acid-phosphatase and geranylgeraniol

Bisphosphonates have been known to directly inhibit bone resorption and promote apoptosis in mature osteoclasts. Although bisphosphonates have been recognized as the most effective drugs to treat osteoporosis and bone cancer metastasis, the exact effects and mechanism(s) of bisphosphonates on osteoclastogenesis are unclear. The aim of this study was to clarify whether nitrogen-containing bisphosphonates affect recruitment and differentiation in osteoclasts. We examined the effects of zoledronic acid on receptor activator of NF-κB (RANK) expression and cell migration during osteoclastogenesis in two types of osteoclast precursors, RAW264.7 cells and Bone marrow cells (BMCs). Tumor necrosis factor-α (TNF-α) and RANK ligand (RANKL) upregulated RANK expression in RAW264.7 and BMCs in the presence of macrophage colony stimulating factor in a time-dependent manner. Zoledronic acid (30 and 50 μM) had no effect on cell viability in osteoclast precursors after 36 h of cultivation. Zoledronic acid (10 and 30 μM) strongly inhibited TNF-α- and RANKL-induced upregulation of RANK in a dose-dependent manner. The inhibitory effects on RANK expression were likely to be associated with the suppression of the NF-κB pathway, but not other downstream signaling pathways. Zoledronic acid (30 μM) also suppressed the TNF-α- and RANKL-induced migration of precursors by inhibiting the mevalonic acid pathway. Our results suggest that nitrogen-containing bisphosphonates not only inhibit mature osteoclasts but also prevent osteoclast precursors from differentiating and migrating towards inflammatory osteolysis lesions.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Bone Resorption; Cell Differentiation; Cell Line, Tumor; Cell Movement; Diphosphonates; Diterpenes; Dose-Response Relationship, Drug; Gene Expression; I-kappa B Proteins; Imidazoles; Isoenzymes; Macrophages; Mice; Mice, Inbred Strains; NF-kappa B; NF-KappaB Inhibitor alpha; Osteoclasts; Phosphorylation; Protein Prenylation; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Stem Cells; Tartrate-Resistant Acid Phosphatase; Transcription, Genetic; Tumor Necrosis Factor-alpha; Up-Regulation; Zoledronic Acid

Multinucleated bone-resorbing osteoclasts (Ocl) are cells of hematopoietic origin that play a major role in osteoporosis pathophysiology. Ocl survival and activity require M-CSF and RANK ligand (RANKL). M-CSF signals to Akt, while RANKL, like TNFalpha, activates NF-kappaB. We show here that although these are separate pathways in the Ocl, signaling of all three cytokines converges on mammalian target of rapamycin (mTOR) as part of their antiapoptotic action. Accordingly, rapamycin blocks M-CSF- and RANKL-dependent Ocl survival inducing apoptosis, and suppresses in vitro bone resorption proportional to the reduction in Ocl number. The cytokine signaling intermediates for mTOR/ribosomal protein S6 kinase (S6K) activation include phosphatidylinositol-3 kinase, Akt, Erks and geranylgeranylated proteins. Inhibitors of these intermediates suppress cytokine activation of S6K and induce Ocl apoptosis. mTOR regulates protein translation acting via S6K, 4E-BP1 and S6. We find that inhibition of translation by other mechanisms also induces Ocl apoptosis, demonstrating that Ocl survival is highly sensitive to continuous de novo protein synthesis. This study thus identifies mTOR/S6K as an essential signaling pathway engaged in the stimulation of cell survival in osteoclasts.

Topics: Acid Phosphatase; Actins; Adaptor Proteins, Signal Transducing; Alendronate; Alkyl and Aryl Transferases; Animals; Apoptosis; Blotting, Western; Bone Marrow Cells; Bone Resorption; Carrier Proteins; Caspase 3; Caspase 9; Caspases; Cell Cycle Proteins; Cell Survival; Collagen; Collagen Type I; Cytokines; Diterpenes; Enzyme Inhibitors; Eukaryotic Initiation Factors; Immunohistochemistry; Interleukin-1; Isoenzymes; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Mice; Models, Biological; NF-kappa B; Osteoclasts; Peptides; Phosphoproteins; Protein Kinases; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; Serine-Threonine Kinase 3; Signal Transduction; Sirolimus; Tartrate-Resistant Acid Phosphatase; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

It is still not certain what the direct effect of menatetrenone is on osteoclast precursors. In the present study, we investigated whether menatetrenone has a direct effect on circulating osteoclast precursors to influence osteoclast differentiation. Monocytes isolated from human peripheral blood were cultured with receptor-activated NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Menatetrenone or vitamin K1 was then added to the cultures. Geranylgeraniol or phytol (the respective side chain) was also added to the cultures instead of menatetrenone or vitamin K1, respectively. After 7 and 14 days incubation, cultures were evaluated for cytochemical and functional evidence of osteoclast formation. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the percentage area of lacunar resorption induced by RANKL and M-CSF were decreased when menatetrenone or geranylgeraniol was added to the cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when the cultures were treated with menatetrenone or geranylgeraniol. In contrast, vitamin K1 or phytol did not affect the number of TRAP-positive MNCs nor the percentage area of lacunar resorption. These results indicate that menatetrenone not only influences osteoclast formation via bone stromal cells but also acts directly on circulating osteoclast precursors to influence osteoclast differentiation. These findings also suggest that geranylgeraniol, the side chain of menatetrenone, plays an important role in this inhibitory effect.

Topics: Acid Phosphatase; Antifibrinolytic Agents; Bone Resorption; Cell Count; Cell Differentiation; Cells, Cultured; Diterpenes; Dose-Response Relationship, Drug; Drug Combinations; Giant Cells; Humans; Isoenzymes; Leukocytes, Mononuclear; Osteoclasts; Phytol; Stem Cells; Tartrate-Resistant Acid Phosphatase; Vitamin K 1; Vitamin K 2

Although the effects of vitamin K2 and vitamin K1 on bone metabolism have been reported, the difference between them has not been investigated. We now show the effects of menatetrenone, one of the vitamin K2 homologues, and vitamin K1 on bone resorption. Menatetrenone at greater than 3 x 10(-6) M significantly inhibited the calcium release from mouse calvaria induced by 3 x 10(-10) M of 1,25(OH)2D3 or 10(-7) M of prostaglandin E2, and it also inhibited osteoclast-like multinucleated cell (MNC) formation induced by 10(-8) M of 1,25(OH)2D3 in co-culture of spleen cells and stromal cells at the same concentrations. In contrast, the same doses of vitamin K1 had no effects on bone resorption and MNC formation in these in vitro systems. The inhibitory effect of menatetrenone on the calcium release from calvaria was not affected by the addition of 3 x 10(-5) M of warfarin, an inhibitor of vitamin K cycle. The same concentration of geranylgeraniol, the side-chain component of menatetrenone at the 3-position of the naphthoquinone, inhibited tartrate-resistant acid phosphatase (TRACP) activity and MNC formation to the same degree as menatetrenone. Phytol, the side-chain component of vitamin K1, did not affect TRACP activity at all doses tested, but weakly inhibited MNC formation. Moreover, multi-isoprenyl alcohols of two to seven units, except geranylgeraniol which contains four units, did not effect MNC formation. These findings suggest that the inhibitory effect of menatetrenone on bone resorption is not due to gamma-carboxylation and that the side chain of menatetrenone may play an important role in this inhibitory effect.

Topics: Acid Phosphatase; Analysis of Variance; Animals; Bone Resorption; Calcitriol; Calcium; Cells, Cultured; Dinoprostone; Diterpenes; Giant Cells; Male; Mice; Mice, Inbred ICR; Organ Culture Techniques; Osteoclasts; Phytol; Spleen; Stromal Cells; Structure-Activity Relationship; Vitamin K; Vitamin K 1; Vitamin K 2; Warfarin