Calcif Tissue Int (1992) 51(Suppl 1):87--810
Calcified Tissue International © 1992 Springer-Verlag New York Inc.
Ipriflavone Inhibits Murine Osteoclast Formation In Vitro Ikuo Morita, Kouji S a k a g u c h i , T o s h i a k i K u r a c h i , and Sei-itsu M u r o t a Department of Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan
Summary. Ipriflavone, one of the isoflavone derivatives, is a therapeutic drug for osteoporosis. The mechanism is thought to be the inhibition of bone resorption. In the present paper, we report that ipriflavone inhibited formation of osteoclasts from murine spleen cells co-cultured with stromal cells cloned from murine bone marrow. In this system, ipriflavone inhibited osteoclast g e n e r a t i o n in a dosedependent manner (10-7-10 -5 M). Ipriflavone also inhibited prostaglandin E 2 production in MC3T3-E1 cells, which are widely employed as osteoblasts. Moreover, ipriflavone inhibited the proliferation of stromal cells ( 1 0 - 6 1 0 -5 M), but not osteoblastic cells. These results suggest that one mechanism for the inhibitory effects of ipriflavone on bone resorption is the inhibition of osteoclast formation through inhibiting prostaglandin E 2 production in osteoblasts and thereby suppressing proliferation of stromal cells. Key words: Ipriflavone - Osteoblast - Prostaglandin E2 Stromal cells - Proliferation.
ously by Sakaguchi et al., unpublished data. Briefly, the bone marrow of 7-week-old male ddy mice (Nisseizai, Tokyo, Japan) was flushed from femurs and tibiae by ice-cold c~-MEM. The cells were collected, pelleted, resuspended in ct-MEM supplemented with 10% FCS, and subsequently incubated for 6 hours (37°C, 5% CO2). Adherent cells were incubated for an additional 7 days. Cloning by alkaline phosphatase (ALP) staining was carried out twice. Twentytwo ALP-positive clones (TMS) were obtained and characterized. Among them, TMS-14 and TMS-12 supported osteoclast formation.
Spleen Cell Isolation Murine spleen cells were prepared by a modified method described previously [9]. Briefly, the spleens of 6- to 8-week-old male ddy mice were minced and a single cell suspension was prepared. The cells were purified using a density gradient between Ficoll (type 400, Sigma, St. Louis, MO) and 60% Urografin (Scbering AG, Germany). A layer of monocytes was collected and used for the following experiments.
Co-culture o f Spleen Cells and Stromal Cells Iprifiavone (IP), 7-isopropoxy-3-phenyl-4H-1-benzopyran-4one, has been shown to prevent the decrease of bone mass in several models of experimental osteoporosis [1-3]. Beneficial clinical effects of IP in osteoporotic patients have also been reported [4, 5]. In regard to the mechanism, it has been recognized that IP increases the uterotrophic activity of estrogens [6] and stimulates estrogen-induced calcitonin secretion [7]. Tsuda et al. [8] reported that IP directly inhibited 45Ca release from bones in tissue culture experiments. Though this observation is certainly novel, the mechanism by which IP suppresses bone resorption has remained enigmatic. To more clearly understand the mechanism, we have examined the effect of IP in an in vitro system involving the co-culture of spleen cells and a stromal cell line. In the present study, it was demonstrated that IP inhibits formation of osteoclasts in a dose-dependent manner. Furthermore, this effect is caused by an inhibition of both prostaglandin E 2 production and the proliferation of stromal cells. Materials and Methods a-MEM (minimal essential medium) and fetal calf serum (FCS) were purchased from Gibco (Grand Island, NY). IP was supplied by Takeda Chemical Industries (Osaka, Japan). Other reagents used were of analytical grade.
Stromal Cells Murine stromal cells were isolated and cloned as described previ-
Offprint requests to: I. Morita
TMS-14 stromal cells (5 × 103 cells/well, 24-well tissue culture plates) were cultured for 24 hours. After this period, purified spleen cells (5 × 105 cells/well) were seeded onto the TMS cells. The cultures were maintained for 8 days, with the medium being changed twice a week (only 80% of the medium was changed each time).
Osteoclast Formation Numerous osteoclasts were formed in our co-culture system, and osteoclast formation was measured by tartrate-resistant acid phosphatase (TRACP) activity according to a modified method of Kind and King [10]. In brief, the cells were washed by phosphate-buffered saline (PBS[-]) and fixed by ethanol-acetone (l:l) for 1 minute. Then 0.215% phenylphosphate and 0.6% 4-aminoantipyrine in 0.25 M tartrate-0.1% citrate buffer (pH 4.9) were added to and incubated with the fixed cells for 10 minutes. The reaction was stopped by the addition of 1.2% KaFe(CN)6, 1% NaOH, and 2.1% NaHCO 3. The absorbance of the solution was measured at 500 nm by spectrophotometry (UV-160, Shimadzu, Kyoto, Japan).
Prostaglandin E z Synthesizing Activity and Content The prostaglandin E2 synthesizing activity in the cell-free system was measured by the conversion of [14C]-arachidonic acid to prostaglandin E2. Osteoblasts were scraped and placed in a container with 1/15 M phosphate buffer (pH 7.4) and sonicated. The solution was then incubated with 0.2 ~Ci (1 ~.g/ml) of [14C]-arachidonicacid for 20 minutes at 37°C. The reaction was stopped by the addition of 0.1 N HC1 and extracted with ethyl acetate. The extract was evaporated and the residue dissolved in EtOH. The product was subjected to thin layer chromatography (TLC) and developed by using
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Calcified Tissue International © 1992 Springer-Verlag New York Inc.
Ipriflavone Inhibits Murine Osteoclast Formation In Vitro Ikuo Morita, Kouji S a k a g u c h i , T o s h i a k i K u r a c h i , and Sei-itsu M u r o t a Department of Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan
Summary. Ipriflavone, one of the isoflavone derivatives, is a therapeutic drug for osteoporosis. The mechanism is thought to be the inhibition of bone resorption. In the present paper, we report that ipriflavone inhibited formation of osteoclasts from murine spleen cells co-cultured with stromal cells cloned from murine bone marrow. In this system, ipriflavone inhibited osteoclast g e n e r a t i o n in a dosedependent manner (10-7-10 -5 M). Ipriflavone also inhibited prostaglandin E 2 production in MC3T3-E1 cells, which are widely employed as osteoblasts. Moreover, ipriflavone inhibited the proliferation of stromal cells ( 1 0 - 6 1 0 -5 M), but not osteoblastic cells. These results suggest that one mechanism for the inhibitory effects of ipriflavone on bone resorption is the inhibition of osteoclast formation through inhibiting prostaglandin E 2 production in osteoblasts and thereby suppressing proliferation of stromal cells. Key words: Ipriflavone - Osteoblast - Prostaglandin E2 Stromal cells - Proliferation.
ously by Sakaguchi et al., unpublished data. Briefly, the bone marrow of 7-week-old male ddy mice (Nisseizai, Tokyo, Japan) was flushed from femurs and tibiae by ice-cold c~-MEM. The cells were collected, pelleted, resuspended in ct-MEM supplemented with 10% FCS, and subsequently incubated for 6 hours (37°C, 5% CO2). Adherent cells were incubated for an additional 7 days. Cloning by alkaline phosphatase (ALP) staining was carried out twice. Twentytwo ALP-positive clones (TMS) were obtained and characterized. Among them, TMS-14 and TMS-12 supported osteoclast formation.
Spleen Cell Isolation Murine spleen cells were prepared by a modified method described previously [9]. Briefly, the spleens of 6- to 8-week-old male ddy mice were minced and a single cell suspension was prepared. The cells were purified using a density gradient between Ficoll (type 400, Sigma, St. Louis, MO) and 60% Urografin (Scbering AG, Germany). A layer of monocytes was collected and used for the following experiments.
Co-culture o f Spleen Cells and Stromal Cells Iprifiavone (IP), 7-isopropoxy-3-phenyl-4H-1-benzopyran-4one, has been shown to prevent the decrease of bone mass in several models of experimental osteoporosis [1-3]. Beneficial clinical effects of IP in osteoporotic patients have also been reported [4, 5]. In regard to the mechanism, it has been recognized that IP increases the uterotrophic activity of estrogens [6] and stimulates estrogen-induced calcitonin secretion [7]. Tsuda et al. [8] reported that IP directly inhibited 45Ca release from bones in tissue culture experiments. Though this observation is certainly novel, the mechanism by which IP suppresses bone resorption has remained enigmatic. To more clearly understand the mechanism, we have examined the effect of IP in an in vitro system involving the co-culture of spleen cells and a stromal cell line. In the present study, it was demonstrated that IP inhibits formation of osteoclasts in a dose-dependent manner. Furthermore, this effect is caused by an inhibition of both prostaglandin E 2 production and the proliferation of stromal cells. Materials and Methods a-MEM (minimal essential medium) and fetal calf serum (FCS) were purchased from Gibco (Grand Island, NY). IP was supplied by Takeda Chemical Industries (Osaka, Japan). Other reagents used were of analytical grade.
Stromal Cells Murine stromal cells were isolated and cloned as described previ-
Offprint requests to: I. Morita
TMS-14 stromal cells (5 × 103 cells/well, 24-well tissue culture plates) were cultured for 24 hours. After this period, purified spleen cells (5 × 105 cells/well) were seeded onto the TMS cells. The cultures were maintained for 8 days, with the medium being changed twice a week (only 80% of the medium was changed each time).
Osteoclast Formation Numerous osteoclasts were formed in our co-culture system, and osteoclast formation was measured by tartrate-resistant acid phosphatase (TRACP) activity according to a modified method of Kind and King [10]. In brief, the cells were washed by phosphate-buffered saline (PBS[-]) and fixed by ethanol-acetone (l:l) for 1 minute. Then 0.215% phenylphosphate and 0.6% 4-aminoantipyrine in 0.25 M tartrate-0.1% citrate buffer (pH 4.9) were added to and incubated with the fixed cells for 10 minutes. The reaction was stopped by the addition of 1.2% KaFe(CN)6, 1% NaOH, and 2.1% NaHCO 3. The absorbance of the solution was measured at 500 nm by spectrophotometry (UV-160, Shimadzu, Kyoto, Japan).
Prostaglandin E z Synthesizing Activity and Content The prostaglandin E2 synthesizing activity in the cell-free system was measured by the conversion of [14C]-arachidonic acid to prostaglandin E2. Osteoblasts were scraped and placed in a container with 1/15 M phosphate buffer (pH 7.4) and sonicated. The solution was then incubated with 0.2 ~Ci (1 ~.g/ml) of [14C]-arachidonicacid for 20 minutes at 37°C. The reaction was stopped by the addition of 0.1 N HC1 and extracted with ethyl acetate. The extract was evaporated and the residue dissolved in EtOH. The product was subjected to thin layer chromatography (TLC) and developed by using
$8
I. Morita et al.: Ipriflavone Inhibits Murine Osteoclast Formation In Vitro 1.0
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