Tag Archives: in vitro

Butyrate Stimulates Bone Formation and Suppresses Osteoclasts In Vitro

Abstract

Sodium butyrate stimulates mineralized nodule formation and osteoprotegerin expression by human osteoblasts.

Butyric acid (sodium butyrate; BA) is a major metabolic by-product of main periodontopathic bacteria present in subgingival plaque. In the present study, we examined the effects of BA on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, extracellular matrix protein expression, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) in normal human osteoblasts.
The cells were cultured with 0, 10(-8), 10(-6) or 10(-4)M BA for up to 12 days. Mineralized nodule formation was detected by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium assay kit. The gene and protein expression levels for type I collagen, bone sialoprotein (BSP), osteopontin (OPN), M-CSF, and OPG were examined using real-time PCR and ELISA, respectively.
Mineralized nodule formation and the calcium content of mineralized nodules were increased by BA in a dose-dependent manner. Cell proliferation and ALPase activity were not affected by the addition of BA. Following the addition of 10(-4)M BA, the expression levels of BSP, OPN, and OPG increased, whereas the expression levels of type I collagen and M-CSF were not markedly affected.
These results suggest that BA stimulates bone formation by increasing the production of BSP and OPN, whereas it suppresses osteoclast differentiation by increasing the production of OPG by human osteoblasts.

Katono T, Kawato T, Tanabe N, Suzuki N…
Arch. Oral Biol. Oct 2008
PMID: 18406397

Phloretin Reduces Osteoclast Size by Inhibiting Aquaporin 9 In Vitro

Abstract

Involvement of aquaporin 9 in osteoclast differentiation.

Aquaporins (water channels) selectively enhance water permeability of membranes. Since osteoclast differentiation includes a dramatic increase in cell volume, we hypothesize that aquaporin(s) is/are critical for the formation of the multinucleated osteoclast from its mononuclear precursor. Our studies employ two cell models, bone marrow macrophages (BMMs) and the murine macrophage-like cell line, RAW264.7, as osteoclast precursors. Receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) and macrophage-colony-stimulating factor or RANKL alone were used to induce osteoclast differentiation in BMMs or RAW264.7 cells, respectively. We first used qualitative reverse transcription (RT)-PCR to examine which of the aquaporins are expressed in osteoclasts and in their precursor cells. Out of the 10 aquaporins examined, only aquaporin 9 (AQP9) was expressed in osteoclast-lineage cells. AQP9 has unique aqueous pore properties mediating the passage of a wide variety of non-charged solutes in addition to water. Western analyses using specific antibodies revealed a higher AQP9 level in RANKL-treated than in untreated cells. Quantitative real-time RT-PCR analyses also demonstrated higher AQP9 mRNA levels in RANKL-treated cells. Finally, we examined the effect of phloretin, an AQP9 inhibitor, on RANKL-induced osteoclast differentiation. Cells were incubated with RANKL for 5 days, and phloretin was added for the last 2 days, when most fusion occurs. A dramatic reduction in osteoclast size and in the number of nuclei per osteoclast was observed in cultures containing phloretin. The inhibitor did not have a significant effect on the number and size of mononuclear phagocytes in cultures not treated with RANKL. Our results suggest a role for AQP9 in osteoclast differentiation, specifically in the fusion process.

Aharon R, Bar-Shavit Z
J. Biol. Chem. Jul 2006
PMID: 16698796 | Free Full Text

Strontium Prevents Inhibitory Effect of AGEs on Osteoblasts

Abstract

Strontium ranelate prevents the deleterious action of advanced glycation endproducts on osteoblastic cells via calcium channel activation.

Accumulation of advanced glycation endproducts (AGEs) in bone tissue occurs in ageing and in Diabetes mellitus, and is partly responsible for the increased risk of low-stress bone fractures observed in these conditions. In this study we evaluated whether the anti-osteoporotic agent strontium ranelate can prevent the deleterious effects of AGEs on bone cells, and possible mechanisms of action involved. Using mouse MC3T3E1 osteoblastic cells in culture we evaluated the effects of 0.1mM strontium ranelate and/or 100 μg/ml AGEs-modified bovine serum albumin (AGEs-BSA) on cell proliferation, osteogenic differentiation and pro-inflammatory cytokine production. We found that AGEs-BSA alone decreased osteoblastic proliferation and differentiation (P<0.01) while increasing IL-1β and TNFα production (P<0.01). On its own, strontium ranelate induced opposite effects: an increase in osteoblast proliferation and differentiation (P<0.01) and a decrease in cytokine secretion (P<0.01). Additionally, strontium ranelate prevented the inhibitory and pro-inflammatory actions of AGEs-BSA on osteoblastic cells (P<0.01). These effects of strontium ranelate were blocked by co-incubation with either the MAPK inhibitor PD98059, or the calcium channel blocker nifedipine. We also evaluated by Western blotting the activation status of ERK (a MAPK) and b-catenin. Activation of both signaling pathways was decreased by AGEs treatment, and this inhibitory effect was prevented if AGEs were co-incubated with strontium ranelate (P<0.01). On its own, strontium ranelate increased both pERK and activated b-catenin levels. In conclusion, this study demonstrates that strontium ranelate can prevent the deleterious in vitro actions of AGEs on osteoblastic cells in culture by mechanisms that involve calcium channel, MAPK and b-catenin activation.

Fernández JM, Molinuevo MS, Sedlinsky C, Schurman L…
Eur. J. Pharmacol. Apr 2013
PMID: 23499695

Apigenin Inhibits Osteoclasts in Mouse Cells

Abstract

Attenuation of osteoclastogenesis and osteoclast function by apigenin.

The physiological effects of the flavone, apigenin on bone cells were studied. We first show that apigenin inhibits tumor necrosis factor alpha (TNFalpha)- and interferon gamma (IFNgamma)-induced secretion of several osteoclastogenic cytokines from MC3T3-E1 mouse calvarial osteoblast cell line. Ligands of the TNF receptor family constitute the most potent osteoclastic cytokines. In MC3T3-E1 cells, apigenin dose-dependently (from 5 to 20 microM) inhibits TNFalpha-induced production of the osteoclastogenic cytokines, IL-6 (interleukin-6), RANTES (regulated upon activation, normal T cell-expressed and -secreted), monocyte chemoattractant protein-1 (MCP-1) and MCP-3. In addition, apigenin inhibits IFNgamma-stimulated secretion of monokines, CXCL-9, and -10 in MC3T3-E1 cells. Next, we show that apigenin strongly inhibits differentiation of 3T3-L1 preadipocytes to adipocytes with attendant inhibition of adipocyte differentiation-induced IL-6, MCP-1, and leptin production. Inhibition of adipogenic differentiation by apigenin could be due to induction of osteogensis as it robustly upregulates mRNA levels of bone morphogenetic protein-6 (BMP-6). Finally, the presence of apigenin inhibited osteoclast differentiation from the RAW 264.7 cell line by reducing receptor activator of nuclear factor kappa ligand (RANKL)-induced expression of tartrate-resistant acid phosphatase (TRAP), RANK, and calcitonin receptor but not CCR1, resulting in the inhibition of multinucleated osteoclast formation. Similarly, apigenin inhibited expression of the osteoclast differentiation markers TRAP, RANK, and c-Fms in osteoclast precursor cells obtained from mouse bone marrow following treatment with RANKL and macrophage colony stimulating factor (MCSF). Furthermore, apigenin induced apoptosis of mature osteoclasts obtained from rabbit long bone and inhibited bone resorption. In all instances, a structurally related compound, flavone had no significant effect. These data suggest that apigenin has multiple effects on all three bone cells that could prevent bone loss in vivo.

Bandyopadhyay S, Lion JM, Mentaverri R, Ricupero DA…
Biochem. Pharmacol. Jul 2006
PMID: 16750176

Horny Goat Weed Inhibits Osteoclasts In Vitro

Abstract

Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappaB signaling pathways.

Osteoclasts are specialized bone-resorbing cells derived from multipotent myeloid progenitor cells. They play a crucial homeostatic role in skeletal modeling and remodeling and destroy bone in many pathologic conditions. Receptor activator of NF-kappaB ligand (RANKL) is essential to osteoclastogenesis. In this study, we investigated the effects of Ikarisoside A, isolated from Epimedium koreanum (Berberidaceae), on osteoclastogenesis in RANKL-treated murine monocyte/macrophage RAW 264.7 cells. The results indicate that Ikarisoside A is a potent inhibitor of osteoclastogenesis in RANKL-stimulated RAW 264.7 cells as well as in bone marrow-derived macrophages. The inhibitory effect of Ikarisoside A resulted in decrease of osteoclast-specific genes like matrix metalloproteinase 9 (MMP9), tartrate-resistant acid phosphatase (TRAP), receptor activator of NF-kappaB (RANK), and cathepsin K. Moreover, Ikarisoside A blocked the resorbing capacity of RAW 264.7 cells on calcium phosphate-coated plates. Ikarisoside A also has inhibitory effects on the RANKL-mediated activation of NF-kappaB, JNK, and Akt. Finally, Ikarisoside A clearly decreased the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1) as well as the transcriptional activity of NFATc1, the master regulator of osteoclast differentiation. The data indicate that Ikarisoside A has potential for use in treatment of diseases involving abnormal bone lysis such as osteoporosis, rheumatoid arthritis, and periodontal bone erosion.

Choi HJ, Park YR, Nepal M, Choi BY…
Eur. J. Pharmacol. Jun 2010
PMID: 20353769

Vitamin K2 (MK4) Inhibits Bone Resorption Through Inhibition of PGE2 In Vitro

Abstract

Menatetrenone inhibits bone resorption partly through inhibition of PGE2 synthesis in vitro.

We studied the effect of menatetrenone, a vitamin K2 homolog, on bone resorption stimulated by interleukin-1 alpha (IL-1 alpha), prostaglandin E2 (PGE2), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone-resorbing activity was assessed by measurement of calcium and hydroxyproline in the media and calvariae. IL-1 alpha (0.1-100 U/ml), 1,25-(OH)2D3 (10(-10)-10(-7) M), PGE2 (10(-9)-10(-6) M), and PTH (3 x 10(-8)-3 x 10(-7) M) dose dependently increased the levels of calcium and hydroxyproline in the medium. Indomethacin (10(-6) M) completely inhibited bone resorption induced by IL-1 alpha and partially inhibited bone resorption induced by 1,25-(OH)2D3. However, indomethacin did not affect the action of PGE2 or PTH. Menatetrenone (3 x 10(-6)-3 x 10(-5) M) inhibited the bone resorption induced by IL-1 alpha (2 U/ml), PGE2 (10(-7) M), PTH (3 x 10(-7) M), and 1,25-(OH)2D3 (3 x 10(-10) M) in a dose-dependent manner. Menatetrenone also inhibited the PGE2 production stimulated by IL-1 alpha. These results indicate that menatetrenone may inhibit bone resorption through at least two different mechanisms; one possibly is an inhibitory effect on prostaglandin production.

Hara K, Akiyama Y, Tajima T, Shiraki M
J. Bone Miner. Res. May 1993
PMID: 8511981

PGE2 Stimulates Bone Formation and Resorption In Mouse Cells

Abstract

Prostaglandin E2 stimulates osteoclast-like cell formation and bone-resorbing activity via osteoblasts: role of cAMP-dependent protein kinase.

Prostaglandin E2 (PGE2) is an important local regulator in bone. The present study was performed to investigate the effect of PGE2 on osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts, PGE2 (10(-8) to 10(-6) M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE2 (10(-8) and 10(-6) M) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells. PGE2 also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures. In contrast, PGE2 significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts. Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE2-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE2-stimulated osteoclast-like cell formation. In conclusion, PGE2 stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE2-stimulated osteoclast-like cell formation and bone-resorbing activity.

Kaji H, Sugimoto T, Kanatani M, Fukase M…
J. Bone Miner. Res. Jan 1996
PMID: 8770698

PGE2 Stimulates Bone Resorption and Formation In Vitro

Abstract

PGE2 stimulates both resorption and formation of bone in vitro: differential responses of the periosteum and the endosteum in fetal rat long bone cultures.

The ability of PGE2 to stimulate bone resorption in vitro and in vivo is well established but the effects of this compound on bone formation are still controversial. Recent clinical reports have suggested that long-term infusion of PGE in infants with cyanotic heart diseases led to a stimulation of periosteal bone formation and to hyperostosis. In the present report, we describe the effects of PGE2 (10(-5) M) in bone organ cultures on bone resorption, measured by the release of 45Calcium and the number of osteoclasts in sections of cultured bones, and bone volume, by measuring separately medullary and cortical areas. PGE2 induced a marked increase in 45Ca release and in cortical and medullary osteoclast numbers over 4 days in vitro; despite this increase in bone resorption, cortical bone volume remained constant, indicating a parallel increase in bone resorption and formation at this site. Morphological and quantitative data demonstrated a higher extent of osteoblastic surface along the periosteum of PGE2-treated bones when compared with control cultures. Medullary bone volume, on the other hand, decreased sharply during the culture period, demonstrating a lack of parallel increase in bone formation at this site. It is concluded that, under these experimental conditions, prostaglandin E2 stimulated both resorption and formation along the periosteum and only bone resorption along the endosteum of the cultured bones. The overall effect of PGE2 on bone as a whole, however, was net bone loss.

Nefussi JR, Baron R
Anat. Rec. Jan 1985
PMID: 3985383

Phloretin Inhibits Osteoclasts In Vitro

Abstract

Novel antiosteoclastogenic activity of phloretin antagonizing RANKL-induced osteoclast differentiation of murine macrophages.

Bone-remodeling imbalance resulting in more bone resorption than bone formation is known to cause skeletal diseases such as osteoporosis. Phloretin, a natural dihydrochalcone compound largely present in apple peels, possesses antiphotoaging, and antiinflammatory activity.
Phloretin inhibited receptor activator of NF-κB ligand (RANKL)-induced formation of multinucleated osteoclasts and diminished bone resorption area produced during the osteoclast differentiation process. It was also found that ≥ 10 μM phloretin reduced RANKL-enhanced tartrate-resistance acid phosphatase activity and matrix metalloproteinase-9 secretion in a dose-dependent manner. The phloretin treatment retarded RANKL-induced expression of carbonic anhydrase II, vacuolar-type H(+) -ATPase D2 and β3 integrin, all involved in the bone resorption. Furthermore, submicromolar phloretin diminished the expression and secretion of cathepsin K elevated by RANKL, being concurrent with inhibition of TRAF6 induction and NF-κB activation. RANKL-induced activation of nuclear factor of activated T cells c1 (NFATc1) and microphthalmia-associated transcription factor was also suppressed by phloretin.
These results demonstrate that the inhibition of osteoclast differentiation and bone resorption by phloretin entail a disturbance of TRAF6-NFATc1-NF-κB pathway triggered by RANKL. Therefore, phloretin may be a potential therapeutic agent targeting osteoclast differentiation and bone resorption in skeletal diseases such as osteoporosis.

Kim JL, Kang MK, Gong JH, Park SH…
Mol Nutr Food Res Aug 2012
PMID: 22700286

Glabridin Inhibits Osteoclasts In Vitro

Abstract

The inhibitory effect and the molecular mechanism of glabridin on RANKL-induced osteoclastogenesis in RAW264.7 cells.

Osteoblastic bone formation and osteoclastic bone resorption are in balance to maintain a constant, homeostatically controlled amount of bone. Excessive bone resorption by osteoclasts is involved in the pathogenesis of bone-related disorders. In the present study, we evaluated the inhibitory effects of glabridin, a flavonoid purified from licorice root, on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and its molecular mechanisms in murine osteoclast progenitor RAW264.7 cells. Glabridin significantly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity, the formation of multinucleated osteoclasts and resorption-pit formation. In mechanistic studies of the anti-osteoclastogenic potential of glabridin, we found that glabridin inhibited RANKL-induced expression of c-Fos and subsequent expression of NFATc1, which is a master regulator of osteoclastogenesis. Interestingly, glabridin inhibited the RANKL-induced expression of signaling molecules (TRAF6, GAB2, ERK2, JNK1 and MKK7) and osteoclast survival-related signaling pathways such as c-Src, PI3K and Akt2. Glabridin also inhibited the bone resorptive activity of mature osteoclasts by inhibiting osteoclast-associated genes (cathepsin K, MMP-9, CAII, TCIRG1, OSTM1 and CLCN7). Taken together, our data suggest that glabridin holds great promise for use in preventing osteoclastogenesis by inhibiting RANKL-induced activation of signaling molecules and subsequent transcription factors in osteoclast precursors and these findings may be useful for evaluating treatment options in bone-destructive diseases.

Kim HS, Suh KS, Sul D, Kim BJ…
Int. J. Mol. Med. Feb 2012
PMID: 22038020