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Keywords:

  • fibroblast growth factor;
  • bone;
  • osteoclast;
  • prostaglandin;
  • cyclooxygenase

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10−9 M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10−8 M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (≥10−12 M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10−11 M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10−9 M − 10−8 M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (≥10−12 M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (≥10−9 M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently.

Among many growth factors regulating bone metabolism, fibroblast growth factor 2 (FGF-2 or basic FGF) is recognized as a potent mitogen for a variety of mesenchymal cells.(1,2) In bone tissues, FGF-2 is produced by cells of osteoblastic lineage, accumulated in bone matrix, and acts as an autocrine/paracrine factor for bone cells.(3–10) FGF-2 shows variable regulations of proliferation and differentiation of osteoblastic cells so that it modulates bone formation.(5–10) Regarding its pharmacologic action, numerous investigations have shown that its exogenous application has stimulatory effects on bone formation in many in vivo models. Aspenberg et al. reported that a local application of FGF-2 increased bone yield in implanted demineralized bone matrix and enhanced bone-graft incorporation.(11,12) Two separate groups have shown that daily systemic administrations of FGF-2 increased endosteal bone formation.(13–15) We have reported that a single local injection of FGF-2 stimulates intraosseous bone formation and facilitates fracture healing, and that anabolic actions of FGF-2 are mainly caused by its mitogenic effects on immature mesenchymal cells.(16–20)

On the other hand, FGF-2 has been reported to stimulate bone resorption in bone organ cultures and osteoclastogenesis in a mouse bone marrow culture.(21–23) A histomorphometric analysis of the fracture healing revealed that in vivo application of FGF-2 stimulated not only callus formation but also osteoclastic callus resorption.(24) Regarding the clinical relevance of its resorptive effect, we recently reported that FGF-2 in the synovial fluid played an important role in the joint destruction of rheumatoid arthritis (RA) patients.(25) We have also reported that FGF-2 stimulates prostaglandin (PG) production through a transcriptional induction of cyclooxygenase-2 (COX-2) in neonatal mouse calvarial culture and in mouse osteoblastic MC3T3-E1 cell culture and suggested the mediation of PG production in the bone resorptive effect of FGF-2.(22)

The present study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone resorption. The effects of FGF-2 on osteoclast formation and mature osteoclast activation were investigated using three different culture systems. Well-known 45Ca release from prelabeled cultured neonatal mouse calvariae reflects the overall bone resorption through both osteoclast formation and mature osteoclast activation. Tartrate-resistant acid phosphatase (TRAP)–positive multinucleated osteoclast-like (OCL) cell formation in the coculture of mouse osteoblastic cells and bone marrow cells reflects osteoclast formation. These two assays reflect both direct and indirect (mainly through cells of osteoblastic lineage) actions of FGF-2 on osteoclastic bone resorption. On the other hand, resorbed pit formation assay by mature osteoclasts purified from rabbit long bones (more than 99% purity) reflects the direct activity of mature osteoclasts.(26) In this study, direct and indirect actions of FGF-2 on mature osteoclasts were further investigated by comparing its effect on resorbed pit formation by purified osteoclasts with that by unfractionated bone cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Materials

Immortal mouse macrophage cell line C7 cells were generously provided by Dr. S.I. Hayashi at Tottori University, Japan. Recombinant human FGF-2 was purchased from Scios Nova (Mountain View, CA, U.S.A.). NS-398 and etodolac were provided by Taisho Pharmaceutical Co., Ltd. (Tokyo, Japan) and Nihon-shin-yaku Pharmaceutical Co. (Kyoto, Japan), respectively. Recombinant human macrophage colony–stimulating factor (M-CSF) was purchased from Austral Biologicals (San Ramon, CA, U.S.A.). Recombinant human soluble RANK ligand was purchased from PeproTech, Inc. (London, U.K.). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Neonatal mouse calvarial culture

Seven-day-old ddY mice delivered by timed pregnant mother mice (6–7 weeks old, Nihon Kurea, Tokyo) were killed, and calvariae were harvested aseptically and dissected free of suture tissue without disturbing the periosteum. Bones were precultured for 16–24 h in BGJb medium containing 100 μg/ml l-ascorbic acid phosphate and 1 mg/ml bovine serum albumin (BSA). They were further cultured in the experimental medium in 24-multiwell dishes on a rocking platform. Treatment of each animal was conducted in accordance with the guidelines for the care and use of laboratory animals established at the Tokyo University, Japan.

Histomorphometric analyses

Calvariae cultured for 24 h and 96 h were cut into 6-μm sections, fixed, and stained with Villanueva-Goldner. The specimens were subjected to histomorphometric analyses using a microscope with a video camera connected to an image analysis system (SP-1000, Olympus, Tokyo, Japan).

45Ca release assay from cultured neonatal mouse calvariae

Bone resorption was measured as the release of previously incorporated 45Ca into neonatal mouse calvariae as previously described.(27) Briefly, timed pregnant mice were injected with 0.05 mCi 45Ca on the 16th day of gestation. Seven-day-old neonatal mouse calvariae were dissected and cultured as described above. After preculture periods, calvariae were cultured with experimental agents for 96 h with a medium change after 48 h. 45Ca in medium and trichloracetic acid extracts of bone were determined by liquid scintillation counting, and the cumulative percentage of 45Ca release was calculated.

Mouse primary osteoblastic cell culture

Calvariae dissected from 1- to 4-day-old mice as described above were washed in phosphate-buffered saline (PBS) and digested with 1 ml of trypsin/EDTA (Gibco BRL, Rockville, MD, U.S.A.) containing 10 mg collagenase (Sigma Chemical Co., St. Louis, MO, U.S.A., type 7) for 10 minutes × 5 times, and cells from fractions 3–5 were pooled. Cells were plated in 6-multiwell dishes at a density of 5000 cells/cm2 and grown to confluence in modified essential medium (α-MEM) containing 10% fetal bovine serum (FBS).

TRAP-positive–multinucleated OCL cell formation assay in the coculture of mouse primary osteoblastic cells and bone marrow cells

Mouse primary osteoblastic (POB) cells (2 × 104 cells/well) and bone marrow cells (1 × 106 cells/well) prepared from 8-week-old mice were cocultured in 24-miltiwell dishes with α-MEM containing 10% FBS in the presence and absence of FGF-2 for 5 days with a medium change at 3 days. After 5 days of culture, the cells were fixed with 3.7% (vol/vol) formaldehyde in PBS and ethanol-acetone (50:50, vol:vol), and stained at pH 5.0 in the presence of l(+)-tartaric acid using naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, MO, U.S.A.) in N,N-dimethyl formamide as the substrate. TRAP-positive-multinucleated cells containing more than three nuclei were counted as OCL cells.

Reverse-transcription polymerase chain reaction for COX-2 expression in mouse POB, C7, and OCL cells

C7 cells (6 × 104 cells/dish) were cultured on 100-mm dishes in α-MEM containing 10% FBS, 10 ng/ml of M-CSF, and 30 ng/ml of soluble RANK ligand for 6 days with a medium change at 3 days. Total RNA was extracted from cells cultured for 2, 3, 4, and 6 days. OCL cells were prepared as described above by the coculture of mouse POB cells (2 × 106 cells/dish) and bone marrow cells (2 × 107 cells/dish) on 100-mm culture dishes in α-MEM containing 10% FBS, 1,25(OH)2 vitamin D3 (10−8 M), and PGE2 (10−6 M) for 7 days with a medium change at 2 days. Then the dishes were treated with PBS containing 0.001% pronase E and 0.02% EDTA for 10 minutes to remove osteoblastic cells. After treatment, more than 99% of the adherent cells on the dishes were ascertained to be TRAP-positive-multinucleated OCL cells. After purification, OCL cells were incubated for 2 h with α-MEM containing 10% FBS. POB, C7, and OCL cells were further cultured in the presence and absence of FGF-2 for the indicated period, and total RNA was extracted from these cells by the acid guanidinium thiocyanate-phenol-chloroform method, and then 2-μg aliquots of total RNA were reverse-transcribed using oligo(dT) as a primer (1.5 μM final concentration) in a final 30-μl reverse-transcription (RT) solution.(28) Three microliters of RT solution (containing complementary DNA [cDNA] from 0.2 μg of total RNA) was then amplified by polymerase chain reaction (PCR) within the exponential phase of the amplification using specific primer pairs: 5′-TCAGCCAGGCAGCAAATCCTTG-3′ and 5′-TAGTCTCTCCTATGAGTATGAGTC-3′ for COX-2; 5′-CATGTAGGCCATGAGGTCCACCAC-3′ and 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ for G3PDH. PCR consisted of 25 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 1.5 minutes, and the PCR product was 939 base pair (bp).

Resorted pit formation assay by purified mature osteoclasts and unfractionated cells from rabbit long bones

Long bones from 10-day-old rabbits (Japanese White, Saitama Experimental Animal, Saitama, Japan) were minced with scissors and agitated with a vortex mixer.(26). An aliquot of unfractionated bone cells was seeded onto 0.24% collagen gel (Nitta Gelatin, Tokyo, Japan) coated on 100-mm tissue culture dishes and incubated. Four hours later, nonadherent cells were washed off and osteoclasts were then removed from the gels with 0.1% collagenase solution (Wako Pure Chemical Co., Osaka, Japan). By staining with TRAP, we ascertained that more than 99% of isolated cells were pure osteoclasts. Isolated osteoclasts (150 cells/well) or unfractionated bone cells (5 × 104 cells/well) were cultured on a dentine slice placed in each well of 96-multiwell dishes. FGF-2 was added to the cultures at 1 h after the seeding. After 24 h of culture, cells on dentine slices were removed in IN NH4OH solution and stained with 0.5% toluidine blue for 1 minute. Total area of pits and the number of pits on the dentine slice were estimated under a light microscope with a micrometer using an image analyzer (System Supply Co., Nagano, Japan).

Statistical analysis

Means of groups were compared by analysis of variance (ANOVA) and significance of differences was determined by post hoc testing using Bonferroni's method.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Histological examination of cultured mouse calvariae

FGF-2 (10−8 M) stimulated not only osteoblastic cell proliferation mainly at the periosteum but also osteoclastic bone resorption at the endosteum in cultured neonatal mouse calvariae for 96 h (Fig. 1A). Histomorphometric analyses of cultured calvariae indicated that FGF-2 significantly increased osteoclast number 1.6-fold at 24 h and 8.1-fold at 96 h and eroded surface formation 2.0-fold at 96 h of culture (Fig. 1B).

Effects of FGF-2 on OCL cell formation in the coculture system

Dose-response examination of effects of FGF-2 (10−14 − 10−8 M) on TRAP-positive-multinucleated OCL cell formation in the coculture system of mouse POB cells and bone marrow cells showed that FGF-2 at high concentrations (10−9 M and 10−8 M) induced OCL cell formation (Fig. 2). The OCL cells formed were ascertained to have bone resorbing activity because they produced resorbed pits when further cultured on dentine slices (data not shown). These results show good correlation with a previous study using cultured mouse bone marrow cells alone by Hurley et al.(23) Because we previously reported that FGF-2 at 10−9 M and higher concentrations stimulates PG production through a transcriptional induction of COX-2 in mouse calvaria and mouse osteoblastic MC3T3-E1 cell cultures, the contribution of PG production was examined by adding nonsteroidal anti-inflammatory drugs (NSAIDs) to the cultures.(22) Indomethacin and NS-398 at 10−8 M and higher concentrations abrogated OCL cell formation induced by FGF-2 (10−8 M) (Fig. 2). These results indicate that high concentrations of FGF-2 (≥10−9 M) stimulate bone resorption at least in part through the induction of osteoclast formation and that this induction is dependent on PG production.

Mediation of COX-2 induction in FGF-2-stimulated 45Ca release from cultured mouse calvariae

We previously reported that FGF-2 (≥10−10 M) stimulated 45Ca release from prelabeled cultured calvariae dose dependently.(22) Here, we examined the contribution of PG production and COX-2 induction to resorptive effects of FGF-2. We used four NSAIDs that have different selectivities for inhibition of COX-1 and COX-2. Indomethacin is more selective for COX-1 than COX-2; flurbiprofen is almost equally selective for COX-1 and COX-2; etodolac is more selective for COX-2 than COX-1; and NS-398 is a specific inhibitor of COX-2.(29–31) All NSAIDs inhibited 45Ca release stimulated by FGF-2 (10−8 M) dose dependently with the maximum inhibition of 70–80%, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of COX-2 than COX-1 (Fig. 3). Fifty percent inhibition was achieved by 5 × 10−8 M indomethacin and by 3 × 10−9 M NS-398. NSAIDs alone showed little inhibition (<10%) in the absence of FGF-2 (data not shown). This result indicates that resorptive effects of FGF-2 are dependent on PG production, especially on COX-2 induction.

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Figure FIG. 1.. (A) Histological findings Villanueva-Goldner staining at 96 h and (B) histomorphometric analyses of cultured mouse calvariae. Neonatal mouse calvariae cultured for 24 h and 96 h in the presence and absence of FGF-2 (10−8 M) were cut into 6-μm sections, fixed, and stained with Villanueva-Goldner. The specimens were subjected to histomorphometric analyses. N. Oc/B. Pm, number of osteoclasts/bone perimeter (10 cm); ES/BS, eroded surface/bone surface. Data are expressed as means (bars) ± SEMs (error bars) for 8 cultures/group. ap < 0.01, bp < 0.05, significant effect of FGF-2.

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Figure FIG. 2.. Dose-response of effects of FGF-2 on mouse OCL cell formation in the presence and absence of NSAIDs (indomethacin and NS-398). POB cells from neonatal mouse calvariae and bone marrow cells from 8-week-old mouse long bones were cocultured for 5 days with a medium change at 3 days. After 5 days of culture, TRAP-positive–multinucleated cells containing more than three nuclei were counted as OCL cells. Data are expressed as means (symbols) ± SEMs (error bars) for 8–10 cultures/group. Symbols indicate FGF-2 alone (◯); FGF (10−8 M) + indomethacin at 10−9 M (△) and 10−8 M (▵); FGF (10−8 M) + NS-398 at 10−9 M (▪) and 10−8 M (□). All these inhibitions are statistically significant, and higher concentrations (10−7 M and 10−6 M) of indomethacin and NS-398 also abrogated OCL cell formation (data not shown).

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COX-2 induction in cells of osteoblast and osteoclast lineages

To identify the target cells of FGF-2 on COX-2 induction, COX-2 messenger RNA (mRNA) levels in cultured mouse POB cells and OCL cells were compared in the presence and absence of FGF-2 (10−8 M) at 2, 4, and 24 h of culture (Fig. 4). In addition to these cells, we used immortal mouse macrophage cell line C7 cells, which are known to differentiate into TRAP-positive–multinucleated OCL cells after 5–6 days of culture in the presence of soluble RANK ligand and M-CSF without support of osteoblastic cells.(32,33) Because we extracted RNA from C7 cells at 2, 3, 4, and 6 days of culture, various differentiation stages of osteoclastic cells were assumed to be included. As a result, COX-2 was highly expressed and its mRNA level was stimulated by FGF-2 in POB cells at each time point as previously reported by Northern blot analysis.(22) On the other hand, COX-2 mRNA level was undetectable or not stimulated by FGF-2 either in OCL cells or C7 cells. This level did not change even when the amount of template cDNA or the amplification cycle was increased (data not shown). Although results of C7 cells cultured for 3 days are shown in Fig. 4, similar results were seen when cells cultured for 2, 4, and 6 days were used. Thus, it was concluded that FGF-2 at least partially stimulates bone resorption indirectly through COX-2 induction in osteoblastic cells.

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Figure FIG. 3.. Dose-response of effects of NSAIDs with different selectivities for inhibition of COX-1 and COX-2 on FGF-2-stimulated 45Ca release from cultured mouse calvariae. Seven-day-old neonatal mouse calvariae prelabeled with 45Ca were cultured with experimental agents for 4 days with a medium change after 2 days in the presence of FGF-2 (10−8 M). Ratio of selectivity for inhibition of COX-2 to that of COX-1 is NS-398 > etodolac > flurbiprofen > indomethacin. Data are expressed as means (symbols) ± SEMs (error bars) for 7–10 cultures/group. NSAIDs alone showed little inhibition (<10%) in the absence of FGF-2 (data not shown). ap < 0.05, bp < 0.01, significant inhibition compared with that by indomethacin at the same concentration.

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Direct effect of FGF-2 on osteoclasts

To determine the direct action of FGF-2 on osteoclasts, the pit area resorbed by purified osteoclasts from rabbit long bones on dentine slice was measured and was compared with that by unfractionated bone cells. FGF-2 at 10−12–10−8 M stimulated pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10−11 M and no further stimulations were observed at higher concentrations (Table 1, Fig. 5). This effect was dose-dependent at concentrations between 10−13 M and 10−11 M (Table 1, experiment 2), and significant stimulation was seen at 10−12 M and higher concentrations. This stimulation was mainly caused by the activation of mature osteoclasts, but it was not caused by the increase in osteoclast number because the area of each pit (total pit area/number of pits) also was increased by FGF-2. In addition, this direct action of FGF-2 was not inhibited by NS-398 (Table 1). However, FGF-2 at 10−9–10−8 M further stimulated pit formation by unfractionated bone cells up to 9.7-fold (Fig. 5). This stimulatory effect of high concentrations of FGF-2 on pit formation by unfractionated bone cells was 70–80% inhibited by NS-398. These results suggest that FGF-2 at low concentrations (≥10−12 M) stimulates bone resorption moderately through its direct action on osteoclasts, whereas at high concentrations (≥10−9 M) it stimulates bone resorption potently through its indirect action mediated by COX-2 induction in osteoblastic cells.

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Figure FIG. 4.. COX-2 induction in mouse POB cells, C7 cells, and OCL cells by FGF-2 (RT-PCR). POB cells were extracted from neonatal mouse calvariae by sequential digestions with collagenase. Immortal mouse macrophage cell line C7 cells were cultured in the presence of M-CSF and soluble RANK ligand for 6 days when part of the cells differentiated to OCL cells. Cells cultured for 2, 3, 4, and 6 days were used. The data from C7 cells cultured for 3 days are shown, and similar results were seen in cells cultured for 2, 4, and 6 days. OCL cells were prepared by the coculture of mouse POB cells and bone marrow cells in the presence of 1,25(OH)2 vitamin D3 and PGE2 for 7 days and isolated by removing stromal cells with 0.001% pronase E and 0.02% EDTA. After treatment, more than 99% of the adherent cells on the dishes were ascertained to be TRAP-positive-multinucleated OCL cells. Total RNA was extracted from POB, C7, and OCL cells cultured in the presence and absence of FGF-2 for the indicated period. After RT, cDNA from 0.2 μg of total RNA was amplified by PCR within the exponential phase of the amplification.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Histological examination of cultured neonatal mouse calvariae disclosed the stimulatory effects of FGF-2 on osteoblast proliferation mainly at the periosteal site and osteoclastic bone resorption at the endosteal site. Although osteoclastic cells were also found in bone marrow cavities that were seldom seen in the neonatal calvariae, these cells seemed not to be stimulated by FGF-2. This may be because the cavities were not exposed to the culture medium enough for FGF-2 to act on these cells during the culture. Osteoblastic cells also were found at the endosteal site and a part of them were stimulated by FGF-2. However, most of them appeared highly differentiated and included lining cells that were not influenced by FGF-2. This selectivity could be ascribed to the nature of FGF-2 mitogenic action, which is stronger on immature osteoblastic cells than on mature osteoblasts as previously reported.(7,10)

Table Table 1.. Dose Response of Effects of FGF-2 on Resorption Pit Formation by Purified Osteoclasts in the Presence and Absence of NS-398 (10−6 M)
 Pit area/dentine (×103 μm2) 
 Control+NS-398Pit area/pit number (μm2)
  1. The area of each pit (pit area/pit) was expressed by the total pit area/the number of pits/dentine. Values are the mean ± SEM for 8–12 cultures/group in experiment 1 and for 8 cultures/group in experiment 2.

  2. *p < 0.05.

  3. p < 0.01; significant effects of FGF-2.

Experiment 1
Control24.57 ± 3.6523.98 ± 4.07812.9 ± 84.4
10−17 M FGF-227.83 ± 3.13 779.1 ± 91.3
10−16 M29.72 ± 3.64 808.4 ± 93.1
10−15 M29.48 ± 6.85 747.6 ± 81.2
10−14 M31.99 ± 6.74 883.1 ± 166.1
10−13 M25.92 ± 4.91 832.3 ± 98.2
10−12 M43.19 ± 4.94* 1161.7 ± 128.0
10−11 M50.27 ± 7.3949.43 ± 10.741364.0 ± 139.6
10−10 M46.69 ± 3.4647.82 ± 7.53*1332.5 ± 156.3*
10−9 M47.88 ± 5.7151.71 ± 8.951308.1 ± 140.1
10−8 M48.25 ± 4.99 1404.2 ± 126.7
Experiment 2
Control26.10 ± 3.57 791.9 ± 90.2
10−14 M FGF-229.62 ± 5.91 817.1 ± 118.8
3 × 10−14 M27.49 ± 3.92 806.3 ± 103.6
10−13 M33.53 ± 2.80 827.6 ± 88.1
3 × 10−13 M37.69 ± 4.23 949.7 ± 97.0
10−12 M43.25 ± 4.58* 1178.2 ± 138.1
3 × 10−12 M50.47 ± 6.02 1315.5 ± 136.8*
10−11 M50.98 ± 5.36 1435.1 ± 169.1

All three assays used in the present study have shown that COX-2 induction and PG production in osteoblastic cells are involved in the stimulation of osteoclast formation and bone resorption by high concentrations of FGF-2. Although 10−8 M of indomethacin and NS-398 abrogated OCL cell formation in the coculture system (Fig. 2), inhibitions of 45Ca release by both NSAIDs were only partial (Fig. 3). In addition, NS-398, a specific inhibitor of COX-2, did not decrease FGF-2–stimulated pit formation by isolated osteoclasts but significantly decreased that by unfractionated bone cells. Taken together, induction of COX-2 and PGs in osteoblastic cells by FGF-2 may contribute to osteoclast formation, but not to mature osteoclast activation. In fact, previous studies on effects of PGs have shown that PGs act to inhibit isolated osteoclasts while they stimulate OCL cell formation in bone marrow cultures.(34–38)

FGF-2 and PGs are known to show similar actions on bone metabolism, and both stimulate bone formation as well as bone resorption. Because their anabolic effects are seen when applied exogenously, they may be pharmacologic actions that can be achieved at high concentrations. FGF-2 anabolic action has been reported to be primarily through its mitogenic effect on low-differentiated mesenchymal cells.(7,10,16,17) Because we have reported that the mitogenic effect of FGF-2 was not inhibited by NSAIDs in in vitro cultures, FGF-2 anabolic effect, unlike its resorptive effect, may be independent of PG production.(22) Mediation of PGs in FGF-2 effects on bone formation and resorption in vivo has not yet been studied.

Little is known about the direct stimulator of mature osteoclasts; however, receptor tyrosine kinases (RTKs) recently have been identified on mature osteoclasts. Nakamura et al. have reported the existence of eight RTKs, which include FGF receptor type 1 (FGFR1) on mature osteoclasts isolated from rabbit long bones.(39) Our preliminary study has shown that among FGFRs (FGFR1–4) only FGFR1 was identified on mouse osteoclasts whereas FGFR1–4 could be identified on osteoblastic cells as previously reported by Debiais et al.(10,40) This difference in distribution of FGFRs between osteoblasts and osteoclasts might explain the difference of affinities and concentrations of FGF-2 affecting these cells. Further studies about signaling pathways through FGFRs on osteoblasts and osteoclasts will lead to further understanding of the regulation of bone metabolism by FGFR2.

Regarding the clinical relevance of the direct action of FGF-2 on osteoclasts, we have recently found that FGF-2 concentrations in the synovial fluid are correlated positively to the severity of joint destruction in RA patients.(25)

However, the concentrations of FGF-2 were much lower, on the order of 10−13–10−12 M, than other cytokines such as interleukin 6 (IL-6) and soluble IL-6 receptor, on the order of 10−11–10−10 M. These levels of FGF-2 are not enough to affect osteoblastic cells but possibly may affect osteoclasts directly. Thus, FGF-2 in the synovial fluid might play a role in the final step of osteoclastic bone resorption in RA joint destruction, which is preceded by recruitment and differentiation of osteoclasts by other factors. Other than the well-known pharmacologic action of FGF-2 on bone formation, endogenous FGF-2 may possibly function in the pathogenesis of bone resorptive diseases through its direct action on osteoclasts.

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Figure FIG. 5.. Dose-response of effects of FGF-2 on resorbed pit area on dentine slice by purified osteoclasts (10−17–10−8 M, direct effects) and unfractionated bone cells (10−13–10−8 M, direct and indirect effects) from rabbit long bones in the presence and absence of NS-398 (10−6 M). Bone cells were extracted from long bones of 10-day-old rabbits, and were seeded onto collagen gel. Nonadherent cells were washed off and osteoclasts then were removed from the gels with collagenase solution. By staining with TRAP, we ascertained that more than 99% of the isolated cells were pure osteoclasts. FGF-2 and NS-398 were added to the cultures at 1 h after the seeding. Data are expressed as means (symbols) ± SEMs (error bars) of the ratio of treated/control (8–12 cultures/group). The values of pit area by purified osteoclasts are shown in Table 1 (experiment 1), and the mean control value of that by unfractionated bone cells was 14,409 μm2/dentine. aSignificant effect of FGF-2; bsignificant inhibition by NS-398; both p < 0.01.

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Taken together with our previous studies on anabolic action of FGF-2, we hereby propose that FGF-2 at high concentrations acts on osteoblastic cells and stimulates not only bone formation but also bone resorption potently through COX-2 induction and PG production, whereas at low concentrations it acts directly on mature osteoclasts to resorb bone.(16–20,22) It is speculated that this differential regulation of FGF-2 action on osteoblastic cells and osteoclastic cells might explain its various effects—physiological, pathological, and pharmacologic—on bone metabolism.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We are grateful to Yumiko Nagai, Kazumi Obara, and Hideyuki Yamato of the Hard Tissue Research Team at Kureha Chemical Co., Ltd. for their expert technical assistance and to Dr. Shin-ichi Hayashi at Tottori University for providing C7 cells and helpful discussion. This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (no. 09307033 to H.K. and no. 10470302 to K.N.) and a Bristol-Myers Squibb/Zimmer Unrestricted Research grant (K.N.).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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