has been demonstrated that inactivation of P. gingivalis fimA, the gene for the major fimbrial subunit of the organism, blocks. P. gingivalis-induced alveolar bone ...
INFECTION AND IMMUNITY, June 1995, p. 2374–2377 0019-9567/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 63, No. 6
Porphyromonas gingivalis Fimbria-Stimulated Bone Resorption In Vitro Is Inhibited by a Tyrosine Kinase Inhibitor SHIGEMASA HANAZAWA,* YASUHIRO KAWATA, YUKIO MURAKAMI, KATSUYUKI NAGANUMA, SHIGERU AMANO, YUKO MIYATA, AND SHIGEO KITANO Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Japan Received 5 December 1994/Returned for modification 6 February 1995/Accepted 28 March 1995
Our previous study (Y. Kawata, S. Hanazawa, S. Amano, Y. Murakami, T. Matsumoto, K. Nishida, and S. Kitano, Infect. Immun. 62:3012–3016, 1994) showed that Porphyromonas gingivalis fimbriae stimulate bone resorption in vitro. Since it has recently been demonstrated that tyrosine kinase encoded by the c-src gene plays an important role in osteoclastic bone resorption, in the present study we examined the effect of a tyrosine kinase inhibitor on the fimbria-stimulated bone resorption. Genistein, a potent inhibitor of tyrosine kinase, markedly inhibited bone resorption stimulated by the fimbriae. Genistein also inhibited induction of several tyrosine-phosphorylated proteins in the fimbria-treated calvarial bone cells from mouse embryos. The bone resorption assay was described in detail in our previous study (1). In brief, bone cells were liberated from the calvaria of ICR mouse embryos at the age of 14 days (CLEA Japan, Tokyo, Japan) with bacterial collagenase and trypsin. The calvarial bone cells were incubated at a cell density of 5 3 105 cells per 15 ml of 10% fetal calf serum (FCS) (Flow Laboratories, McLean, Va.)-containing a-Eagle’s minimum essential medium (a-MEM) (Flow Laboratories) on a bovine femoral bone slice (4 by 4 mm). One hour later, they were washed and then cultured with or without the fimbriae or genistein. After various incubation times, the bone slices were scraped with a rubber policeman to remove cells to enable visualization of the bone surface. The scraped bone slices were dehydrated with ethanol and sputter coated with gold. Then, the slices were examined with a T-200 scanning electron microscope (Japan Electronics Co., Tokyo, Japan). The resorbed area on the bone slices was traced to polyethylene film and then measured with a Shonic graphic analyzer (Showa Denko Co., Tokyo, Japan). These results were expressed as the means 6 standard errors of four replicate cultures. Tyrosine-phosphorylated proteins were detected by an immunoprecipitation assay using antiphosphotyrosine antibody. The calvarial bone cells (107) were incubated for 1 h in 1 ml of low-phosphate Eagle’s MEM (Sigma) with or without genistein at 1 mg/ml in 3-cm plastic dishes and then were either treated with the fimbriae or left untreated. Thereafter, 32Pi (20 mCi; Amersham Japan, Tokyo, Japan) was added to the cultures, and after an incubation at 378C for 6 h the cultures were placed on ice and washed three times with cold phosphate-buffered saline containing 1 mM sodium vanadate. The washed cells were solubilized with homogenizing buffer (2 mM sodium vanadate, 5 mM Tris-HCl [pH 7.9], 150 mM NaCl, 20 mM EDTA, 10 mM NaF, and 10 mg of aprotinin per ml). The cell lysates were then centrifuged for 1 h at 150,000 3 g at 48C, treated for 6 h at 48C with anti-mouse immunoglobulin G antibody (Zymed Laboratories, South San Francisco, Calif.), and then incubated with protein A-Sepharose beads (30 ml; PharmaciaLKB Japan, Tokyo, Japan). After having been washed four times in homogenizing buffer as described above, the cell lysates were treated for 6 h at 48C with antiphosphotyrosine mouse monoclonal antibody (Zymed). Immune complexes were recovered by incubation with protein A-Sepharose beads, washed five times with homogenizing buffer, and then treated with the SDS-PAGE sample buffer (1.3% SDS, 2.5% 2-mer-
Porphyromonas gingivalis fimbriae play an important role in the pathogenesis of adult periodontitis associated with this organism (4, 5, 7–10, 12, 16, 17). A recent study (5) has shown that gnotobiotic rats immunized with P. gingivalis fimbriae are afforded protection from resorption of the alveolar bone caused by infection with this bacterium. More recently, it also has been demonstrated that inactivation of P. gingivalis fimA, the gene for the major fimbrial subunit of the organism, blocks P. gingivalis-induced alveolar bone loss in gnotobiotic rats (14). These studies suggest that P. gingivalis fimbriae may play a pivotal role in initiation and development of the alveolar bone loss in adult periodontal disease. It has been observed that mice deficient in the expression of the c-src gene develop osteoporosis (18). Since the src gene product has tyrosine kinase activity, this demonstration suggests the possibility that tyrosine kinase plays an important role in osteoclastic function. Several other studies (2, 6, 11, 18–20) have also shown the importance of tyrosine kinase in osteoclastic cell function. We (12) demonstrated that P. gingivalis fimbriae stimulate bone resorption in vitro, although the mechanism(s) responsible for this activation was not defined. In the present study, we examined, by using a tyrosine kinase inhibitor, the role of tyrosine kinase in fimbria-stimulated bone resorption in vitro. We provide evidence that tyrosine kinase functions as a key enzyme in the process of bone resorption stimulated by P. gingivalis fimbriae. P. gingivalis ATCC 33277 fimbriae were prepared and purified according to the method of Yoshimura et al. (21) as described previously (8). Lipopolysaccharide contamination in the purified fimbria preparation at 4 mg of protein per ml used in this study was not detected by silver staning following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and was not involved in the fimbria-stimulated bone resorption as demonstrated previously (12). Protein content of the fimbriae was measured by the method of Bradford (3). Genistein (Sigma Chemical Co., St. Louis, Mo.) was used as a potent inhibitor of tyrosine kinase, as described previously (15).
* Corresponding author. Mailing address: Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan. 2374
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FIG. 1. Genistein inhibits pit formation and bone resorption of mouse embryonic calvarial bone cells treated with P. gingivalis fimbriae. Calvarial bone cells (5 3 105 per bone slice) prepared from 14-day-old mouse embryos were inoculated onto a bone slice and incubated in 10% FCS-containing a-MEM with or without the fimbriae at 4 mg of protein per ml and with or without genistein. After 7 days, pits were counted (A) and the resorption areas were measured (B). The data are means with standard errors of four replicate cultures. Three identical experiments, independently performed, gave similar results.
captoethanol, 0.09 M Tris [pH 6.8], 5% glycerol). 32P activity in the recovered immune complex was determined, and an amount of immune complex with a radioactivity of 104 cpm was analyzed on SDS–10% polyacrylamide gels by the method of Laemmli (13). As molecular markers, we used the following: lysozyme (Mr, 14,300), trypsin inhibitor (Mr, 21,500), carbonic anhydrase (Mr, 30,000), ovalbumin (Mr, 46,000), bovine serum albumin (Mr, 69,000), phosphorylase (Mr, 97,400), and myosin (Mr, 200,000). Following the electrophoresis, the gels were dried and then, for autoradiography, were exposed to Kodak X-Omat film at 2708C. We (12) showed previously that the fimbriae at 4 mg of protein per ml significantly stimulated osteoclastic bone resorption by calvarial bone cells from 14-day-old mouse embryos and that the stimulatory action was markedly neutralized by monoclonal antibody specific for fimbriae. However, we have observed that the monoclonal antibody did not affect P. gingivalis lipopolysaccharide-stimulated bone resorption (unpublished data). These findings indicate that any lipopolysac-
charide contaminant in the purified fimbria preparation was not involved in the fimbria-stimulated bone resorption. Firstly, we examined the effect of genistein on the fimbriastimulated bone resorption. As shown in Fig. 1, genistein at 0.1 and 1 mg/ml inhibited significantly the fimbria-stimulated bone resorption. We observed by the trypan blue dye exclusion assay that the genistein doses used were not cytotoxic for the calvarial bone cells (data not shown). Next, we examined the kinetics of the inhibitory action of genistein at 1 mg/ml on the fimbria-stimulated bone resorption. The fimbriae were inoculated simultaneously with or without the inhibitor into the cultures of calvarial bone cells on bone slices. After the selected times, the area of bone resorption lacunae was measured. As shown in Fig. 2, the inhibitor markedly inhibited bone resorption stimulated by the fimbriae on days 3, 5, and 7 after initiation of the treatment. We also examined the effect of increasing the time interval between addition of the fimbriae and genistein on the fimbriastimulated bone-resorbing activity. Genistein at 1 mg/ml was
FIG. 2. Inhibitory kinetics of genistein for P. gingivalis fimbria-induced bone-resorbing activity. Calvarial bone cells (5 3 105 per bone slice) prepared from 14-day-old mouse embryos were inoculated onto a bone slice and incubated in 10% FCS-containing a-MEM with the fimbriae (4 mg of protein per ml) with or without genistein at 1 mg/ml. After the selected incubation times, pits were counted (A) and the resorption areas were measured (B). The data are means with standard errors of four replicate cultures. Three identical experiments, independently performed, gave similar results.
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FIG. 3. Effect of increasing the time interval between addition of genistein and addition of the fimbriae on bone-resorbing activity. Calvarial bone cells (5 3 105 per bone slice) prepared from 14-day-old mouse embryos were inoculated onto a bone slice and incubated in 10% FCS-containing a-MEM with fimbriae (4 mg of protein per ml). At the selected incubation times, the cultured cells were treated (– – – –) with genistein at 1 mg/ml or were left untreated (———). The numbers of pits (A) and the resorption areas (B) were measured on day 7 after initiation of the fimbria treatment. Arrows, genistein addition. The data are mean with standard errors of four replicate cultures. Two identical experiments, independently performed, gave similar results.
added to the calvarial bone cells with or without the fimbriae simultaneously or with delays of different amounts of time. The area of bone resorption lacunae was measured on day 7 after initiation of the fimbria treatment. Figure 3 shows that maximum inhibition was observed when genistein was added at the beginning of the cell culture. When the inhibitor was added later, the inhibitory effect gradually decreased, and no significant inhibition was observed when the inhibitor was added on day 5 after initiation of the cell culture. Finally, we examined the inhibitory effect of genistein on induction of tyrosine-phosphorylated protein in the mouse embryonic calvarial bone cells by the fimbriae. As shown in Fig. 4, the fimbriae induced several tyrosine-phosphorylated proteins in the calvarial bone cells. Genistein markedly inhibited induction of the phosphorylated proteins corresponding to apparent masses of 36, 59, 69, 82, and 103 kDa in the fimbria-treated cells.
FIG. 4. Inhibitory effect of genistein on induction of tyrosine-phosphorylated proteins in the calvarial bone cells by the fimbriae. Calvarial bone cells (107) prepared from 14-day-old mouse embryos were inoculated into 3-cm plastic dishes and incubated in 10% FCS-containing a-MEM with or without genistein at 1 mg/ml. After incubation for 1 h, the cell cultures were treated with the fimbriae at 4 mg of protein per ml or were left untreated. Then, 32Pi at 20 mCi per plate was added to the cultures. The tyrosine-phosphorylated proteins were detected at 6 h after the initiation of the fimbrial treatment by an immunoprecipitation assay with antiphosphotyrosine antibody as described in Materials and Methods.
Recent studies (4, 5, 12) have strongly suggested that P. gingivalis fimbriae are involved in the pathogenesis of the alveolar bone in periodontal disease. Although the mechanism of the fimbria-stimulated bone resorption has not yet been defined in detail, in the present study we demonstrated that genistein, a potent inhibitor of tyrosine kinase, dramatically inhibits the fimbria-stimulated bone resorption. This demonstration is supported by several recent studies (2, 6, 18, 19) indicating that the c-src gene encoded tyrosine kinase plays an important role in the function of osteoclastic cells. Thus, our present study suggests that tyrosine kinase plays a functional role in the fimbria-stimulated bone resorption. Several studies have indicated that tyrosine kinase also plays an important role in osteoclastic cell differentiation. In the experiment involving certain time intervals between addition of the fimbria and genistein in the bone resorption assay, we showed that significant inhibition was observed when the inhibitor was added on day 1 or 3 after the fimbria addition. Thus, tyrosine kinase may also be involved in differentiation of osteoclasts induced by the fimbriae. Our previous study (12) suggested that formation of the fimbria-stimulated osteoclasts was mediated in part via interleukin-1 and granulocyte-macrophage colony-stimulating factor. Although we now do not know whether tyrosine kinase acts as a signal for induction of both of these cytokines by the fimbriae, in further experiments we shall examine this point. We examined using antiphosphotyrosine antibody which tyrosine-phosphorylated proteins in the fimbria-treated bone cells are inhibited by genistein. In this experiment, we observed that genistein inhibited induction of several phosphorylated proteins by the fimbria. Therefore, these phosphorylated proteins may play an important role in the fimbria-stimulated bone resorption, though the precise function of these proteins is yet unknown. In conclusion, the present study suggests the possibility that a tyrosine kinase inhibitor such as genistein may have therapeutic potential as an inhibitor of alveolar bone loss in adult periodontal disease. REFERENCES 1. Amano, S., S. Hanazawa, Y. Kawata, K. Ohta, H. Kitami, and S. Kitano. 1992. An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors. J. Bone Miner. Res. 7:321–328. 2. Boyce, B. F., T. Yoneda, C. Lowe, P. Soriano, and G. R. Mundy. 1992. Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. J. Clin. Invest. 90:1622–1627.
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