Tatsuro Miyahara

Activation of peroxisome proliferator-activated receptor γ inhibits TNF-α-mediated osteoclast differentiation in human peripheral monocytes in part via suppression of monocyte chemoattractant protein-1 expression

Tumor necrosis factor-alpha (TNF-alpha) plays critical roles in bone resorption at the site of inflammatory joints. The aim of this study is to evaluate the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists, a new class of anti-inflammatory compounds, on TNF-alpha-mediated osteoclastogenesis in human monocytes. Human monocytes were differentiated into osteoclasts in the presence of TNF-alpha and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit formation assay using dentin were used for the identification of activated osteoclasts. The protein and gene expressions of transcription factors were determined by immunofluorescence and real-time RT-PCR analysis, respectively. TNF-alpha-induced osteoclast generation from human peripheral monocytes in a dose-dependent manner, and the induction was not inhibited by osteoprotegerin, a decoy receptor for receptor activator of NF-kappaB ligand. The addition of PPAR-gamma agonists, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, to the culture resulted in a remarkably reduced number of generated osteoclasts. In addition, both agonists inhibited the protein and gene expressions of nuclear factor of activated T-cell isoform c1 (NFATc1), c-Fos, c-Jun and NF-kappaB p65, which are known to be associated with osteoclastogenesis. GW9662, an antagonist of PPAR-gamma, fully rescued ciglitazone-induced inhibition, but did not affect 15d-PGJ2-induced inhibition. Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine related to osteoclastogenesis, was induced during TNF-alpha-mediated osteoclast differentiation, and the neutralizing antibody to MCP-1 reduced osteoclast formation by about 40%. 15d-PGJ2 and ciglitazone blocked the induction of MCP-1 by TNF-alpha. Moreover, the addition of MCP-1 rescued the inhibition of TRAP-positive multinucleated cell (TRAP-MNCs) formation by 15d-PGJ2 and ciglitazone, although generated TRAP-MNCs had no capacity to resorb dentin slices. Our data demonstrate that 15d-PGJ2 and ciglitazone down-regulate TNF-alpha-mediated osteoclast differentiation in human cells, in part via suppression of the action of MCP-1. These PPAR-gamma agonists may be a promising therapeutic application for rheumatoid arthritis and inflammatory bone-resorbing diseases.

Effects of Cimicifugae rhizoma on serum calcium and phosphate levels in low calcium dietary rats and on bone mineral density in ovariectomized rats.

We studied the effects of ethyl acetate-(EtOAc) soluble fractions from methanol (MeOH) extracts of Cimicifugae rhizoma derived from two species - Cimicifuga heracleifolia Komarov and C. foetida L. - and four triterpenoids (1-4) isolated from them on the serum calcium (Ca) and phosphate (P) levels in low - Ca dietary rats. The EtOAc-soluble fraction from C. heracleifolia Komarov (HE) significantly drecreased Ca levels when administered. Similarly, the EtOAc-soluble fraction from C. foetida L. (FE) significantly lowered serum Ca levels at doses of 100 and 200 mg/kg/day, while the four triterpenoids (1-4) did the same at a dose of 25mg/kg/day. Interestingly only 7,8-didehydro-24-0-acetylhydroshengmanol-3-0-β-xyloside (4) showed a significant influence on serum P levels. The effects of HE and FE on the bone mineral density (BMD) of the lumbar spine (L 2-4) in ovariectomized rats were measured by dual energy X-ray absorptiometry (DXA). Rats treated with HE and FE showed a significant increase in BMD compared to untreated ovariectomized rats. BMD was lower in the latter than in sham-operated rats.

Stimulative effects of lead on bone resorption in organ culture

To clarify whether hypercalcemia after injection of Pb to rats is due to biological bone resorption or physicochemical mineral dissolution, the effect of lead (Pb) on release of previously incorporated 45Ca in organ culture was investigated. Pb at 50 microM and above stimulated the release of 45Ca and hydroxyproline (Hyp). Pb did not stimulate 45Ca release from the bones inactivated by freezing and thawing. Eel calcitonin (ECT), bafilomycin A1 and scopadulcic acid B (SDB) inhibited Pb-stimulated 45Ca release. These results indicate that Pb-induced 45Ca release is due to osteoclastic bone resorption. Pb-stimulated bone resorption was inhibited by indomethacin and flurbiprofen. Pb stimulated the release of prostaglandin E2 (PGE2) from the bones into the media. There was significantly high correlation between 45Ca and PGE2 release. Pb-induced bone resorption was inferred to be mediated by PGE2. From these results, it was suggested that hypercalcemia after Pb injection might be caused by biological bone resorption.

Inhibitory effects of scopadulcic acid B and its derivatives on bone resorption and osteoclast formation in vitro

Abstract Scopadulcic acid B and its structurally related compounds showed inhibitory effects on bone resorption and osteoclast formation in vitro . Scopadulciol was the most potent inhibitor among 21 compounds tested.

Synthesis and anti-influenza virus activity of dihydrofuran-fused perhydrophenanthrenes with a benzyloxy-type side-chain.

As one of our ongoing research project concerning development of a novel anti-influenza virus agent, dihydrofuran-fused perhydrophenanthrenes were derivatized by means of Williamson ether synthesis and Suzuki-Miyaura cross coupling reactions. Newly synthesized compounds were subjected to evaluation of anti-influenza virus activity using influenza A/Aichi/2/68 (H3N2 subtype) virus strain by a plaque titration method. These investigations revealed that incorporation of benzyl-type ether substituents was effective for exerting the inhibition activity of influenza virus proliferation.

The effect of low molecular weight chitosan on bone resorption in vitro and in vivo.

We studied the effect of low molecular weight chitosan (LMWC) on the formation of osteoclast-like multinucleated cells (OCLs) in the co-culture of mouse osteoblastic cells and bone marrow cells in the presence of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. LMWC at 440 microg/ml inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive OCLs induced by 1alpha,25(OH)2D3. We prepared OCLs in the co-culture of osteoblastic cells and bone marrow cells. The effect of LMWC on pit formation by OCLs was examined using dentin slices, and LMWC inhibited pit formation at 440 microg/ml. Oral administration of the LMWC to ovariectomized rats prevented a decrease in bone mineral density (BMD) of the lumbar vertebra without affecting the body and uterus weights. These results suggested that LMWC prevented a decrease in BMD in vivo by inhibiting osteoclastic bone resorption.

Influence of Poisonous Metals on the Bone Metabolism. II. The Effect of Cadmium on the Metabolism of Chondroitin Sulfate in Embryonic Chicken Limb Bone

The effect of cadmium on chondroitin sulfate synthesizing and releasing activities in embryonic chicken limb bone was studied. At the concentration more than 20 ppm CdCl2, cadmium enhanced the release of chondroitin sulfate into the medium. However, at the concentration of 5 to 10 ppm CdCl2, cadmium inhibited the incorporation of 35S into chondroitin sulfate. These findings suggest that the primary action of cadmium to the chondroitin sulfate metabolism is the inhibition of the synthesis. p-Nitrophenyl-β-D-xyloside did not reverse the inhibition of chondroitin sulfate synthesis brought about by cadmium. Furthermore, cadmium inhibited the incorporation of [3H]-acetate into UDP-N-acetyl hexosamine. From these results, we suppose that cadmium inhibits mainly the synthesis of nucleotides, even though the inhibition of cadmium to core protein synthesis and chain elongation may be extensively excluded.