Acid Phosphatase-positive Multinuclear Cells Research Articles

Inhibitory effect of oolonghomobisflavan B on osteoclastogenesis by suppressing p38 MAPK activation

Suppression of differentiation and/or function of osteoclasts is considered an effective therapeutic strategy for osteolytic bone diseases such as periodontitis and osteoporosis. Evidence regarding the health benefits of oolong tea consumption is accumulating, and tea polyphenols have various pharmacological properties such as anti-cancer and anti-diabetes effects. In this study, we investigated the effect of oolonghomobisflavan B (OFB), a polyphenolic compound in oolong tea, on osteoclast differentiation. OFB suppressed receptor activator of nuclear factor-κB (RANKL)-induced formation of tartate-resistant acid phosphatase-positive multinuclear cells without cytotoxicity. OFB also significantly attenuated p38 phosphorylation, which is essential for RANKL-induced osteoclastogenesis, and inhibited the expressions of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and osteoclast-specific target genes, including dendritic cell-specific transmembrane protein and cathepsin K. Our findings suggest that OFB exhibits an anti-osteoclastogenic activity by inhibiting RANKL-mediated p38 activation, which is useful for the prevention and treatment of osteolytic bone diseases.

Inhibitory effect of Chaenomelis Fructus ethanol extract on receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenesis

The fruit of Chaenomeles sinensis (Thouin) Koehne (Chaenomelis Fructus) known as “Mo-Gua” in Korea has been commonly used in traditional medicine to treat inflammatory diseases, such as sore throat. However, its effect on bone metabolism has not been elucidated yet. Here, we examined the effect of Chaenomelis Fructus ethanol extract (CFE) on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation and formation. CF-E considerably inhibited osteoclast differentiation and tartrate-resistant acid phosphatase-positive multinuclear cell formation from bone marrow-derived macrophages and osteoclast precursor cells in a dose-dependent manner. In addition, the formation of actin rings and resorption pits were significantly suppressed in CF-E-treated osteoclasts as compared with the findings in non-treated control cells. Consistent with these phenotypic inhibitory results, the expressions of osteoclast differentiation marker genes (Acp5, Atp6v0d2 , Oscar, CtsK, and Tm7sf4) and Nfatc1 , a pivotal transcription factor for osteoclastogenesis, were markedly decreased by CF-E treatment. The inhibitory effect of CF-E on RANKL-induced osteoclastogenesis was associated with the suppression of NFATc1 expression, not by regulation of mitogen-activated protein kinases and NF-κB activation but by the inactivation of phospholipase C gamma 1 and 2. These results indicate that CF-E has an inhibitory effect on osteoclast differentiation and formation, and they suggest the possibility of CF-E as a traditional therapeutic agent against bone-resorptive diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.

Plectin stabilizes microtubules during osteoclastic bone resorption by acting as a scaffold for Src and Pyk2.

Osteoclasts are multinuclear cells which maintain bone homeostasis by resorbing bone. During bone resorption, osteoclasts attach to the bone matrix via a sealing zone formed by an actin ring. Rous sarcoma oncogene (Src) is essential for actin ring formation and bone resorption. Recently, we demonstrated that plectin, a cytolinker protein, is a Src-binding protein in osteoclasts. However, the function of plectin in osteoclasts remains unknown. In this study, we demonstrated that shRNA knockdown of plectin in RAW 264.7 cells resulted in tartrate resistant acid phosphatase positive multinuclear cells (TRAP (+) MNCs) with impaired actin ring formation and bone resorption activity. Moreover, we found that in plectin-silenced TRAP (+) MNCs, Src and protein tyrosine kinase 2 beta (Pyk2), two critical kinases in osteoclastic bone resorption, were inactivated and microtubule polarity was disturbed. These results suggest that plectin plays a critical role in osteoclast biology by acting as a scaffold to facilitate Src and Pyk2 activation during microtubule organization.

Inhibitory effect of Thai propolis on human osteoclastogenesis.

Avulsed teeth should be immediately replanted into the socket or otherwise kept in a physiologic storage medium to maintain periodontal ligament cell viability. A previous study has demonstrated that Thai propolis extract can maintain viability of human periodontal ligament cells. However, root resorption by osteoclasts often occurs when the avulsed teeth are replanted. The aim of this study was to determine the inhibitory effect of Thai propolis extract on human osteoclastogenesis in vitro. Human peripheral blood mononuclear cells were isolated for osteoclast precursors and cultured in the presence or absence of various non-toxic concentrations of propolis extract, as determined by the alamarBlue® assay, during in vitro induction of osteoclastogenesis. Osteoclast formation was examined by tartrate-resistant acid phosphatase staining, actin ring formation, and real-time polymerase chain reaction. The resorption pit assay was performed to determine osteoclast function. Non-toxic concentrations of propolis extract suppressed osteoclast formation by significantly decreasing the percentages of tartrate-resistant acid phosphatase-positive multinuclear cells and the ratios of cells with F-actin ring formation (P<.01) in a dose-dependent fashion. Expression of several osteoclast-specific genes was significantly downregulated by propolis in a dose-dependent manner (P<.05). The percentages of resorption areas on dentin slices were significantly decreased by propolis (P<.05). Thai propolis can inhibit human osteoclast formation and function, which may be beneficial for prevention of root resorption following replantation of avulsed teeth.

Acidosis environment promotes osteoclast formation by acting on the last phase of preosteoclast differentiation: A study to elucidate the action points of acidosis and search for putative target molecules

Acidosis promoted tartaric acid-resistant acid phosphatase-positive multinuclear cell (TRAP + MNC) or osteoclast formation. Large osteoclast or TRAP + LMNC formation was observed far more in an acidosis environment than in a physiologically neutral environment. One of the major action points of acidosis was determined to be located in the last phase of preosteoclast differentiation using a co-culture system and a soluble RANKL-dependent bone marrow cell culture system. On-going osteoclast formation in an acidosis environment markedly deteriorated when the medium was replaced with physiologically neutral medium within the first 6 h; however, bone marrow cells previously stimulated in an acidosis environment for 9 h differentiated into TRAP + LMNC in pH 7.4 medium. Messenger RNA (mRNA) expression levels of DC-STAMP, a key molecule in cell fusion, and NFATc1 did not increase in the acidosis environment compared with those under physiologically neutral conditions. Ruthenium red, a general TRP antagonist, deteriorated acidosis-promoted TRAP + LMNC formation. 4-Alpha-PDD, a TRPV4-specific agonist, added in the last 21 h of preosteoclast differentiation, potentiated TRAP + LMNC formation in a mild acidosis environment, showing synergism between TRPV4 activation and acidosis. RN1734, a TRPV4-specific antagonist, partly inhibited acidosis-promoted TRAP + LMNC formation. We thus narrowed down the major action points of acidosis in osteoclast formation and elucidated the characteristics of this system in detail. Our results show that acidosis effectively uses TRPV4 to drive large-scale cell fusion and also utilizes systems independently of TRPV4.

Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells

The present study was designed to investigate whether chondroitin sulfate (CS)-E, a CS structural isomer variant, alter the differentiation of macrophage cell line RAW264 cells to osteoclast-like cells. CS-B, CS-E, low molecular weight CS-E, synthetic chondroitin polysulfate (CPS) and heparin significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinuclear cells and pit formation on calcium phosphate (CaP)-coated plates. CS-E pre-coated on the CaP plate also inhibited pit formation. Digestion of CS on the cell surface by chondroitinase showed no effect on the osteoclastic differentiation of RAW264 cells whereas inhibitory effect on the differentiation of osteoblastic cell line MC3T3-E1. On the other hand, exogenously added fluorescein-labeled CS-E directory bound to fibronectin and RAW264 cells. These results suggest that CS-E structure on the surface of osteoblasts or bone matrix binds to cell adhesion molecule such as integrin on the pre-osteoclastic cells and inhibits the differentiation into osteoclasts. CS-E may have a potential in treating bone defect if combined with CaP materials.

Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts and their precursors using both in vivo and in vitro experiments. The in vivo osteolysis experiment showed that LPS (lipopolisaccharide)-induced osteolysis occurred more rapidly and extensively in bgn deficient mice compared to wild type ( WT) mice. To further understand the mechanism of action, we determined the effects of Bgn on 1α, 25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3)-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate-resistant acid phosphatase-positive multinuclear cells appeared earlier and more extensively in the co-cultures containing calvarial cells from bgn deficient mice than WT mice, regardless of the genotype of osteoclast precursors. The osteoblast abnormality that stimulated osteoclast formation appeared to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte protease inhibitor (slpi) in bgn deficient osteoblasts treated with 1,25-(OH) 2D 3. These findings point to a novel molecular factor made by osteoblasts that could potentially be involved in LPS-induced osteolysis.

β1 Integrin/Focal Adhesion Kinase-mediated Signaling Induces Intercellular Adhesion Molecule 1 and Receptor Activator of Nuclear Factor κB Ligand on Osteoblasts and Osteoclast Maturation

We have assessed characteristics of primary human osteoblasts, shedding light on signaling mediated by beta1 integrin. beta1 integrins are major receptors for these matrix glycoproteins. 1) Integrins beta1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphav were highly expressed on primary osteoblasts. 2) Engagement of beta1 integrins on osteoblasts by cross-linking with specific antibody or ligand matrices, such as fibronectin or collagen, augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on the surface. 3) Up-regulation of ICAM-1 and RANKL on osteoblasts by beta1 stimulation was completely abrogated by pretreatment with herbimycin A and genistein, tyrosine kinase inhibitors, or transfection of dominant negative truncations of focal adhesion kinase (FAK). 4) Engagement of beta1 integrins on osteoblasts induced tartrate-resistant acid phosphatase-positive multinuclear cell formation in the coculture system of osteoblasts and peripheral monocytes. 5) Up-regulation of tartrate-resistant acid phosphatase-positive multinuclear cell formation by beta1 stimulation was completely abrogated by transfection of dominant negative truncations of FAK. Our results indicate that beta1 integrin-dependent adhesion of osteoblasts to bone matrices induces ICAM-1 and RANKL expression and osteoclast formation via tyrosine kinase, especially FAK. We here propose that beta1 integrin/FAK-mediated signaling on osteoblasts could be involved in ICAM-1- and RANKL-dependent osteoclast maturation.

Expression of receptor activator of NF-kappa B ligand and osteoprotegerin in culture of human periodontal ligament cells.

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins implicated in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament (PDL) cells and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated remarkably by application of 10-8 m one-alpha, 25-dihydroxyvitamin D3[1,25-(OH)2D3] and 10-7 m dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated decrease of OPG by the application of 1,25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase-positive multinuclear cells were markedly induced when the PDL cells were cocultured with mouse bone marrow cells in the presence of an anti-OPG antibody together with 1,25-(OH)2D3 and Dex. These results indicate that PDL cells synthesize both RANKL and OPG and that inactivation of OPG may play a key role in the differentiation of osteoclasts.

High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture.

The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood. We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture. Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3). High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs). This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG). This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL. In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA. High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin. In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system.

The Inhibitory Effects of Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide on Osteoclast Formation Are Associated with Upregulation of Osteoprotegerin and Downregulation of RANKL and RANK

The presence of a network of peptidergic nerve fibers in the skeleton, expressing several neuropeptides including vasoactive intestinal peptide (VIP), has been demonstrated. This observation, together with our findings in vitro showing that VIP can regulate the activities of osteoblasts and osteoclasts as well as the recruitment of osteoclasts, has suggested the existence of a neuro-osteogenic interplay in bone metabolism. In the present study, the effects of VIP and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon superfamily, on osteoclast formation and mRNA expression of three key regulatory proteins involved in osteoclast formation have been investigated. VIP, PACAP-27, and PACAP-38, at concentrations of 10−6 M, all significantly inhibited formation of tartrate-resistant acid phosphatase-positive multinuclear cells (TRAP + MNC) in mouse bone marrow cultures stimulated by 1,25(OH)2-vitamin D3 (D3; 10−8 M). By using semiquantitative RT-PCR, it was found that D3 upregulated the mRNA expressions of receptor activator of NF-κB ligand (RANKL) and receptor activator of NF-κB (RANK), whereas the expression of osteoprotegerin (OPG) was downregulated in mouse bone marrow cultures stimulated by D3 for 7 days. Both VIP and PACAP-38 decreased the stimulatory effects of D3 on RANKL and RANK expression, whereas the inhibitory effect of D3 on OPG expression was reversed by VIP and PACAP-38. These observations indicate that the inhibitory effects of VIP and PACAP on osteoclast recruitment are due to regulation of the expression of key proteins involved in later stages of osteoclast differentiation.

CD9 molecule expressed on stromal cells is involved in osteoclastogenesis

Osteoclasts are derived from hematopoietic stem cells and their development is dependent on the products of stromal cells. CD9, a member of the tetraspan transmembrane-superfamily, is expressed on both hematopoietic cells and stromal cells. Addition of antagonistic rat anti-mouse CD9 antibody (KMC8.8) to cultures inhibited osteoclastogenesis on established stromal cell layers. When rat bone marrow cells depleted of adherent stromal cells were cultured on mouse stromal cells, numerous tartrate-resistant acid phosphatase-positive multinuclear cells were observed, and KMC8.8, which recognizes mouse but not rat CD9, completely prevented the generation of osteoclasts, suggesting that the CD9 expressed on the stromal cell is essential for osteoclastogenesis. Possibly for the same reason, KMC8.8 pretreatment of the mouse macrophage-like cell line C7, which is able to differentiate into mature osteoclasts, did not inhibit subsequent C7 cell differentiation, whereas the addition of KMC8.8 to cocultures of C7 cells with stromal cells inhibited the differentiation of C7 cells into osteoclasts. Moreover, we found that blockage of a signal via CD9 on stromal cells reduced transcription of the osteoclast differentiation factor (Odf) gene, which, together with macrophage colony-stimulating factor, is essential for osteoclastogenesis. These results revealed that CD9 molecules on stromal cells play a critical role in osteoclast development, possibly by modulating the expression of Odf.

Ipamorelin, a new growth-hormone-releasing peptide, induces longitudinal bone growth in rats.

Ipamorelin is a new and potent synthetic pentapeptide which has distinct and specific growth hormone (GH)-releasing properties. With the objective of investigating the effects on longitudinal bone growth rate (LGR), body weight (BW), and GH release, ipamorelin in different doses (0, 18, 90 and 450 microg/day) was injected s.c. three times daily for 15 days to adult female rats. After intravital tetracycline labelling on days 0, 6, and 13, LGR was determined by measuring the distance between the respective fluorescent bands in the proximal tibia metaphysis. Ipamorelin dose-dependently increased LGR from 42 microm/day in the vehicle group to 44, 50, and 52 microm/day in the treatment groups (P<0.0001). There was also a pronounced and dose-dependent effect on BW gain. The treatment did not affect total IGF-I levels, IGFBPs, or serum markers of bone formation and resorption. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the metaphysis of the tibia did not change significantly with treatment. The responsiveness of the pituitary to a provocative i.v. dose of ipamorelin or GHRH showed that the plasma GH response was marginally reduced (P<0.03) after ipamorelin, but unchanged after GHRH. The pituitary GH content was unchanged by ipamorelin treatment. Whether ipamorelin or other GH secretagogues may have a place in the treatment of children with growth retardation requires demonstration in future clinical studies.

Effects of CP-336,156, a new, nonsteroidal estrogen agonist/antagonist, on bone, serum cholesterol, uterus and body composition in rat models.

We have discovered a new, nonsteroidal, potent estrogen agonist/antagonist, CP-336,156. CP-336,156 binds selectively and with high affinity to the human estrogen receptor-alpha with a half-inhibition concentration of 1.5 nM, which is similar to that seen with estradiol (4.8 nM). When given orally to immature (3-week-old) female Sprague-Dawley rats for 3 days at doses of 0.1, 1.0, 10, or 100 microg/kg x day, unlike 17alpha-ethynyl estradiol, CP-336,156 had no effect on uterine wet or dry weight. Similarly, no uterine hypertrophy was observed in aged (17-month-old) female rats treated (p.o.) with CP-336,156 at 10 or 100 microg/kg x day for 28 days. We also found that CP-336,156 decreased total serum cholesterol and fat body mass and had no effect on lean body mass in these aged female rats. In 5-month-old ovariectomized (OVX) Sprague-Dawley female rats, CP-336,156 completely prevented OVX-induced increases in body weight gain, total serum cholesterol, and serum osteocalcin at doses between 10 and 1000 microg/kg x day after 4 weeks. At these doses, CP-336,156 completely prevented OVX-induced bone loss and inhibited the increased bone turnover associated with estrogen deficiency in lumbar vertebrae, proximal tibiae, and distal femora. Similar to estrogen, CP-336,156 induced apoptosis and p53 expression with a concomitant decrease in the number of tartrate-resistant acid phosphatase-positive multinuclear cells in rat bone marrow cell cultures in vitro, suggesting that the induction of apoptosis may be a mechanism for the estrogenic activities of CP-336,156 in bone. In summary, CP-336,156 is a new, orally active, nonsteroidal, potent estrogen agonist/antagonist that has similar effects in bone as estradiol but without the uterine-stimulating effects associated with estradiol in rats.

Macrophage colony stimulating factor down-regulates MCSF-receptor expression and entry of progenitors into the osteoclast lineage.

Macrophage colony-stimulating factor (MCSF), although necessary for entry of precursors into the early preosteoclast pathway, inhibits osteoclastogenesis at high doses. To clarify the relationship between MCSF and osteoclast formation, we investigated the effect of exogenous MCSF in murine bone marrow culture. Precursor proliferation and the expression of MCSF-receptor were examined after 4 days of culture in the presence or absence of accessory stromal cells. In both mixed marrow and destromalized cell cultures, exogenous MCSF dose-dependently decreased 125I-MCSF binding (by 65 +/- 5.0% at 3500 and 87 +/- 16.7% at-7000 U/ml, respectively) while enhancing mononuclear cell proliferation after 3 days of exposure (by 2.8- and 6.3-fold, respectively). These effects were maintained 24 h after removal of exogenous MCSF and, as such, likely represented an MCSF-induced change in MCSF receptor-bearing cells. Exposure to exogenous MCSF (3500 U/ml) days 2-4 dose-dependently inhibited tartrate resistant acid phosphatase positive multinuclear cell (TRAP+ MNC) formation counted at the end of day 7, by 64.3 +/- 4.1%. This inhibition of TRAP+ MNC formation was preceded by a 92 +/- 9% decrease in the expression of carbonic anhydrase II mRNA measurable at 4 days. These results indicate that MCSF promotes proliferation of a population of cells expressing lower cognate receptor sites. Changes in MCSF-receptor expression appear to modulate the final lineage selection of the pluripotent monoblastic progenitor.

Effect of retinoic acid on the resorptive activity of chick osteoclasts in vitro

Mixed cell suspensions mechanically isolated from the longbones of day 20 prehatch chicks were cultured for 24 h on bone and sperm whale dentine slices in the presence of 0, 10, 100, and 1000 nM retinoic acid (RA). Significant inhibitions in the numbers of discrete lacunae resorbed per dentine slice, and in the ratio of lacunae per tartrate-resistant acid phosphatase-positive multinuclear cell were observed with all concentrations of RA studied. Semi-automated, 3-dimensional analysis of 733 pits was performed on one series of experiments using a tandem scanning (confocal) microscope, interfaced to an image analyzing computer. The majority of lacunae were small and unilocular; the plan areas of 90% of control pits were below 500 μm 2. Small but statistically significant increases in lacunar areas, but not mean or maximum depths or volumes, were observed in the presence of 10 and 100 nM RA; however, these changes were much smaller than the magnitude of the decrease in pit numbers. Thus, the overall effect of RA in this system was inhibitory. Our findings contrast with the well known stimulatory action of retinoids on bone resorption both in vivo and in organ culture, but may parallel the inhibitory effects of prostaglandins observed in disaggregated osteoclast systems.