Mouse Marrow Culture Research Articles

Regucalcin and metabolic disorders: osteoporosis and hyperlipidemia are induced in regucalcin transgenic rats

Regucalcin transgenic (TG) rat has been generated to determine the role in metabolic disorders. Regucalcin homozygote male and female rats induce a prominent increase in regucalcin protein in the various tissues. Bone loss has been found to induce in regucalcin TG rats with growing (5 weeks old) and aging (50 weeks old). Osteoclastogenesis has been shown to stimulate in culture with the bone marrow cells obtained from regucalcin TG rats. Exogenous regucalcin stimulates osteoclastogenesis in mouse marrow culture in vitro. Regucalcin has a suppressive effect on the differentiation and mineralization in osteoblastic MC3T3-E1 cells in vitro. The mechanism by which regucalcin TG rat induces bone loss may result from the enhancement of osteoclastic bone resorption and the suppression of osteoblastic bone formation. Moreover, regucalcin TG rat has been found to induce hyperlipidemia with increasing age (14-50 weeks); serum triglyceride, high-density lipoprotein (HDL)-cholesterol, free fatty acid, albumin and calcium concentrations are markedly increased in regucalcin TG male and female rats with increasing age. The decrease in lipid and glycogen contents in liver tissues is induced in regucalcin TG rats. The gene expression of leptin and adiponectin is suppressed in the TG rats. Overexpression of regucalcin has been shown to enhance glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells in vitro, and insulin resistance is seen in the cells. The expression of glucose transporter 2 mRNA is increased in the transfectants, while it has been shown to suppress insulin receptor and phosphatidylinositol 3-kinase mRNA expressions that are involved in insulin signaling. This review proposes that regucalcin relates in osteoporosis and hyperlipidemia, and that the regucalcin TG rat model may be useful in determining the pathophysiologic state and the development of therapeutic tool for osteoporosis and hyperlipidemia.

Genistein and zinc synergistically stimulate apoptotic cell death and suppress RANKL signaling‐related gene expression in osteoclastic cells

The effects of the combination of genistein and zinc, which have an anabolic effect on bone metabolism, on osteoclastic cells in mouse marrow culture system in vitro was investigated. The macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages were cultured in the presence of M-CSF (10 ng/ml) and receptor activator of nuclear factor kappaB (NF-kB) ligand (RANKL; 50 ng/ml) for 4 days. The osteoclastic cells formed were further cultured in medium containing either vehicle, genistein, zinc sulfate (zinc), or genistein plus zinc with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 24 or 72 h. The number of osteoclastic cells was significantly decreased with culture of genistein (10(-6) M) plus zinc (10(-5) M) in presence or absence of M-CSF and RANKL for 24 or 72 h as compared with the value for genistein or zinc alone. Agarose gel electrophoresis showed the presence of low-molecular weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with genistein (10(-6) M) plus zinc (10(-5) M) for 24 or 72 h, indicating that the combination of two chemicals induces apoptotic cell death. Such an effect was not seen in the case of each chemical. Genistein plus zinc-induced decrease in osteoclastic cells were significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Culture with genistein (10(-6) M) plus zinc (10(-5) M) for 72 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL as compared with the value for each chemical alone. Genistein plus zinc-induced increase in caspase-3 mRNA expression was completely inhibited in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB; 10(-6) M), an inhibitor of transcription activity. The mRNA expression of tartrate-resistant acid phosphatase (TRACP) or cathepsin K was significantly decreased with culture of genistein plus zinc in the presence of M-CSF and RANKL for 72 h as compared with genistein or zinc alone. Nuclear factor of activated T cells c1 (NFATc1) mRNA expression was significantly decreased with culture of genistein plus zinc in the presence of M-CSF and RANKL for 24 or 72 h as compared with each chemical alone, while NF-kB mRNA expression was significantly changed. This study demonstrates that the combination of genistein and zinc has potent stimulatory effects on apoptotic cell death and suppressive effects on osteoclastic cell function.

β‐cryptoxanthin stimulates apoptotic cell death and suppresses cell function in osteoclastic cells: Change in their related gene expression

The effect of beta-cryptoxanthin, a kind of carotenoid, on osteoclastic cells in mouse marrow culture system in vitro was investigated. The macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages were cultured in the presence of M-CSF (10 ng/ml) and receptor activator of NF-kappaB ligand (RANKL; 25 ng/ml) for 4 days. The osteoclastic cells formed were further cultured in medium containing either vehicle or beta-cryptoxanthin (10(-8)-10(-6) M) with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 24-72 h. Osteoclastic cells were significantly decreased with culture of beta-cryptoxanthin (10(-7) or 10(-6) M) with or without M-CSF and RANKL for 24, 48, or 72 h. beta-Cryptoxanthin (10(-8) M)-induced decrease in osteoclastic cells were significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with beta-cryptoxanthin (10(-7) or 10(-6) M) for 24 or 48 h, indicating that the carotenoid induces apoptotic cell death. Apoptosis-related gene expression was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with beta-cryptoxanthin (10(-7) or 10(-6) M) for 24 or 48 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL, while Bcl-2 and Apaf-2 mRNA expressions were significantly increased with culture of beta-cryptoxanthin (10(-7) or 10(-6) M) without M-CSF and RANKL for 24 or 48 h. Akt-1 mRNA expression was not significantly changed with culture of the carotenoid (10(-7) or 10(-6) M) for 24 or 48 h. Moreover, tartrate-resistant acid phosphatase (TRACP) activity, or TRACP and cathepsin K mRNA expressions were significantly decreased with culture of beta-cryptoxanthin (10(-6) M) in the presence or absence of M-CSF and RANKL for 48 h. This study demonstrates that beta-cryptoxanthin has stimulatory effects on apoptotic cell death and suppressive effects on osteoclastic cell function.

Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model.

Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham), ovariectomized (OVX), OVX given 0.5% (w/w) raw RJ, OVX given 2.0% (w/w) RJ, OVX given 0.5% (w/w) protease-treated RJ (pRJ), OVX given 2.0% (w/w) pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD) by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w) RJ and 0.5–2.0% (w/w) pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH)-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

Regucalcin stimulates osteoclast‐like cell formation in mouse marrow cultures

The effect of regucalcin, a regulatory protein in intracellular signaling, on osteoclastic cell formation in mouse bone marrow culture is investigated. The bone marrow cells were cultured for 7 days in an alpha-minimal essential medium containing either vehicle or regucalcin (10(-10)-10(-8)M). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of regucalcin (10(-10)-10(-8)M) caused a remarkable increase in osteoclast-like multinucleated cells (MNCs). The effect of regucalcin in stimulating osteoclast-like cell formation was significantly inhibited in the presence of calcitonin (CT; 10(-9)M), 17beta-estradiol (10(-9)M), beta-cryptoxanthin (CX; 10(-6)M), or zinc sulfate (10(-4)M), which is an anti-bone resorbing factor. The effect of regucalcin on osteoclast-like cell formation was not significantly blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. The effect of parathyroid hormone (10(-7)M), 1,25-dihydroxyvitamin D(3) (10(-7)M), prostaglandin E(2) (10(-5)M), or tumor necrosis factor-alpha (10 ng/ml) in increasing osteoclast-like cell formation was significantly enhanced in the presence of regucalcin (10(-8)M). Moreover, when rat femoral-diaphyseal or -metaphyseal tissues were cultured for 48 h in the presence of regucalcin (10(-10)-10(-8)M), the diaphyseal or metaphyseal calcium content was significantly decreased in the presence of regucalcin (10(-10)-10(-8)M) in vitro. The consumption of glucose and the production of lactic acid in culture medium by the diaphyseal or metaphyseal tissues was significantly raised in the presence of regucalcin (10(-10)-10(-8)M). This study demonstrates that regucalcin directly stimulates osteoclast-like cell formation in mouse marrow culture in vitro, and that the protein stimulates bone resorption in rat femoral tissues in vitro.

Receptor activator of NF-κB ligand-stimulated osteoclastogenesis in mouse marrow culture is suppressed by zinc in vitro

Zinc has been shown to have an inhibitory effect on osteoclastic bone resorption in vitro. This study was undertaken to determine whether the inhibitory action of zinc on osteoclastogenesis is related to receptor activator of NF-kappaB ligand (RANKL), which plays a pivotal role in differentiation from pre-osteoclasts to osteoclasts. Mouse marrow cells were cultured for 3 days in alpha-minimal essential medium containing lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNFalpha), or RANKL, which stimulates osteoclastogenesis; then zinc sulfate was added to the culture medium containing each osteoclastogenesis-stimulating factor, and the cells were further incubated for 4 days. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of LPS (10 micro g/ml), TNFalpha (10 ng/ml), or RANKL (10 or 20 ng/ml) with M-CSF (10 or 20 ng/ml) induced a remarkable increase in osteoclast-like multinucleated cells (MNCs). The stimulatory effect of LPS was not significantly altered by the addition of zinc sulfate (10(-6)-10(-4) M). Meanwhile, TNFalpha- or RANKL-induced osteoclast-like cell formation was significantly inhibited in the presence of zinc sulfate (10(-6)-10(-4) M). The effect of zinc sulfate (10(-4) M) in inhibiting RANKL-induced osteoclast-like cell formation was completely abolished in the presence of cycloheximide (10(-7) M), an inhibitor of translation in protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB; 10(-6) M), an inhibitor of transcription. These results suggest that the inhibitory action of zinc on osteoclastogenesis is partly due to suppressing signaling pathway which is related to RANKL stimulation in osteoclast development.

Inhibitory effect of β-cryptoxanthin on osteoclast-like cell formation in mouse marrow cultures

The carotenoid β-cryptoxanthin has been shown to have an inhibitory effect on bone-resorping factor-stimulated bone resorption in rat bone tissues in vitro. The effect of β-cryptoxanthin on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in α-minimal essential medium containing a bone-resorbing agent [parathyroid hormone (1–34) (PTH), prostaglandin E 2, 1,25-dihydroxyvitamin D 3, lipopolysaccharide, or tumor necrosis factor-α (TNFα)] with an effective concentration. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of PTH (10 −7 M), prostaglandin E 2 (10 −5 M), 1,25-dihydroxyvitamin D 3 (10 −7 M), lipopolysaccharide (10 μg/mL), or TNFα (10 ng/mL) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of β-cryptoxanthin (10 −8 to 10 −6 M). β-Cryptoxanthin (10 −7 and 10 −6 M) significantly inhibited dibutyryl cyclic adenosine monophosphate (DcAMP) (10 −5 M) or phorbol 12-myristate 13-acetate (PMA) (10 −5 M), an activator of protein kinase C, induced osteoclast-like cell formation. Also, β-cryptoxanthin (10 −7 and 10 −6 M) had a significant inhibitory effect on osteoclast-like formation induced by receptor activator of NF-κB ligand (RANKL) (10 and 20 ng/mL) in the presence of macrophage colony-stimulating factor (M-CSF) (10 and 20 ng/mL). The stimulatory effect of RANKL and M-CSF on osteoclast-like cell formation was significantly enhanced in the presence of PMA, while such an effect was not seen by DcAMP. β-Cryptoxanthin (10 −6 M) significantly inhibited osteoclast-like cell formation induced by RANKL and M-CSF in the presence of PMA or DcAMP. Moreover, the inhibitory effect of β-cryptoxanthin on RANKL plus M-CSF-, PTH-, or TNFα-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10 −7 M), an inhibitor of protein synthesis at translational process, or 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (10 −6 M), an inhibitor of transcription. This study demonstrates that β-cryptoxanthin has a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture. The inhibitory action of β-cryptoxanthin may partly involve in a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.

Inhibitory effect of genistein on osteoclast-like cell formation in mouse marrow cultures

The effect of genistein on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in α-minimal essential medium containing a well-known bone resorbing agent [parathyroid hormone (1–34) (PTH), prostaglandin E 2 (PGE 2), 1,25-dihydroxyvitamin D 3 (VD 3), or lipopolysaccharide (LPS)] with an effective concentration. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of PTH (10 −8 M), PGE 2 (10 −6 M), VD 3 (10 −8 M), or LPS (1 μg/mL) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were inhibited significantly in the presence of genistein (10 −7 to 10 −5 M). The inhibitory effect of genistein (10 −5 M) was equal to that of 17 β-estradiol (10 −8 M), calcitonin (10 −9 M), or zinc sulfate (10 −5 M). Genistein (10 −5 M) significantly inhibited dibutyryl cyclic adenosine monophosphate (10 −5 M)-induced osteoclast-like cell formation. However, genistein (10 −5 M) did not inhibit phorbol 12-myristate 13-acetate-induced osteoclast-like cell formation. The present study demonstrated that genistein has a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture. The inhibitory action of genistein may involve in cyclic AMP signaling.

Important role of EP4, a subtype of prostaglandin (PG) E receptor, in osteoclast-like cell formation from mouse bone marrow cells induced by PGE2.

Of various PGs, PGE1 and PGE2 are shown to be the most potent stimulators of osteoclastogenesis in vitro. PGE receptors have been classified into four subtypes, EP1-EP4. Little is known about PGE receptors functioning in bone cells. In this study, using mouse marrow culture, we investigated which PGE receptors are important in osteoclast-like cell (OCL) formation induced by PGE. 11-deoxy-PGE1 (EP2, EP3 and EP4 agonist) stimulated OCL formation potently. Butaprost (EP2 agonist) stimulated it slightly, while sulprostone (EP1 and EP3 agonist) and ONO-AP-324-01 (EP3 agonist) did not. AH23848B (EP4 antagonist) inhibited PGE2-induced OCL formation in a dose-dependent manner. The expression of EP4 mRNA in mouse bone marrow was confirmed by RT-PCR. The results indicate an important role of EP4 in PGE2-induced OCL formation in marrow cultures and suggest therapeutic potential of EP4 antagonists in some clinical conditions with accelerated bone resorption.

TR1, a new member of the tumor necrosis factor receptor superfamily, induces fibroblast proliferation and inhibits osteoclastogenesis and bone resorption.

A newly identified member of the tumor necrosis factor receptor (TNFR) superfamily shows activities associated with osteoclastogenesis inhibition and fibroblast proliferation. This new member, called TR1, was identified from a search of an expressed sequence tag database, and encodes 401 amino acids with a 21-residue signal sequence. Unlike other members of TNFR, TR1 does not contain a transmembrane domain and is secreted as a 62 kDa glycoprotein. TR1 gene maps to chromosome 8q23-24.1 and its mRNA is abundantly expressed on primary osteoblasts, osteogenic sarcoma cell lines, and primary fibroblasts. The receptors for TR1 were detected on a monocytic cell line (THP-1) and in human fibroblasts. Scatchard analyses indicated two classes of high and medium-high affinity receptors with a kD of approximately 45 and 320 pM, respectively. Recombinant TR1 induced proliferation of human foreskin fibroblasts and potentiated TNF-induced proliferation in these cells. In a coculture system of osteoblasts and bone marrow cells, recombinant TR1 completely inhibited the differentiation of osteoclast-like multinucleated cell formation in the presence of several bone-resorbing factors. TR1 also strongly inhibited bone-resorbing function on dentine slices by mature osteoclasts and decreased 45Ca release in fetal long-bone organ cultures. Anti-TR1 monoclonal antibody promoted the formation of osteoclasts in mouse marrow culture assays. These results indicate that TR1 has broad biological activities in fibroblast growth and in osteoclast differentiation and its functions.

Combination of β-cryptoxanthin and zinc has potent effects on apoptotic cell death and suppression of bone resorption-related gene expression in osteoclastic cells

We investigated whether the effect of beta-cryptoxanthin (CRP) on osteoclastic cells formed in the mouse marrow culture system in vitro is enhanced by culture with zinc. Bone marrow cells were isolated from mice. The macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages were cultured in the presence of M-CSF (10 ng/ml) and receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL; 50 ng/ml) for 96 h. The osteoclastic cells formed were further cultured for 24 or 72 h in a medium containing either vehicle, CRP, zinc sulfate (zinc), or CRP plus zinc with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml). The number of osteoclastic cells was significantly decreased after culture with the combination of CRP (10(-7) M) and zinc (10(-5) M) in the presence or absence of M-CSF and RANKL for 24 or 72 h as compared with the value for CRP or zinc alone. Agarose gel electrophoresis showed the presence of low-molecular weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with CRP (10(-7) M) plus zinc (10(-5) M) for 24 or 72 h in the presence of M-CSF and RANKL, indicating that the combination of the two chemicals induces apoptotic cell death. CRP plus zinc-induced decrease in osteoclastic cells was significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Culture with CRP (10(-7) M) plus zinc ((10(-5) M) for 24 or 72 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL as compared with the value for each chemical alone. CRP plus zinc-induced increase in caspase-3 mRNA expression was completely inhibited in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DBR; 10(-6) M), an inhibitor of transcription activity. The mRNA expression of tartrate-resistant acid phosphatase (TRACP) and cathepsin K was significantly decreased after culture with CRP plus zinc in the presence or absence of M-CSF and RANKL for 72 h as compared with CRP or zinc alone. Nuclear factor of activated T cells c1 (NFATc1) mRNA expression was significantly decreased after culture with CRP plus zinc in the presence or absence of M-CSF and RANKL for 72 h as compared with each chemical alone, while NF-kappaB mRNA expression was not significantly changed. This study demonstrated that the combination of CRP and zinc has potent suppressive effects on osteoclastic cells in vitro.

Regulation of osteoclast function.

Regulation of osteoclast function.

Characterization of zinc effect to inhibit osteoclast-like cell formation in mouse marrow culture: interaction with dexamethasone.

The inhibitory effect of zinc compounds on osteoclast-like cell formation in mouse marrow culture in vitro was characterized. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone resorbing agent, 1,25-dihydroxyvitamin D3[1,25(OH)2D3] or prostaglandin E2 (PGE2). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1,25(OH)2D3 (10(-8) M) or PGE2 (10(-6) M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were enhanced by the presence of dexamethasone (10(-9) to 10(-6) M). The dexamethasone (10(-7) M)-enhanced osteoclast-like cell formation was not inhibited by the presence of zinc sulfate (10(-6) M) or zinc-chelating dipeptide (beta-alanyl-L-histidinato zinc; 10(-6) M), although the zinc compounds had an inhibitory effect on osteoclastic formation in the absence of the steroid. The effect of dexamethasone was not seen, when the steroid was added at the later stage of culture with bone-resorbing agents. In this case, the inhibitory effect of zinc compounds was clearly revealed. This effect of zinc compounds disappeared in the presence of Ca2+-chelating agent (0.5 mM EGTA). The present study suggests that zinc compounds have an inhibitory effect at the stage of differentiation of preosteoclastic cells in bone marrow cell culture system.

Differential effects of transforming growth factor-beta on osteoclast-like cell formation in mouse marrow culture: relation to the effect of zinc-chelating dipeptides.

The effect of transforming growth factor-beta (TGF-beta) on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone resorbing agent. Osteoclast-like cell formation was estimated with staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of TGF-beta (10(-13)-10(-11) M) caused a significant increase in the number of osteoclast-like multinucleated cells (MNCs); the maximum effect was seen with 10(-12) MTGF-beta. With a higher concentration (10(-10) M) of TGF-beta, the growth factor dramatically inhibited the 1,25-dihydroxyvitamin D5 [1,25(OH)2D3; 10(-8) M]-induced formation of osteoclast-like MNCs. This inhibitory effect was also seen in the formation of osteoclast-like MNCs stimulated by parathyroid hormone (10(-8) M), prostaglandine E2 (10(-6) M), and interleukin-1 alpha (50 U/ml). The stimulatory effect of TGF-beta (10(-12) M) on osteoclast-like MNCs formation was inhibited by zinc sulfate (10(-6) M) or zinc-chelating dipeptide [beta-alanyl-L-histidinato zinc (AHZ), 10(-6) M]. The stimulating effect of TGF-beta was markedly weakened by the presence of EGTA (0.5 mM), a chelator of Ca2+. The inhibitory effect of zinc compounds was not seen in the presence of EGTA. Moreover, the inhibitory effect of TGF-beta (10(-10) M), zinc sulfate (10(-6) M), or AHZ (10(-6) M) on osteoclast-like MNCs formation was not demonstrated in mature osteoclastic cells, although calcitonin (3 x 10(-8) M) significantly inhibited the osteoclastic formation. The present study demonstrates that TGF-beta has a stimulating and an inhibiting effect on osteoclast-like cell formation in mouse marrow culture, and that zinc can inhibit the stimulatory effect of TGF-beta.

Inhibitory effect of zinc-chelating dipeptide on parathyroid hormone-stimulated osteoclast-like cell formation in mouse marrow cultures: involvement of calcium signaling.

A possible mechanism of zinc action inhibiting the PTH-induced osteoclast-like cell formation in mouse marrow culture system in vitro was investigated. Bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone-resorbing agent parathyroid hormone (1-34) (PTH). Osteoclast-like cell formation was estimated with staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The effect of zinc sulfate (10(-6) M) or beta-alanyl-L-histidinato zinc (AHZ; 10(-6) M) inhibiting the PTH (10(-8) M)-induced osteoclast-like cell formation was clearly seen in the absence or presence of theophylline (10(-4) M). However, zinc compounds did not inhibit the stimulatory effect of dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP; 10(-4) M) on osteoclast-like cell formation. The stimulating effect of PTH (10(-8) M) on osteoclast-like cell formation was clearly weakened (about 50%) in the presence of EGTA (1.0 mM) or dibucaine (10(-5) M). Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator (10(-8) to 10(-6) M), clearly stimulated osteoclast-like cell formation. PMA effect was inhibited by the presence of AHZ (10(-6) M) or zinc sulfate (10(-8) M). However, the inhibitory effect of zinc compounds was not seen in the presence of both PTH (10(-8) M) and PMA (10(-6) M). The present findings suggest that zinc compounds inhibit PTH-stimulated osteoclast-like cell formation mediated through the Ca(2+)-dependent activation of protein kinase C.

Inhibitory effect of zinc compounds on osteoclast-like cell formation in mouse marrow cultures

The effect of zinc compounds on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in α-minimal essential medium containing a well-known bone resorbing agent [1,25-dihydroxyvitamin D 3, parathyroid hormone (1–34), interleukin-1α or prostaglandin E 2]. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1,25-dihydroxyvitamin D 3 (10 −8M), parathyroid hormone (10 −8M), interleukin-1α- (50U/mL) or prostaglandin E 2 (10 −6M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (β-alanyl- l-histidinato zinc; AHZ) in the concentration range of 10 −8 to 10 −5 M. The inhibitory effect of AHZ (10 −8 and 10 −7M) was more intensive than that of zinc sulfate. Furthermore, the presence of Ni 2+, Cu 2+, Mn 2+ or Co 2+ (10 −7 and 10 −6 M) did not have an effect on parathyroid hormone (10 −8 M)-induced osteoclast-like cell formation. The present study clearly demonstrates that zinc compounds have a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture.

P65. Pyrrole and pyridinium cross-links in osteoporotic bone collagen

Throm☐ane A2 (TXA2) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, throm☐ane B2 (TXB2). TXA2 has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA2 have not yet been proved because of its highly unstable nature.Recently, a chemically stable analogue of TXA2, 9,11-epithio-11,12-methanothrom☐ane A2 (STA2), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E2 (PGE2), a well-known bone resorbing agent. STA2 time-and dose-dependently stimulated the release of 45Ca from prelabelled mouse calvariae. Both STA2 and PGE2 induced the accumulation of cAMP in mouse calvariae. The TXA2, agonist. ONO-3708, inhibited STA2-induced release of 45Ca, TXB2 induced neither bone resolor cAMP accumulation. When mouse marrow cells were cultured with STA2 for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA2 added. Again TXB2 showed no effect on osteoclast-like cell formation. These results indicate a role for TXA2 in some form of bone resorption.

Participation of oxidative stress in the process of osteoclast differentiation

In the present paper, the involvement of active oxygen species in bone resorption has been studied. In order to compare the production of active oxygen by mouse marrow culture cells, fluorescence due to peroxides reacted with 2,7-dichlorofluorescin was measured. After marrow cells were cultured with 1,25-(OH) 2D 3 for 8 days, there were tartrate resistant acid phosphatase positive multinucleated cells (TRACP(+)MNCs), TRACP positive mononucleated cells, macrophage-like cells and marrow derived stromal cells. Among these cells, TRACP(+) cells could produce almost the equivalent amount of peroxides as could the macrophage-like cells. In order to examine the role of active oxygen in bone metabolism, the amount of oxidative stress was altered during the culture period in the same marrow culture system. Catalase, a catabolic enzyme of hydrogen peroxide (H 2O 2), significantly suppressed the formation of TRACP(+)MNCs in a dose dependent manner. This suppression was limited in the early stage of the culture period and was reduced by the addition of exogenous H 2O 2 to culture. Moreover, when superoxide dismutase, a converting enzyme from superoxide anion to H 2O 2, was added in this system, the formation of TRACP(+)MNCs was significantly increased. These results strongly suggest that active oxygen species, especially H 2O 2, may be involved in the regulation of osteoclast formation.

Role of colony-stimulating factors in osteoclast development

Effects of various colony-stimulating factors (CSFs) [interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF), and granulocyte CSF (G-CSF)] on osteoclast-like cell formation were examined in two different culture systems: the one-step mouse marrow culture system and the two-step coculture system of mouse primary osteoblastic cells with the bone marrow cells collected from the colonies that formed in the methylcellulose in the presence of the CSFs. In the one-step mouse marrow cultures, none of the CSFs stimulated the formation of tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts)-positive multinucleated cells (MNCs). Furthermore, the CSFs other than G-CSF inhibited in a dose-dependent manner the TRAP-positive MNC formation induced by 1 alpha-25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. In contrast, when marrow cells were first cultured in semisolid methylcellulose in the presence of a CSF and the recovered marrow cells from the semisolid cultures were subsequently cocultured with primary osteoblastic cells in the presence of 1 alpha,25-(OH)2D3, numerous TRAP-positive MNCs were formed. [125I]salmon calcitonin specifically bound to TRAP-positive cells formed in this two-step culture system. Over 90% of the TRAP-positive mononuclear cells and MNCs accumulated [125I]calcitonin. M-CSF was the most potent in inducing TRAP-positive MNCs, followed by GM-CSF, IL-3, and G-CSF in that order. No TRAP-positive cells were formed in the absence of either osteoblastic cells or 1 alpha,25-(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)

A stable analogue of throm☐ane A 2, 9,11-epithio-11,12-methanothrom☐ane A 2, stimulates bone resorption in vitro and osteoclast-like cell formation in mouse marrow culture

Throm☐ane A 2 (TXA 2) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, throm☐ane B 2 (TXB 2). TXA 2 has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA 2 have not yet been proved because of its highly unstable nature. Recently, a chemically stable analogue of TXA 2, 9,11-epithio-11,12-methanothrom☐ane A 2 (STA 2), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E 2 (PGE 2), a well-known bone resorbing agent. STA 2 time-and dose-dependently stimulated the release of 45Ca from prelabelled mouse calvariae. Both STA 2 and PGE 2 induced the accumulation of cAMP in mouse calvariae. The TXA 2, agonist. ONO-3708, inhibited STA 2-induced release of 45Ca, TXB 2 induced neither bone resolor cAMP accumulation. When mouse marrow cells were cultured with STA 2 for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA 2 added. Again TXB 2 showed no effect on osteoclast-like cell formation. These results indicate a role for TXA 2 in some form of bone resorption.