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Endocrinology Vol. 142, No. 8 3632-3637
Copyright © 2001 by The Endocrine Society

ARTICLES

Reciprocal Control of Expression of mRNAs for Osteoclast Differentiation Factor and OPG in Osteogenic Stromal Cells by Genistein: Evidence for the Involvement of Topoisomerase II in Osteoclastogenesis

Takumi Yamagishi, Eri Otsuka and Hiromi Hagiwara

Research Center for Experimental Biology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

Address all correspondence and requests for reprints to: Hiromi Hagiwara, Ph.D., Research Center for Experimental Biology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. E-mail: hhagiwar{at}bio.titech.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells with monocyte/macrophage lineage in response to a combination of osteoclast differentiation factor (RANKL) and OPG, a decoy receptor for RANKL, produced by ST2 cells in response to 1{alpha},25-dihydroxyvitamin D3. Treatment of ST2 cells with the natural isoflavonoid genistein for 6 h before coculture with spleen cells inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. When we measured levels of RANKL mRNA in ST2 cells, we found that genistein decreased the level of this mRNA. By contrast, the level of OPG mRNA was enhanced by genistein. Genistein is a specific inhibitor of topoisomerase II (topo II) and an inhibitor of protein tyrosine kinase, as well as being a potent phytoestrogen. To characterize the mode of action of genistein, we examined the effects of an inactive form of genistein (daidzein), 17ß-estradiol, inhibitors of topo II, and inhibitors of tyrosine kinases on the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. Among the compounds tested, two inhibitors of topo II, amsacrine and etoposide, attenuated the formation of osteoclast-like cells via reciprocal regulation of the expression of mRNAs for RANKL and OPG in ST2 cells, acting similarly to genistein. Our findings indicate that genistein might inhibit the formation of osteoclast-like cells via inhibition of the activity of topo II, suggesting the novel possibility that topo II might play an important role in osteoclastogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCLASTS ARE MULTINUCLEATED cells that are responsible for the resorption of bone. They are formed from hematopoietic cells of the monocyte/macrophage lineage. The processes of differentiation and fusion can be reproduced in a culture system in which mouse bone marrow cells (1) or spleen cells (2) are cocultured with mouse osteoblasts or osteogenic stromal cells. Various osteotropic hormones and cytokines, including 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25(OH)2D3], macrophage colony-stimulating factor (M-CSF), PTH, PG E2, IL-1, and IL-6, affect osteoclastogenesis at distinct stages of the development of osteoclasts.

The genes for osteoclast differentiation factor (RANKL) (3) and its decoy receptor OPG (4, 5) were recently cloned and characterized. RANKL [also known as TNF-related activation-induced cytokine (TRANCE) (6), osteoclast differentiation factor (ODF) (7), and OPG ligand (OPGL) (8)] is produced by osteoblasts or osteogenic stromal cells and plays important roles in osteoclastogenesis. It is involved in the formation of osteoclast-like cells (OCLs), the fusion of osteoclastic precursor cells, and the resorption of bone (7, 9). By contrast, OPG [also known as osteoclastogenesis-inhibitory factor (OCIF)] inhibits osteoclastogenesis by masking RANKL on osteoblasts or osteogenic stromal cells (4). Rankl-deficient mice and opg-deficient mice develop osteopetrosis (10) and osteoporosis (11, 12), respectively. Thus, RANKL and OPG appear to be essential for the development of osteoclasts both in vivo and in vitro.

Genistein (4', 5, 7-trihydroxyisoflavone) is a natural isoflavonoid found in Leguminosae. It has remarkable estrogenic activity as a phytoestrogen and inhibits the activities of topoisomerase II (topo II) (13)and protein tyrosine kinases (14, 15). At the cellular level, genistein induces apoptosis and differentiation of cancer cells, inhibits cell proliferation, and modulates angiogenesis (16). Furthermore, it has been reported that genistein is an important nutrient in the prevention of osteoporosis; it might have such an effect by stimulating the formation of bone (17) and/or by inhibiting the resorption of bone (18). However, the mechanism of action of genistein is not fully understood.

In the present study, we found that ST2 cells treated with genistein failed to induce formation of OCLs in coculture with spleen cells and we observed that genistein controlled the levels of expression of mRNAs for RANKL and OPG in ST2 cells. We also showed that inhibitors of topo II inhibited the formation of OCLs. Thus, an analysis of the mechanism of action of genistein revealed the probable involvement of topo II in the formation of OCLs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Genistein, L-ascorbic acid, 1{alpha},25(OH)2D3, and etoposide were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Daidzein, 17ß-estradiol, tyrophostin 25 [an inhibitor of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor], wortmannin (an inhibitor of PI3 kinase), and amsacrine (an inhibitor of topo II) were obtained from Sigma (St. Louis, MO). PD 098059 (an inhibitor of MEK) and lavendustin A (an inhibitor of the tyrosine kinase activity of the EGF receptor) were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA) and Nacalai Tesque (Kyoto, Japan), respectively. 32P- Labeled nucleotides were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). FBS and penicillin/streptomycin antibiotic mixture were obtained from Life Technologies, Inc. (Grand Island, NY).

Formation of OCLs in vitro
Spleen cells were collected from splenic tissues of 6-wk-old male ddY mice. Erythrocytes contaminating the spleen cell fraction were eliminated by addition of 0.83% (wt/vol) ammonium chloride in 10 mM Tris-HCl (pH 7.4) to the cell pellet. Mouse bone marrow-derived stromal ST2 cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). ST2 cells (2 x 104 cells/well) in 48-well plates (0.98 cm (2)/well; IWAKI, Tokyo, Japan) were treated with genistein and other compounds at various concentrations for 6 h, washed twice with {alpha}-MEM, and then cocultured with mouse spleen cells (5 x 104 cells/well) in {alpha}-MEM that contained 10% FBS, and 10-8 M 1{alpha},25(OH)2D3 (2). Genistein and other compounds were dissolved in dimethylsulfoxide (final concentration, 0.1%, vol/vol). Dimethylsulfoxide at 0.1% did not affect the formation of OCLs. The cocultures were maintained at 37 C in a humidified atmosphere of 5% CO2 in air. Fresh {alpha}-MEM that contained 10% FBS, and 10-8 M 1{alpha},25(OH)2D3 was supplied at 3-d intervals.

Localization of tartrate-resistant acid phosphatase (TRAP)
After coculture for 8 d, adherent cells were fixed in 10% formalin for 5 min and then in a mixture of ethanol and acetone (1:1, vol/vol) for 1 min. Then they were stained for TRAP activity as described by Udagawa et al. (19). TRAP-positive mononuclear cells and TRAP-positive multinucleated cells (with three or more nuclei) were counted under a microscope (IX70; Olympus Corp., Tokyo, Japan).

Cell viability
We used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) (Dojindo, Kumamoto, Japan) to examine the viability of ST2 cells. ST2 cells (1 x 104 cells/well; 96-well plates), exposed to genistein at various concentrations for 6 h, were subcultured for several days. Cells were treated with MTT (50 µg/well) and then absorbance at 570 nm was measured.

Semiquantitative RT-PCR
We detected mRNAs for RANKL, OPG, and M-CSF in ST2 cells by semiquantitative RT-PCR. RNA was extracted from ST2 cells that had been exposed to genistein or inhibitors of topo II by the acid guanidinium-phenol-chloroform method (20). Total RNA (5 µg) was reverse transcribed by Moloney murine leukemia virus reverse transcriptase, Superscript (200 U; Life Technologies, Inc.), with random primers (50 ng) in a 20-µl reaction mixture. The cDNA was amplified in 20 µl of Taq DNA polymerase mixture (Takara, Tokyo, Japan) that contained 1 µM sense primer, 5'-CAGGTTTGCAGGACTCGAC-3', and antisense primer, 5'-AGCAGGGAAGGGTTGGACA-3', for mouse RANKL (accession no. AF013170; positions 434–1,034; 601 bp); 1 µM sense primer, 5'-CCACTCTTATACGGACAGCT-3', and antisense primer, 5'-TCTCGGCATTCACTTTGGTC-3', for mouse OPG (accession number U94331; positions 291–796; 506 bp); 1 µM sense primer, 5'-TTGCCAAGGAGGTGTCAGAA-3', and antisense primer, 5'-TATTGGAGAGTTCCTGGAGC-3', for mouse M-CSF (accession number M21952; positions 251–511; 261 bp); or 1 µM sense primer, 5'-ACTTTGTCAAGCTCATTTCC-3', and antisense primer, 5'-TGCAGCGAACTTTATTGATG-3', for mouse glyceraldehyde-3-phosphase dehydrogenase (GAPDH; accession number M32599; positions 957-1223; 267 bp). Each reaction cycle, performed 23, 23, 19, and 19 times for amplification of the cDNA for RANKL, OPG, M-CSF, and GAPDH, respectively, consisted of incubation at 94 C for 30 sec, at 60 C for 40 sec, and at 72 C for 30 sec. Products of PCR were subjected to electrophoresis on a 2% agarose gel and visualized by staining with ethidium bromide. DNA marker fragments (molecular weight marker V; Roche Molecular Biochemicals, Mannheim, Germany) were used as size markers.

For quantitative analysis of mRNAs for RANKL, OPG, M-CSF, and GAPDH, the products of PCR were blotted onto nylon membranes (MagnaGraph; Micron Separation Inc., Westborough, MA) after electrophoresis. Blots were prehybridized at room temperature for 2 h in 6x SSPE (1x SSPE consists of 0.15 M NaCl, 8.65 mM NaH2PO4·2H2O, and 1.25 mM EDTA, pH 7.4) that contained 2x Denhardt’s solution (1x Denhardt’s solution consists of 0.1% each of BSA, polyvinylpyrrolidone, and Ficoll), 50% formamide, 100 µg herring sperm DNA, and 0.5% SDS. Then blots were allowed to hybridize at 42 C for 16 h in the same solution with a 32P-labeled cDNA probe specific for RANKL, OPG, M-CSF or GAPDH at 2 x 106 cpm/ml. Blots were washed twice in 0.1x SSC and 0.1% SDS at 60 C for 1 h. Washed blots were analyzed with a Bioimage Analyzer (BAS 2000; Fuji Photo Film Co., Ltd. Film, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We used a coculture system that consisted of mouse spleen cells and mouse osteogenic stromal cells, ST2 cells, for induction of the formation of OCLs. Spleen cells do not include cells that produce RANKL and OPG. Furthermore, by treating ST2 cells with genistein before coculture, we were able to examine the effects of genistein on the formation of OCLs by ST2 cells and levels of mRNAs for RANKL and OPG expressed in ST2. Fig. 1CGo shows that the formation of TRAP-positive OCLs was inhibited when ST2 cells had been exposed to genistein for 6 h before coculture with spleen cells for 8 d in the presence of 10-8 M 1{alpha},25(OH)2D3. The formation of TRAP-positive mononuclear (A) and multinucleated (B) OCLs was inhibited by genistein in a dose-dependent manner. Inhibition of the formation of OCLs was observed upon inclusion of genistein at 20 to 50 µM in the culture medium of ST2 cells. The formation of OCLs was also inhibited even after exposure of ST2 cells to genistein for only 1 h. We confirmed, using MTT, that the effects of compounds tested in this study were not attributable to generalized toxicity in ST2 cells (Fig. 2Go).



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  		Figure 1. Inhibition of the formation of TRAP-positive OCLs by genistein. ST2 cells that had been treated with genistein at the indicated concentrations for 6 h were cocultured with mouse spleen cells in 48-well plates for 8 d in {alpha}-MEM that contained 10% FBS and 10-8 M 1{alpha},25(OH)2D3. Control cultures were supplemented with 0.1% dimethylsulfoxide. Fresh medium was supplied every 3 d. Cells were stained for TRAP activity and TRAP-positive mononuclear cells (A) and TRAP-positive multinucleated cells (B) were counted under a microscope. Data are means ± SE of results from four determinations. The photographs in C show typical results of staining for detection of TRAP activity of OCLs in cocultures of cells. Bars, 100 µm.

 


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  		Figure 2. The effects of genistein, amsacrine, and etoposide on cell viability. ST2 cells that had been treated with compounds at the indicated concentrations for 6 h were cultured in 48-well plates for the indicated periods in {alpha}-MEM that contained 10% FBS. Cells were treated with MTT and then absorbance at 570 nm were measured. Data are means ± SE of results from four determinations.

 
We examined the expression of mRNAs for RANKL, OPG, and M-CSF in ST2 cells by semiquantitative RT-PCR. Cells that had been treated with 0, 10, 30, and 50 µM genistein for 6 h were then subcultured for 48 h in the presence and in the absence of 10-8 M 1{alpha},25(OH)2D3, an inducer of RANKL and an inhibitor of OPG. As shown in Fig. 3Go, the level of mRNA for RANKL induced in response to 1{alpha},25(OH)2D3 was progressively reduced in ST2 cells with increasing concentrations of genistein and 50 µM genistein inhibited 70% of the 1{alpha},25(OH)2D3-induced expression of RANKL mRNA. We could not detect any mRNA for RANKL in the absence of 1{alpha},25(OH)2D3. By contrast, the levels of mRNA for OPG, both in the presence and in the absence of 1{alpha},25(OH)2D3, were strongly enhanced upon exposure of ST2 cells to genistein. At 30 µM genistein, levels of OPG mRNA (after normalization by reference to levels of GAPDH mRNA) increased 220% and 600%, as compared with the control, in the presence and in the absence of 1{alpha},25(OH)2D3, respectively. After subculture for 8 d, the effect of genistein on the expression of RANKL and OPG was lost (data not shown). Genistein had no effect on the level of mRNA for M-CSF in ST2 cells.



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  		Figure 3. The effects of genistein on the expression of mRNAs for RANKL, OPG, and M-CSF in ST2 cells. Total RNA was isolated from ST2 cells that had been treated for 6 h with 0, 10, 30, 50 µM genistein and then cultured for 2 d in the presence and in the absence of 10-8 M 1{alpha},25(OH)2D3 (VD3). We examined the effects of genistein on the expression of mRNAs for RANKL, OPG, and M-CSF. The products of RT-PCR were subjected to electrophoresis in a 2% agarose gel and were allowed to hybridize with 32P-labeled cDNAs for RANKL, OPG, M-CSF, and GAPDH as described in Materials and Methods. The results shown are representative of the results of three experiments.

 
To examine that inhibition of expression of RANKL mRNA by genistein is critical in the formation of OCLs, we added soluble RANKL (sRANKL) to the medium of coculture system with 50 µM genistein-treated ST2 cells and spleen cells. As shown in Fig. 4Go, addition of sRANKL to the medium formed TRAP-positive OCLs, and the effect of sRANKL was dose dependent. The formation of OCLs was restored 50% of control by the addition of 100 ng/ml sRANKL.



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  		Figure 4. The effects of sRANKL on the formation of TRAP-positive OCLs in coculture with genistein-treated ST2 cells and spleen cells. ST2 cells that had been treated with genistein at 50 µM for 6 h were cocultured with mouse spleen cells in 48-well plates for 8 d in {alpha}-MEM that contained 10% FBS, 10-8 M 1{alpha},25(OH)2D3, and sRANKL at various concentrations. Fresh medium and sRANKL was supplied every 3 d. Cells were stained for TRAP activity and TRAP-positive multinucleated cells were counted under a microscope. Data are means ± SE of results from four determinations.

 
Genistein acts as a phytoestrogen, an inhibitor of topo II, and an inhibitor of protein tyrosine kinase (the tyrosine kinase activity of the EGF receptor). Therefore, to clarify the mode of action of genistein, we examined the effects on the formation of OCLs of an inactive form of genistein, daidzein, and of 17ß-estradiol, inhibitor of topo II such as amsacrine and etoposide, and inhibitors of protein tyrosine kinase such as tyrphostin 25 and lavendustin A (inhibitors of the tyrosine kinase activity of the EGF receptor), PD 098059 (an inhibitor of MEK), and wortmannin (an inhibitor of PI3 kinase). As shown in Fig. 5Go, exposure of ST2 cells for 6 h to daidzein (0.1 µM to 50 µM; Fig. 5AGo), to 17ß-estradiol (0.1 nM to 50 µM; Fig. 5BGo), and to inhibitors of protein tyrosine kinase at optimal concentrations (Fig. 5CGo) had no effects on the formation of OCLs. By contrast, the inhibitors of topo II, namely, amsacrine and etoposide, each inhibited the formation of TRAP-positive multinucleated OCLs in a dose-dependent manner (Fig. 6Go). Amsacrine at 3 µM and etoposide at 0.5 µM each completely inhibited the formation of OCLs. Furthermore, amsacrine (5 µM) and etoposide (0.5 µM) decreased the level of expression of mRNA for RANKL and increased that for OPG in ST2 cells to a similar extent to genistein (Fig. 7Go).



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  		Figure 5. The effects of daidzein, 17ß-estradiol, and inhibitors of tyrosine kinase on the formation of OCLs. ST2 cells that had been treated with daidzein at indicated concentrations (A), 17ß-estradiol at indicated concentrations (B), and inhibitors of tyrosine kinase, namely, 50 µM tyrophostin 25 (an inhibitor of the tyrosine kinase activity of the EGF receptor; 15 ), 50 µM lavendustin A (an inhibitor of the tyrosine kinase activity of the EGF receptor; 15 ), 20 µM PD 098059 (an inhibitor of MEK; 38 ), and 100 nM wortmannin (an inhibitor of PI3 kinase; 39) (C), for 6 h were cocultured with mouse spleen cells in 48-well plates for 8 d in {alpha}-MEM that contained 10% FBS and 10-8 M 1{alpha},25(OH)2D3. Controls were treated with 0.1% of dimethylsulfoxide. Fresh medium was supplied every 3 d. The cells were stained for TRAP activity and TRAP-positive multinucleated cells were counted under a microscope. Data are means ± SE of results from four determinations.

 


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  		Figure 6. Inhibition of the formation of OCLs by inhibitors of topo II. ST2 cells that had been treated with amsacrine (A) and etoposide (B) at indicated concentrations for 6 h were cocultured with mouse spleen cells in 48-well plates for 8 d in {alpha}-MEM that contained 10% FBS and 10-8 M 1{alpha},25(OH)2D3. Controls were treated with 0.1% of dimethylsulfoxide. Fresh medium was supplied every 3 d. The cells were stained for TRAP activity and TRAP-positive multinucleated cells were counted under a microscope. Data are means ± SE of results from four determinations.

 


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  		Figure 7. Inhibition of the expression of mRNAs for RANKL and OPG by inhibitors of topo II. Total RNA was isolated from ST2 cells that had been treated for 6 h with 5 µM amsacrine (Amsa) or 0.5 µM etoposide (Etopo) and then cultured for 2 d in the presence and in the absence of 10-8 M 1{alpha},25(OH)2D3 (VD3). Then we examined the effects of amsacrine and etoposide on expression of mRNAs for RANKL and OPG by RT-PCR. The products of RT-PCR were subjected to electrophoresis in a 2% agarose gel and were allowed to hybridize with 32P-labeled cDNAs for RANKL, OPG, and GAPDH as described in Materials and Methods. The results shown are representative of the results of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Osteoporosis is accepted as a major public-health problem. In particular, postmenopausal estrogen deficiency often results in osteoporosis and nutritional and pharmacological factors are needed to prevent bone loss. Genistein is a natural isoflavone (16) that prevents bone loss caused by estrogen deficiency in ovariectomized rats (21, 22, 23, 24). In experiments in vitro, this compound promotes osteoblastic differentiation (17). When genistein was added continuously to the culture medium of mouse bone marrow cells, the formation of OCLs was inhibited as a result, supposedly, of the estrogen-like action of this compound (18). Thus, genistein might be an efficient agent for retention of bone mass. However, little information is available about the mechanism of action of genistein in bone metabolism. In an effort to clarify the mode of action of genistein in osteoclastogenesis, we focused on the expression of mRNAs for RANKL, OPG, and M-CSF in ST2 cells that support the formation of OCLs.

RANKL is a membrane-bound protein that induces formation of OCLs in combination with M-CSF (3, 6, 7, 8). By contrast, OPG, which is a decoy receptor for RANKL, inhibits formation of OCLs (4, 5). The RANKL and OPG produced by osteoblasts or osteogenic stromal cells are key regulators of osteoclastogenesis. The ratio of levels of RANKL and OPG in the microenviroment is critical in the regulation of the formation of OCLs. Therefore, analysis of compounds that regulate the expression of RANKL and/or OPG is important for a full understanding of bone metabolism and metabolic diseases of bones. Our findings suggest that a low level of mRNA for RANKL and a high level of mRNA for OPG, expressed in ST2 cells in response to genistein, might attenuate the formation of OCLs in our coculture system. In fact, the addition of sRANKL to the medium of coculture system with genistein-treated ST2 cells and spleen cells restored the formation of TRAP-positive OCLs. However, the formation of OCLs by 100 ng/ml of sRANKL was approximately 50% of control. We consider that increased expression of OPG by genistein might attenuate the effect of sRANKL. The reciprocal control of the expression of mRNAs for RANKL and OPG by genistein has not previously been reported and is quite unusual. To date, several factors, such as 1{alpha},25(OH)2D3 (5, 25), PTH (5, 25), PG E2(5, 26), and glucocorticoid (27, 28), are known that stimulate the expression of mRNA for RANKL and depress that of the mRNA for OPG. By contrast, to our knowledge, only TGF-ß acts similarly to genistein, stimulating expression of the mRNA for OPG and depressing expression of the mRNA for RANKL (29, 30). Correlations between the actions of genistein and TGF-ß remain to be elucidated. In preliminary experiments, however, we found that genistein did not affect the expression of mRNA for TGF-ß1 in ST2 cells. Genistein is reported to induce differentiation and apoptosis of cells. Nevertheless, we have obtained the results that genistein did not affect the expression of differentiation marker genes for osteoblasts, such as type I-collagen, alkaline phosphatase, osteopontin, and osteocalcin, and those for adipocytes, such as peroxisome proliferator activated receptor {gamma} and {alpha}-glycerophosphate-3-dehydrogenase, in ST2 cells. Furthermore, this compound did not also induce apoptosis of ST2 cells under our condition of culture.

To determine how genistein might reciprocally regulate expression of the mRNAs for RANKL and OPG, we examined the effects of 17ß-estradiol, inhibitors of topo II, and inhibitors of tyrosine kinase on the formation of OCLs. Exposure of ST2 cells to 17ß-estradiol (0.1 nM to 50 µM) for 6 h before coculture did not inhibit the formation of OCLs, indicating that genistein does not act via an estrogen receptor-mediated mechanism in ST2 cells. The inhibitors of certain tyrosine kinase tested also did not affect the formation of OCLs at optimal concentrations but other tyrosine kinases might be involved in the formation of OCLs. By contrast, the inhibitors of topo II amsacrine and etoposide inhibited the formation of OCLs; they suppressed the expression of the mRNA for RANKL and simultaneously increased expression of the mRNA for OPG, being as effective as genistein. Thus, our findings show that the effects of genistein in ST2 cells might be due to inhibition of topo II and that the effects of genistein on bone health might be not limited to agonism at estrogen receptor.

The topo II family consists of highly conserved nuclear enzymes that are involved in the replication, transcription, and repair of DNA (31, 32, 33, 34). The mechanism of action of topo II in the regulation of transcription is not fully understood. We found in the present study that two inhibitors of topo II, amsacrine and etoposide, suppressed expression of the gene for RANKL and stimulated expression of the gene for OPG in ST2 cells. This is the first report, to our knowledge, that topo II might play a key role in osteoclastogenesis. Inhibitors of topo II are important targets for anticancer drugs (35, 36, 37). Our findings also suggest that genistein and other inhibitors of topo II might be useful tools in the prevention and treatment of postmenopausal osteoporosis. Further investigations, including measurements of levels of topo II during osteoclastogenesis, are needed to examine these possibilities.


   Footnotes
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, and by grants from the Uehara Memorial Foundation.

Abbreviations: EGF, Epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M-CSF, macrophage colony-stimulating factor; MEK, mitogen-activated protein kinase/extracellular regulated kinase kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; OCIF, osteoclastogenesis-inhibitory factor; ODF, osteoclast differentiation factor; OCL, osteoclast-like cell; OPGL, OPG ligand; sRANKL, soluble RANKL; TRANCE, TNF-related activation-induced cytokine; TRAP, tartrate-resistant acid phosphatase.

Received December 28, 2000.

Accepted for publication April 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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