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J Biol Chem, Vol. 275, Issue 11, 8126-8132, March 17, 2000

Cloning and Function of Rabbit Peroxisome Proliferator-activated Receptor / in Mature Osteoclasts^*

Hiroshi Mano^§, Chiharu Kimura^¶, Yukio Fujisawa^¶, Takashi Kameda, Mikiko Watanabe-Mano, Hironori Kaneko, Toshio Kaneda, Yoshiyuki Hakeda, and Masayoshi Kumegawa

From the  Department of Oral Anatomy, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-02, Japan, the ^§ Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156, Japan, and the ^¶ Molecular Pharmacology Laboratory, Pharmaceutical Research Division, Pharmaceutical Group, Takeda Chemical Industries, Ltd., 2-17-85 Juso-Honmachi, Yodogawa, Osaka 532, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Osteoclasts modulate bone resorption under physiological and pathological conditions. Previously, we showed that both estrogens and retinoids regulated osteoclastic bone resorption and postulated that such regulation was directly mediated through their cognate receptors expressed in mature osteoclasts. In this study, we searched for expression of other members of the nuclear hormone receptor superfamily in osteoclasts. Using the low stringency homologous hybridization method, we isolated the peroxisome proliferator-activated receptor / (PPAR/) cDNA from mature rabbit osteoclasts. Northern blot analysis showed that PPAR/ mRNA was highly expressed in highly enriched rabbit osteoclasts. Carbaprostacyclin, a prostacyclin analogue known to be a ligand for PPAR/, significantly induced both bone-resorbing activities of isolated mature rabbit osteoclasts and mRNA expression of the cathepsin K, carbonic anhydrase type II, and tartrate-resistant acid phosphatase genes in these cells. Moreover, the carbaprostacyclin-induced bone resorption was completely blocked by an antisense phosphothiorate oligodeoxynucleotide of PPAR/ but not by the sense phosphothiorate oligodeoxynucleotide of the same DNA sequence. Our results suggest that PPAR/ may be involved in direct modulation of osteoclastic bone resorption.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mature osteoclasts are differentiated from hemopoietic stem cells and fuse each other into multinucleate giant cells. These cells are characterized by a combination of some unique properties. They possess tartrate-resistant acid phosphatase (TRAP)¹ activity, an abundance of cathepsin K, and highly developed ion transport systems (1, 2). Mature osteoclasts are involved in bone metabolism, especially they resorb mineralized bone surfaces under both physiological and pathological conditions (3).

Much evidence obtained from in vitro studies has demonstrated that the differentiation of osteoclasts is controlled by various factors, such as vitamin D₃, osteoclast differentiation factor, osteoclastogenesis inhibitory factor, macrophage colony-stimulating factor, and parathyroid hormone (4-7). The osteoclast is also active in postmenopausal osteoporosis (bone loss), a major health problem (2). Estrogen deficiency has long been recognized as a cause of postmenopausal osteoporosis, and estrogen replacement therapy is an effective treatment for the prevention of bone loss. Research has shown that estrogen loss promotes interleukin-6 secretion in nonosteoclastic cells and that interleukin-6 up-regulates osteoclast differentiation and formation (8, 9). However, the function and metabolism of fully mature osteoclasts remain unclear, especially at the molecular level, because of the difficulty in obtaining large quantities of highly enriched osteoclasts for use in such in vitro studies.

For assessment of the bone-resorbing activity of mature osteoclasts, it would be desirable to have a cell suspension consisting of highly enriched mature osteoclasts that could be cultured on a mineralized substratum such as a bone or dentine slice. Previously, we succeeded in isolating the highly enriched mature osteoclasts by treating unfractionated rabbit bone cells cultured on plastic dishes with Pronase E/EDTA (10). However, it was impossible to detach these isolated osteoclasts from the substratum in order to prepare the cell suspension. Recently, we established a method to isolate mature rabbit osteoclasts with high purity (>99%) from a collagen gel to obtain them in suspension form for subsequent incubation on dentine slices (11). Using these highly enriched mature osteoclasts, we clarified that estrogen directly inhibited bone resorption and cathepsin K gene expression and induced osteoclastic apoptosis through estrogen receptor  (12, 13). Moreover, we demonstrated that retinoids activated both osteoclastic bone resorption and gene expression of cathepsin K, and of retinoic acid receptor  and retinoid X receptor  were expressed in mature osteoclasts (14).

Receptors for steroid hormones, retinoids, vitamin D, thyroid hormone, and prostanoids comprise a superfamily of regulatory proteins that are structurally and functionally related. Previous studies showed that members of the nuclear hormone receptor superfamily shared high homology in their amino acid sequences, with the highest homology conserved among their DNA binding domains. Nuclear hormone receptors bind to cis-acting elements in the promoters of their target genes and modulate gene expression in response to their ligands (15, 16). Molecular cloning studies revealed both new nuclear receptors and orphan nuclear receptors, the natural ligands of which remain unclear. These findings suggest potentially novel signal pathways in the body (17).

In order to examine the possibility of such a novel signal pathway in mature osteoclasts, we screened for nuclear hormone receptors in a rabbit mature osteoclast cDNA library by taking advantage of the structural homology of the DNA-binding domain of nuclear receptors. Thereby, the rabbit cDNA of peroxisome proliferator-activated receptor (PPAR) / was cloned from this cDNA library.

PPAR is known to be activated by arachidonic metabolites. Three types of PPAR are now known to exist, i.e. PPAR, PPAR, and PPAR/. PPAR is abundantly expressed in liver tissue and is activated by leukotriene B₄ (18). PPAR is abundantly expressed in adipose tissue and is activated by 15-deoxy-prostaglandin J₂, 9- and 13-hydroxyoctadecadienoic acid, and thiazolidinediones (19, 20). The distribution of PPAR/ mRNA is the most widespread, and prostacyclin and its analogues activate PPAR/ (21, 22). Although PPAR plays a principal role in adipogenesis, it is unclear what functions PPAR and PPAR/ have in the body.

Here we demonstrate the abundant expression of PPAR/ mRNA in mature osteoclasts and the activation of osteoclastic functions such as bone resorption and osteoclastic gene expression by a ligand of PPAR/. Moreover, antisense S-ODN for PPAR/ blocked this activation in mature osteoclasts. These results suggest that in part PPAR/ plays a role in osteoclastic bone resorption.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of Mature Osteoclasts from Rabbit Long Bones-- Unfractionated bone cells were isolated from tibiae, femora, humeri, ulnae, and radii of 10-day-old rabbits. After the removal of soft tissues, these bones were minced in -minimum essential medium (-MEM). Cells were dissociated from the bone fragments by sedimentation under normal gravity for 1 min. The supernatant was used as unfractionated bone cells (crude osteoclasts). These cells were plated in 10-cm tissue culture dishes coated with 0.24% collagen gel (Nitta Gelatin, Tokyo, Japan) in -MEM supplemented with 5% fetal bovine serum (FBS) and incubated for 3 h. Nonosteoclastic cells were then removed from the gel by sequential treatment with 0.001% Pronase E, 0.02% EDTA, and 0.01% collagenase. The remaining osteoclasts were subsequently collected by treatment with 0.1% collagenase. After having been washed with calcium- and magnesium-free phosphate-buffered saline (PBS), the isolated osteoclasts were replated onto dentine slices in phenol red-free -MEM supplemented with 0.1% bovine serum albumin for generating cDNA libraries and used in the pit assay (11).

The purity of the isolated osteoclasts was confirmed by microscopic scoring of TRAP-positive multinucleate cells in the presence of 50 mM L-tartrate with a leukocyte-acid-phosphatase kit (Sigma). When TRAP-positive multinucleate cells isolated from collagen gels were placed on dentine slices, they showed a compact and round shape, and the morphology of the cells resembled that of osteoclasts in vivo more than the cells cultured on plastic plates (10). These TRAP-positive multinucleate cells (100 cells/culture drop) were cultured in tissue-culture dishes for 7 days to assess formation of colonies of nonosteoclastic stromal cells. Only 0.4 ± 0.49 (n = 5) colonies were formed per culture drop after a 1-week culture. Thus, the purity of the TRAP-positive multinucleate cells was more than 99%. When these cells were cultured on dentine slices, resorption pits were formed around these cells as observed under a scanning electron microscope. Transmission electron microscopic observation revealed a ruffled border-like membrane and clear zone on the side of these cells attached to the surface of the dentine. Thus, cells isolated from collagen gels exhibited the typical morphology of authentic osteoclasts, and the multinucleate cells had the ability to excavate dentine even when stromal cells were absent from the culture. These TRAP-positive multinucleate cells were indeed mature osteoclasts (data not shown).

Molecular Cloning of Rabbit PPAR/ cDNA from Mature Osteoclasts-- Total RNA of mature rabbit osteoclasts cultured on dentine slices was isolated by the acid guanidium thiocyanate-phenol-chloroform method, and the poly(A)⁺ RNA was purified by using an oligo(dT) cellulose column. The cDNA library was synthesized by using a ZAP II-cDNA Synthesis Kit (Stratagene). An EcoT14I-HindIII DNA fragment containing the DNA-binding domain of the human estrogen receptor  (ATCC, Manassas, VA) was used as a probe to screen the ZAP II rabbit mature osteoclast cDNA library under low stringency conditions. Positive clones were sequenced by the dideoxy sequencing method (23, 24).

Pit Assay for Assessment of Bone-resorbing Activity of Mature Osteoclasts-- To determine the bone-resorbing activity of the isolated mature osteoclasts, we measured the area and number of resorption pits formed on dentine slices by them. Briefly, isolated osteoclasts were plated on dentine slices (circular, 6 mm in diameter) in 96-well plates at a density of 150 isolated osteoclasts/slice/well and cultured for 1 h in phenol red-free -MEM supplemented with 0.1% bovine serum albumin at 37 °C in 10% CO₂. The medium was then replaced with fresh medium containing various concentrations of any given reagent and cultured for 20 h (25). The dentine slices were then brushed with a rubber policeman to remove the cells and stained with acid hematoxylin for 5 min. The total number of pits on a dentine slice was counted under a light microscope, and the total area of pits was quantified by densitometric analysis of dentine slice images by videomicroscopy and using of NIH Image software (25).

Oligodeoxynucleotide Uptake by Osteoclasts by Use of Lipofectin-- Antisense or sense S-ODNs of PPAR/ were used to transfect mature osteoclasts by the method of Inui et al. (26). The PPAR/ antisense S-ODN was designed to be composed of 21 bases and to target the region that spans the translation start codon of rabbit PPAR/ mRNA (5'-cggaggctgctccatggctga-3'). The sense counter part of the antisense S-ODN was also designed as a negative control (5'-tcagccatggagcagcctccg-3'). All of these S-ODNs were synthesized (Nippon Flour Mills Co., Ltd., Tokyo, Japan) on an automated solid-phase nucleotide synthesizer and subsequently purified and sterilized. A fraction of the synthesized S-ODNs was labeled with fluorescein at the 5'-ends, in order to confirm the efficiency of S-ODN uptake into mature osteoclasts.

Mature rabbit osteoclasts isolated by the method described above were incubated at 37 °C in 10% CO₂ on dentine slices at a concentration of 150 osteoclasts/dentine slice in 96-well tissue culture plates in the presence or absence of carbaprostacyclin (10⁸ M) (Cayman Chemical Company) in -MEN containing 5% FBS. After a 2-h incubation, the medium was exchanged for the transfection medium, and the cells were incubated in the presence or absence of carbaprostacyclin (10⁸ M) and in the presence or absence of the antisense or sense S-ODNs in serum-free -MEM containing 100 nM Tfx^TM-50 (Promega, Madison, WI). After an 8-h incubation, the medium in each well was exchanged for the identical medium except that the -MEM now contained 5% FBS, and the incubation was then continued for an additional 12 h. After the incubation, the pit number and area were determined.

RNA Expression in Mature Osteoclasts-- Unfractionated bone cells were plated into 90-mm tissue culture dishes with -MEM plus 5% FBS at 10⁸ cells/dish. After an overnight culture period, the plates were washed with PBS to remove nonadherent cells such as hematopoietic cells. The cells were then incubated with PBS containing 0.001% Pronase E, 0.02% EDTA for 20 min at 37 °C to remove nonosteoclastic cells. These detached nonosteoclastic cells were saved as the source of stromal cells. The purity of the osteoclasts in the dishes was more than 99%.

Isolated osteoclasts on the tissue culture dishes were cultured with carbaprostacyclin (1 × 10⁸ M) for 5 h in -MEM containing 0.1% bovine serum albumin for the study of target gene expression elicited by prostacyclin. Other cells were cultured on dishes with antisense or sense S-ODNs and 100 nM Tfx-50 (Promega, WI) as a carrier for 20 h in -MEM containing 0.1% bovine serum albumin for the study of the effect of S-ODN. The total RNA was extracted from the cultured cells by the acid guanidinium-phenol-chloroform method. Total RNA was fractionated on a formaldehyde agarose gel by electrophoresis and then blotted onto a nylon membrane for Northern blot analysis (14). ³²P-Labeled cDNA probes were prepared by the random primer labeling procedure. The cDNAs of rabbit cathepsin K, carbonic anhydrase type II, TARP, and matrix metalloproteinase 9 from osteoclast cDNA library were used as hybridization probes (23, 24, 27). The mouse IP, membrane prostacyclin receptor, cDNA was obtained from S. Narumiya (Kyoto University, Kyoto, Japan). The cDNAs of mouse PPARs were a gift from R. M. Evans (The Salk Institute). A rat glyceraldehyde-3-phosphate dehydrogenase cDNA probe was used as a reference.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Molecular Cloning of the PPAR/ cDNA from Mature Rabbit Osteoclasts and mRNA Expression of PPAR Isoforms in Bone Cells-- A 480-base pair cDNA fragment containing the human estrogen receptor  DNA binding domain was used to screen the rabbit mature osteoclast cDNA library. Positive clones were isolated for sequence analyses. Fig. 1A shows the nucleotide and the deduced amino acid sequences of a positive clone, ROCHER10. It contained a 3.4-kb insert with an open reading frame of 1323 base pairs, encoding a polypeptide of 441 amino acids. Sequence comparison of ROCHER10 against the GenBank^TM data base showed that ROCHER10 was most closely related to the PPAR/ gene with a 94.8% overall identity to human PPAR/, and 89.6%, and 90.5% overall identities to the rat and mouse PPAR/, respectively (Fig. 1B). ROCHER10 shared distant homologies of 67.1, 63.0, 64.6, and 62.9% to the human PPAR, human PPAR, mouse PPAR, and mouse PPAR, respectively. Thus, we concluded that clone ROCHER10 represented the rabbit PPAR/ gene.

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Nucleotide and deduced amino acid sequences of rabbit PPAR/ from mature osteoclasts. A, sequence of a rabbit PPAR/ cDNA clone (ROCHER10). The DNA and ligand-binding domains are boxed. In-frame termination codons are indicated by asterisks. B, schematic comparison between rabbit PPAR/ and other PPARs. Amino acid sequences were aligned by using the program DINASIS (Hitachi Software Engineering, Co., Ltd.). The similarity between rabbit PPAR/ and other receptors is expressed as percentage of amino acid identity.

Next, we examined the mRNA expression levels of PPAR isoforms in rabbit bone cells and mouse clonal osteoblastic MC3T3-E1 cells by Northern blot analysis using the rabbit PPAR/ cDNA, mouse PPAR, and mouse PPAR cDNA as probes (Fig. 2). A 3.5-kb PPAR/ mRNA transcript was detected in MC3T3-E1 cells, rabbit stromal cells, and mature osteoclasts. The expression level of PPAR/ mRNA was most abundant in mature osteoclasts. Similar results were obtained when a mouse PPAR/ cDNA probe was used instead (data not shown). Conversely, no PPAR or PPAR mRNA expression was detectable in mature osteoclasts and stromal cells, whereas weak expression of PPAR (8.5-kb) mRNA was detected in MC3T3-E1 cells. The unique expression patterns of the PPAR isoforms in bone cells suggest that whereas PPAR/ may play a direct role in modulating mature osteoclasts, the PPAR isoform may have specific functions in other cell systems in bone.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Expression of PPARs mRNA in bone cells. Total RNA was isolated from an osteoblastic cell line (MC3T3-E1), bone stromal cells and mature osteoclasts that had been cultured on plastic dishes in -MEM plus 10% FBS, and 20 µg of total RNA was applied in each lane. Northern blot analysis was carried out with 32P-labeled rabbit PPAR/ or with  or  mouse PPAR cDNAs as probes, as described under "Materials and Methods." The experiment was done four times, with similar results each time.

Effect of PPAR/ Ligands on Mature Osteoclasts-- With the abundant expression of PPAR/ in mature osteoclasts, we postulated that ligands of PPAR/ may play a role in regulating osteoclastic cell function. Thus, we treated highly enriched mature osteoclasts with carbaprostacyclin, a prostacyclin analogue, and tested them in the osteoclastic resorption pit assay. As shown in Fig. 3, 10⁹ to 10⁸ M carbaprostacyclin induced an increase to approximately 2-fold greater pit area excavated by highly enriched mature osteoclasts on dentine slices. These concentrations of carbaprostacyclin did not significantly affect the number of pits excavated. In contrast, a high dose of carbaprostacyclin (10⁵ M) decreased the pit area excavated by mature osteoclasts. 10⁷ M leukotriene B₄, an agonist of both PPAR and its own plasma membrane receptor, induced a 1.5-fold greater pit area. WY-14643, a strong agonist of both PPAR and PPAR, did not affect osteoclastic bone resorption significantly.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of carbaprostacyclin on osteoclastic bone-resorbing activity. Highly enriched mature osteoclasts were cultured on dentine slices with leukotriene B4, WY-14643, carbaprostacyclin, or ethanol alone (as a control). Pit area and pit numbers on dentine slices were determined as described under "Materials and Methods." Values are means ± S.D., n = 4. *, p < 0.05 compared with the control group. Data are representative of those obtained in three other independent experiments.

Cathepsin K and matrix metalloproteinase 9 are proteases whose function is to break down bone matrix proteins, and the carbonic anhydrase type II gene product produces [H⁺] to dissolve mineralized bone matrix. TRAP is an osteoclast marker whose function in bone metabolism remains to be further defined. To examine whether a PPAR/ ligand would regulate the expression of genes related to osteoclastic bone resorption, we treated highly enriched mature osteoclasts on plastic culture dishes with carbaprostacyclin (1 × 10⁸ M) for 5 h. Total RNA was extracted from these cells, and mRNA levels of cathepsin K, carbonic anhydrase type II, TRAP, and matrix metalloproteinase 9 were determined by Northern blot analysis. Fig. 4 shows that carbaprostacyclin increased the mRNA level of cathepsin K, carbonic anhydrase type II, and TRAP but not that of matrix metalloproteinase 9 in the mature osteoclasts. This ligand doubled the mRNA level of cathepsin K and tripled the mRNA levels of carbonic anhydrase type II and TRAP in the cells. However, the mRNA level of glyceraldehyde-3-phosphate dehydrogenase was constant in the osteoclasts treated with carbaprostacyclin.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of carbaprostacyclin on osteoclastic gene expression. Total RNA was isolated from mature osteoclasts that had been cultured on plastic dishes with or without carbaprostacyclin (1 × 108 M) for 5 h. Twenty micrograms of total RNA was applied in each lane. Northern blot analysis was carried out with 32P-labeled cathepsin K, carbonic anhydrase type II (CA II), TRAP, matrix metalloproteinase 9 (MMP9), and glyceraldehyde-3-phosphate dehydrogenase (GPDH) cDNAs used as probes, as described under "Materials and Methods." The experiment was done four times, with similar results each time.

Effect of Antisense S-ODN of PPAR/ on Mature Osteoclasts-- To establish a direct role of PPAR/ in carbaprostacyclin-induced osteoclastic bone resorption, we included an antisense S-ODN of PPAR/ in the pit assay. S-ODN uptake by osteoclasts was confirmed by incubating the cells for 24 h at 37 °C with fluorescein-labeled cathepsin K-antisense S-ODN in the culture medium in the presence of Tfx-50 (data not shown; Ref. 26). The carbaprostacyclin-induced increase in pit area was completely blocked by 5-20 µM antisense S-ODN of PPAR/ (Fig. 5A). Even 20 µM sense PPAR/ S-ODN could not inhibit the increase in pit area caused by carbaprostacyclin. Neither of the S-ODNs for PPAR/ affected the number of pits on the dentine slices in either control or carbaprostacyclin-treated cultures (Fig. 5B).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of antisense S-ODN for PPAR/ on the activation of bone-resorbing activity by carbaprostacyclin in mature osteoclasts. Highly enriched mature osteoclasts were cultured on dentine slices with or without carbaprostacyclin (1 × 108 M) and with antisense or sense S-ODN for PPAR/ for 20 h. A, antisense S-ODN for PPAR/ blocked the increase in pit area by carbaprostacyclin in mature osteoclasts. B, pit number was not affected by S-ODNs for PPAR/ in either control or carbaprostacyclin-treated clutters. Pit area (A) and pit numbers (B) formed on dentine slices were determined as described under "Materials and Methods." Values are means ± S.D., n = 4. *, p < 0.05 compared with each control group. Data are representative of those obtained in three other independent experiments.

To confirm that the antisense S-ODN of PPAR/ reduced the endogenous level of PPAR/ expression, we analyzed the PPAR/ mRNA expression level in osteoclasts by Northern blotting. Fig. 6 shows that antisense S-ODN for PPAR/ reduced the mRNA level of PPAR/ in mature osteoclasts cultured on plastic dishes. In contrast, the sense S-ODN for PPAR/ had no effect (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effect of antisense S-ODN for PPAR/ on the level of PPAR/ mRNA in mature osteoclasts. Total RNA was isolated from mature osteoclasts that were cultured on plastic dishes with or without 10 µM antisense S-ODN for PPAR/ for 20 h. Twenty micrograms of total RNA was applied in each lane. Northern blot analysis was carried out with 32P-labeled PPAR/ and glyceraldehyde-3-phosphate dehydrogenase (GPDH) cDNAs as probes, as described under "Materials and Methods." The experiment was performed four times, and similar results were obtained each time.

Prostacyclin also acts through its plasma membrane receptor, IP. To eliminate the involvement of IP in activation of osteoclast functions by prostacyclin, we checked the expression of IP mRNA in the cells. Fig. 7 shows that the typical size (3.5 kb) of IP mRNA was expressed in osteoblastic MC3T3-E1 and stromal cells but not in the osteoclasts and that small (about 2 kb) and smear transcripts were detected in all cell types.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Expression of IP receptor mRNA in bone cells. Total RNA was isolated from osteoblastic cell line (MC3T3-E1), bone stromal cells, and mature osteoclasts that had been cultured on plastic dishes, and 20 µg of total RNA was applied in each lane. Northern blot analysis was carried out using 32P-labeled IP cDNAs as described under "Materials and Methods." GPDH, glyceraldehyde-3-phosphate dehydrogenase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We cloned the full-length cDNA of rabbit PPAR/ from a mature osteoclast cDNA library. Both the amino acid sequence and the mRNA transcript sizes of the rabbit PPAR/ were highly conserved with their human and rodent counterparts. Although previous reports showed that PPAR/ mRNA was widely expressed in mouse organs (22), until now it was unclear whether PPAR mRNA was expressed in bone tissue. Our data show that PPAR/ was more abundantly expressed in mature osteoclasts than in osteoblasts and stromal cells, whereas PPAR mRNA was expressed only in the clonal osteoblastic cells. This cell-specific expression of PPARs in bone cells may suggest a specific role of each PPAR in a given type of bone cells.

It is clear that PPARs are activated by arachidonic acid metabolites such as prostacyclin (prostaglandin I₂) and its analogues, i.e. carbaprostacyclin and iloprost (21). The metabolites produced from arachidonic acid, such as leukotriene and prostaglandin, have complex actions affecting bone metabolism (28, 29). Leukotriene B₄, which is one of the 5-lipoxygenase metabolites, directly activates the bone resorption activity in rat, mouse, and chicken osteoclasts (30-33). Our data show that mature rabbit osteoclasts also were activated by leukotriene B₄ but that nuclear leukotriene B₄ receptor (PPAR) mRNA was not detected in mature rabbit osteoclasts by Northern blot analysis. However, it was reported that leukotriene B₄ binding, which is considered to reflect the leukotriene B₄ receptor, occurred in mature chicken osteoclasts (30). These data suggest that leukotriene B₄ activates osteoclast function through its membrane receptor. Leukotriene B₄ also inactivates osteoblast-mediated bone formation (34). Thus, 5-lipoxygenase metabolites appear to down-regulate bone formation.

Prostaglandin E₂ (PGE₂), which is one of the prostaglandin endoperoxide synthase metabolites, has paradoxical functions in osteoclasts. PGE₂ directly inhibits bone-resorbing activity in rabbit and rat mature osteoclasts (35, 36). On the other hand, PGE₂ indirectly activates osteoclastic bone resorption through accessory cells, such as osteoblasts and stromal cells. Moreover, PGE₂ activates mouse osteoclast differentiation (37, 38) but inactivates human osteoclast-differentiation (39). These paradoxical functions of PGE₂ may be due to variations in experimental conditions, such as the presence of accessory cells, difference in species of osteoclasts, and differences in the differentiation stage of the osteoclasts (40-43).

Previously, it was reported that prostacyclin activated osteoclastic bone resorption through accessory cells in calvaria cultures (44). On the other hand, a millimolar level of prostacyclin inhibited the pit formation by mature osteoclasts (35). Moreover, a high concentration (2.5 × 10⁵ M) of prostacyclin reduced osteoclastic motility and cell size, whereas low concentrations (1.5 × 10⁷ M) increased osteoclast motility and cell size in isolated mature osteoclasts (36). Also, our results show that 1 × 10⁵ M carbaprostacyclin inhibited bone resorption by mature osteoclasts but that 1 × 10⁹ to 1 × 10⁸ M carbaprostacyclin activated the cells. These results indicate a differential effect of various levels of prostacyclin on mature osteoclasts. These may be due to the action of more than one type of receptor for prostacyclin in osteoclasts. It is reported that prostacyclin exerts its function through a nuclear receptor (PPAR/) and a membrane receptor (IP) (21, 45). The plasma membrane receptor for prostacyclin, IP, is abundantly expressed in thymus, lung, heart, and spleen (45, 46). In our present study, typical IP mRNA could not be detected in mature rabbit osteoclasts by Northern blot analysis. These results suggest the possibility of the existence of some other types of membrane receptor and/or nuclear receptor for prostacyclin in mature rabbit osteoclast to response to a high concentration of prostacyclin.

We clearly showed that antisense S-ODN for PPAR/ blocked the induction of osteoclastic bone resorption by carbaprostacyclin. However, antisense and sense S-ODN for PPAR/ or carbaprostacyclin did not affect pit formation number on dentine slice. These data indicate that S-ODN for PPAR/ does not have any toxic effect on mature osteoclasts in the concentrations used. We can thus conclude that PPAR/ has a role in regulation of osteoclast function through prostacyclin. This conclusion is also supported by the data showing that antisense S-ODN for PPAR/ reduced the level of PPAR/ mRNA in osteoclasts and that carbaprostacyclin stimulated an increase in the mRNA levels of several osteoclastic genes.

One of the PPAR subtypes, PPAR, has been implicated as a mediator of adipocyte and monocyte/macrophage differentiation (47-50). Also, it was reported that osteoclasts are differentiated from blood stem cells, especially those of the monocyte/macrophage lineage (1-3). The transcriptional factor c-Fos mediates osteoclast differentiation but not macrophage differentiation (51). Another transcriptional factor, PU. 1, mediates both osteoclast and macrophage differentiation (52). We cannot exclude the possibility that PPARs play a critical role in osteoclastogenesis, because we used only mature osteoclasts in all of our studies.

Our data indicate that PPAR/ mRNA but not IP mRNA is abundantly expressed in mature osteoclasts and that a prostacyclin analogue induced osteoclastic bone resorption in vitro. In addition, the results of antisense S-ODN analysis support the involvement of PPAR/ in prostacyclin-induced osteoclastic bone resorption. These data suggest that, in part, the bone loss mediated by prostacyclin, a metabolite of arachidonic acid, is modulated by PPAR/ in mature osteoclasts.

	ACKNOWLEDGEMENTS

We express our gratitude to Dr. Na N. Yang (Lilly) for useful comments on the manuscript and to Y. Katagiri and S. Ishii for technical assistance. We also thank Drs. M. Kobori (Yamanouchi Pharmaceutical Co., Ltd) and H. Kawashima (Nigata University) for the generous gift of rabbit TRAP cDNA.

	FOOTNOTES

^* This work was supported in part by a grant from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number AB033614.

To whom correspondence should be addressed. Tel.: 81-492-79-2768; Fax: 81-492-71-3523; E-mail: o-anat-1@dent.meikai.ac.jp.

	ABBREVIATIONS

The abbreviations used are: TRAP, tartrate-resistant acid phosphatase; PPAR, peroxisome proliferator-activated receptor; S-ODN, phosphothiorate oligodeoxynucleotide; MEM, minimal essential medium; FBS, fetal bovine serum; kb, kilobase pair(s); PGE₂, prostaglandin E₂.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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