 109 ifu. A, expression of EP4 in purified osteoclasts infected with Ad-LacZ (lane 1) or Ad-EP4 (lane 2). Osteoclasts were purified on 35-mm dishes. Total RNA was extracted from purified osteoclasts and used for RT-PCR with an EP4 receptor-specific primer that hybridized to both endogenous and exogenous EP4 mRNAs. B, effect of PGE2 on cAMP production in osteoclasts infected with Ad-LacZ or Ad-EP4. Purified osteoclasts were preincubated for 5 min with IBMX (1 mM) and incubated for 5 min with PGE2 (10-6 M). The amount of intracellular cAMP was determined by enzyme-linked immunosorbent assay. The values were expressed as the mean ± S.D. of quadruplicate cultures. *, Significantly different from the control culture infected with Ad-LacZ; p < 0.01. #, Significantly different from the control culture infected with Ad-EP4; p < 0.05. G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RT-PCR analysis showed that the level of expression of EP2 and EP4 mRNAs in mature osteoclasts was much lower than that in BMM (Fig. 1). It was reported that PGE₂ suppressed the expression of EP4 mRNA in mouse peritoneal macrophages in a cAMP/PKA-dependent manner (34). In our experiments PGE₂ and 1,25(OH)₂D₃ significantly decreased the expression of EP4 mRNA but not EP2 mRNA in BMM within 24 h (Fig. 1). On the other hand expression of both EP2 and EP4 mRNAs was down-regulated in BMM treated with RANKL for 24 h. TRAP-positive osteoclasts appeared on day 3 but not day 1 in BMM cultures treated with RANKL and M-CSF. These results suggest that osteotropic factors can modulate the expression of EP subtypes in osteoclast precursors, but down-regulation of EP2 and EP4 in osteoclasts is a consequence of the decision to differentiate into osteoclasts, probably for other appropriate purposes.

View larger version (44K): [in this window] [in a new window]   FIG. 6. Effect of forced expression of EP4 in osteoclasts on their actin ring- and pit-forming activities. Co-cultures grown on collagen gel-coated dishes (100-mm diameter) were infected with either Ad-LacZ or Ad-EP4 on day 4 at doses of 108 or 109 ifu. Osteoclast preparations were cultured on dentine slices for 48 h in the presence or absence of PGE2 (10-6 M) or CT (10-10 M). A, some dentine slices were processed for TRAP and F-actin staining. Bars, 25 µm. B and C, the other slices were processed for Mayer's hematoxylin staining to visualize resorption pits. B, resorption pits on dentine slices recovered from Ad-LacZ- or Ad-EP4-infected cultures in the presence or absence of PGE2. Bar, 100 µm. C, percentages of resorbed area on dentine slices. Percent resorbed area relative to the total surface was determined using an image analysis system. The values were expressed as the mean ± S.D. of triplicate cultures. *, Significantly different from the control culture infected with Ad-LacZ; p < 0.01. #, Significantly different from the control culture infected with Ad-EP4 (108 ifu); p < 0.05.

CT receptors are coupled to G_s and G_q proteins that activate cAMP/PKA and Ca²⁺/PKC signals, respectively. Nicholson et al. (41) reported that forskolin, an activator of adenylate cyclase, induced a synergistic effect with CT in stimulating cAMP production in isolated rat osteoclasts and augmented the hypocalcemic response to CT in vivo. We also showed that cAMP/PKA signals were involved in the CT-induced inhibition of actin ring formation and pit formation by osteoclasts (17). Kanaoka et al. (42) reported that CT inhibited nitric oxide-induced apoptosis of osteoclasts in vitro through cAMP/PKA signals. Thus, cAMP/PKA signals play important roles in the inhibition of osteoclast function and the promotion of osteoclast survival induced by CT.

View larger version (28K): [in this window] [in a new window]   FIG. 7. Importance of disappearance of EP2 and EP4 in mature osteoclasts. Osteoclast precursors express functionally active EP1, EP2, and EP4 PGE2 receptors. PGE2 synergistically stimulates RANKL-induced osteoclastic differentiation of the precursors through EP2- and EP4-mediated signals. The precursors down-regulate the expression of their own EP2 and EP4 during their differentiation into osteoclasts. Osteoclasts continuously express EP1, but the role of EP1 in osteoclast function is not known. When EP4 is expressed in osteoclasts, PGE2 directly inhibits the pit-forming activity of the osteoclasts through a cAMP/PKA-dependent mechanism. Thus, the EP4-mediated signals in osteoclasts are similar to those mediated by CT receptors. Thus, PGE2 becomes a potent bone resorption-stimulating factor as a result of the disappearance of EP2 and EP4 from mature osteoclasts. The down-regulation of EP2 and EP4 in osteoclast precursors during osteoclastic differentiation is an important event for escaping the inhibitory effect of PGE2 on bone resorption. CTR, calcitonin receptor.

Several reports have indicated that PGE₂ acts as a direct inhibitor of bone resorption in isolated rat, chick, and rabbit osteoclasts (28, 29, 43). A pharmacological study using specific agonists for each EP subtype showed that isolated rat osteoclasts possess EP2 (44). Rabbit osteoclasts have been shown to express EP4 (29). We used mouse osteoclasts formed in vitro, but not authentic osteoclasts, in this study. Therefore, it is possible that authentic mouse osteoclasts may express EP2 and EP4 and decrease their function in response to PGE₂. However, even if osteoclasts express EP2 and EP4, the expression level must be extremely low. PGE₂ does not inhibit but rather stimulates bone resorption in a mouse organ culture system using fetal or newborn calvariae and long bones (45-48). Unlike PGE₂, calcitonin completely inhibits not only osteotropic factor-induced bone resorption but also spontaneously occurring bone resorption in such organ culture systems. These results further support our conclusion that osteoclast precursors lose EP2 and EP4 to escape the inhibitory effects of PGE₂ on bone resorption (Fig. 7).

We and others have shown that PGE₂ synergistically enhanced RANKL-induced osteoclastic differentiation of BMM through EP2 and EP4 (25-27). Recently, we found that TAK1 (transforming growth factor--activated kinase 1), a signal adapter molecule, plays important roles in cross-talk between cAMP-PKA signals and RANKL-induced signals in PGE₂ enhancement of RANKL-induced osteoclast differentiation (27). Our results suggest that osteoclast precursors express EP2 and EP4, through which PGE₂ synergistically enhances RANKL-induced osteoclast differentiation (Fig. 7). After the differentiation into osteoclasts is decided, osteoclasts would lose EP2 and EP4 to escape the inhibitory effects of PGE₂. In addition, PGE₂ strongly induces RANKL expression in osteoblasts through EP2/EP4-mediated signals. Thus, PGE₂ is a potent bone resorption factor with multiple effects on osteoclast precursors and osteoblasts.

In contrast to the above in vitro studies, many in vivo studies have shown that the PGE series has stimulatory effects on bone formation (49-57). Sibonga et al. (52) and Keila et al. (53) independently reported that administration of PGE₂ even into aged rats increased bone mass due to an increase in osteoblasts over osteoclasts. It was also demonstrated that PGE₂ and bipedal stance exercise synergistically prevented cancellous bone loss induced by ovariectomy in aged rats (54). Local administration of PGE₁ has been shown to increase alveolar bone thickness and bone formation in beagle dogs (55). Paralkar et al. (56) reported that a selective EP2 agonist mimics the anabolic effects of PGE₂ on bone when directly injected into the bone marrow of rats. By infusing PGE₂ to mice lacking each of four PGE receptor subtypes, Yoshida et al. (57) have identified EP4 as the receptor that mediates bone formation in response to this agent. These results suggest that the anabolic action as well as catabolic action of PGE₂ in bone is linked to an elevated level of cAMP. In normal bone remodeling, osteoblastic bone formation follows osteoclastic bone resorption and occurs in a precise and quantitative manner. PGE₂-induced bone resorption may directly induce bone formation. The findings obtained from in vivo studies suggest that EP4 and EP2 are targets for therapeutic intervention in metabolic bone diseases. Further studies will be necessary to elucidate the molecular mechanism of PGE₂-induced bone formation in vivo.

	FOOTNOTES

 
^* This work was supported in part by Grants-in-aid 16659578 and 12137209 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.

^** 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-B; M-CSF, macrophage colony-stimulating factor; PGE₂, prostaglandin E₂; PKA, protein kinase A; PKC, protein kinase C; CT, calcitonin; ifu, infectious unit; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; BMM, bone marrow-derived macrophages; F-actin, filamentous actin; -MEM, -modified minimum essential medium; FBS, fetal bovine serum; TRAP, tartrate-resistant acid phosphatase; IBMX, 3-isobutyl-1-methylxanthine; RT, reverse transcription; Ad-LacZ, adenovirus carrying -galactosidase cDNA; Ad-EP4, adenovirus carrying EP4 cDNA.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Shuh Narumiya (Kyoto University) for the generous gifts of cDNA of EPs.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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