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PubMed2005

Momordin I, an inhibitor of AP-1, suppressed osteoclastogenesis through inhibition of NF-kappaB and AP-1 and also reduced osteoclast activity and survival.

Osteoclasts originating from hematopoietic precursor cells differentiate into multinucleated cells through multiple steps. The essential roles of NF-kappaB and AP-1 in osteoclast differentiation have been clearly demonstrated in numerous studies. c-Fos, a component of AP-1 transcription factor, plays a key role in osteoclast differentiation. Recently, we found a strong inhibitor of AP-1 transcriptional activity, named momordin I, based on the structure of oleanolic acid glycosides and originally isolated from Ampelopsis radix. So, we hypothesized that momordin I might be able to regulate osteoclast formation, activity, and survival. Here, we report the ability of momordin I to suppress osteoclastogenesis in a co-cultured system and a RANKL-induced osteoclast precursor system. Momordin I remarkably inhibited the activation of NF-kappaB as well as AP-1 in RANKL-induced RAW264.7 cells, in which momordin I appeared to target IkappaB degradation and c-Fos expression, respectively, but not MAPK signaling pathways. The ability of momordin I to change the ratio of RANKL and OPG in primary osteoblasts was partially responsible for the reduction of osteoclast formation. Furthermore, pit formation on dentin slices was suppressed by momordin I with stimulating actin ring disruption. Our results also showed that momordin I highly shortened osteoclast lifespan and induced osteoclast apoptosis. In conclusion, the present results demonstrate for the first time that momordin I is a potent inhibitor of osteoclast differentiation via the reduction of NF-kappaB and AP-1, and also suppresses osteoclast function and survival.

Animals↗
PubMed2004

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

The carotenoid beta-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 beta-cryptoxanthin 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 bone-resorbing agent [parathyroid hormone (1-34) (PTH), prostaglandin E(2), 1,25-dihydroxyvitamin D(3), lipopolysaccharide, or tumor necrosis factor-alpha (TNFalpha)] 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 microg/mL), or TNFalpha (10 ng/mL) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of beta-cryptoxanthin (10(-8) to 10(-6)M). beta-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, beta-cryptoxanthin (10(-7) and 10(-6)M) had a significant inhibitory effect on osteoclast-like formation induced by receptor activator of NF-kappaB 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. beta-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 beta-cryptoxanthin on RANKL plus M-CSF-, PTH-, or TNFalpha-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-beta-d-ribofuranosylbenzimidazole (10(-6)M), an inhibitor of transcription. This study demonstrates that beta-cryptoxanthin has a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture. The inhibitory action of beta-cryptoxanthin may partly involve in a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.

Animals↗
PubMed2005

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration.

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.

Animals↗
PubMed2004

Chemically modified tetracyclines act through multiple mechanisms directly on osteoclast precursors.

Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption primarily through their ability to inhibit matrix metalloproteinases (MMPs). We have previously demonstrated that some tetracycline compounds (TCs) induce apoptosis in mature rabbit osteoclasts and inhibit osteoclastic resorption in mouse osteoblast/marrow co-cultures in vitro. In this report, we now show that non-antibiotic analogues of doxycycline (CMT-3) and minocycline (CMT-8) are potent inhibitors of osteoclastogenesis in vitro from human peripheral blood mononuclear cells (PBMC) stimulated with macrophage colony stimulating factor (MCSF) and receptor activator of NF-kappaB ligand (RANKL), through an action that is independent of osteoblast-osteoclast interactions. Osteoclast formation over 20 days was completely abrogated when CMT-3 or CMT-8 were included in PBMC cultures at a concentration of 250 ng/ml, although doxycycline at this concentration reduced osteoclast formation to ca. 50% of control. CMT-3 and CMT-8 also significantly induced apoptosis over 24 h in mature osteoclasts generated over 20 days when added to cultures at 5 microg/ml or more. In a time-course experiment, apoptosis was evident after a delay of 1-2 h following treatment of mature osteoclasts with CMT-3 at 20 microg/ml. The broad-spectrum MMP inhibitor BB94 (Batimastat) did not recapitulate the apoptosis induced by CMT-3, even at a concentration where MMP-13 activity was completely inhibited. There was no evidence for an anabolic effect of any of the TCs on osteoblast lineage cells in a calcifying fibroblastic colony (CFU-f) formation assay, where CMT-3 partially inhibited CFU-f formation at 5 microg/ml. Our data indicate that inhibition of osteoclast formation and induction of osteoclast apoptosis are pharmacologically significant actions of CMTs in inhibiting bone resorption, and that osteoclast apoptosis cannot be attributed to the ability of CMTs to inhibit MMPs or to actions mediated by osteoblastic lineage cells.

Apoptosis↗
PubMed2005

Osteoclastic activity begins early and increases over the course of bone healing.

Osteoclasts are specialised bone-resorbing cells. This particular ability makes osteoclasts irreplaceable for the continual physiological process of bone remodelling as well as for the repair process during bone healing. Whereas the effects of systemic diseases on osteoclasts have been described by many authors, the spatial and temporal distribution of osteoclasts during bone healing seems to be unclear so far. In the present study, healing of a tibial osteotomy under standardised external fixation was examined after 2, 3, 6 and 9 weeks (n = 8) in sheep. The osteoclastic number was counted, the area of mineralised bone tissue was measured histomorphometrically and density of osteoclasts per square millimetre mineralised tissue was calculated. The osteoclastic density in the endosteal region increased, whereas the density in the periosteal region remained relatively constant. The density of osteoclasts within the cortical bone increased slightly over the first 6 weeks, however, there was a more rapid increase between the sixth and ninth weeks. The findings of this study imply that remodelling and resorption take place already in the very early phase of bone healing. The most frequent remodelling process can be found in the periosteal callus, emphasising its role as the main stabiliser. The endosteal space undergoes resorption in order to recanalise the medullary cavity, a process also started in the very early phase of healing at a low level and increasing significantly during healing. The cortical bone adapts in its outward appearance to the surrounding callus structure. This paradoxic loosening is caused by the continually increasing number and density of osteoclasts in the cortical bone ends. This study clearly emphasises the osteoclastic role especially during early bone healing. These cells do not simply resorb bone but participate in a fine adjusted system with the bone-producing osteoblasts in order to maintain and improve the structural strength of bone tissue.

Animals↗
PubMed2004

Involvement of iNOS-dependent NO production in the stimulation of osteoclast survival by TNF-alpha.

Osteoclasts, cells primarily responsible for bone resorption, differentiate from hematopoietic progenitor cells under the influence of various hormones, cytokines, and differentiation factors. Once fully differentiated, osteoclasts rapidly die in the absence of any survival factor. We have previously shown that tumor necrosis factor alpha (TNF-alpha) promotes the survival of differentiated osteoclasts. The expression of inducible nitric oxide synthase (iNOS) and consequent NO production is often stimulated under inflammatory conditions. In this study, we found that TNF-alpha, but not receptor activator of nuclear factor kappa B ligand and interleukin 1, increased the expression of iNOS both at the mRNA and protein levels. Subsequently, an enhanced NO level was detected both inside the cells and the culture medium of TNF-alpha-stimulated osteoclasts. Blocking NOS activity with L-NAME prevented TNF-alpha-induced NO generation by osteoclasts and the osteoclast survival stimulated by TNF-alpha. The iNOS selective inhibitor L-NIL also suppressed TNF-alpha-induced osteoclast survival, whereas low concentrations of NO releaser NOC-18 were sufficient to promote osteoclast survival. Furthermore, antiapoptotic and caspase suppressive effects of TNF-alpha on osteoclasts were abolished by L-NAME. Our findings indicate that iNOS-dependent NO generation contributes to the survival-promoting function of TNF-alpha in osteoclasts.

Animals↗
PubMed2001

Presence of osteoclast precursors in colonies cloned in the presence of hematopoietic colony-stimulating factors.

Osteoclasts are derived from hematopoietic stem cells, but the relationship between osteoclast precursors (OCPs) and hematopoietic colony-forming cells (CFCs) has not yet been clarified. Although osteoclasts share certain cell surface markers and growth factor requirements with their macrophage and monocyte cell lineages, osteoclasts are a different lineage with regard to the requirement for signaling via c-Kit. To investigate whether CFCs are able to differentiate into osteoclasts, we performed in vitro studies of osteoclastogenesis. We performed progenitor assays in the presence of hematopoietic colony-stimulating factors. Primary colonies were plucked and examined for their potential to differentiate into osteoclasts. We found that osteoclasts are present in colonies elicited by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kB ligand (RANKL) in semisolid cultures. Moreover, a part of the cells composing the colonies elicited by granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF alone possessed the potential to differentiate into osteoclasts. These OCPs in the colonies were enriched in the c-Fms+ large-sized cell fraction and had a foamy cell morphology, like mature macrophages. A small number of cells in M-CSF-promoted and GM-CSF-promoted colonies formed secondary colonies in the semisolid medium containing these factors. The frequency of OCPs in these secondary colonies elicited by M-CSF was 10 times higher than that elicited by GM-CSF. Multiple origins of OCPs that differentiate into mature osteoclasts are proposed based on the observation that osteoclasts could be generated from OCPs that emerged from CFCs induced under different conditions or developmental stages.

Animals↗
PubMed2000

[Osteoblastic regulation of osteoclast survival: effect of calcitriol].

Throughout life, bone is remodelled in a dynamic process which results in a balance between bone formation by osteoblasts and bone resorption by osteoclasts. It is now clearly established that osteoblasts/stromal cells are crucial for differentiation of osteoclasts, through a mechanism involving cell-to-cell contact. However, the possible involvement of osteoblasts and stromal cells in the survival of osteoclasts has not yet been clearly demonstrated. In this study, we assessed the influence of cellular microenvironment, especially osteoblasts, on the osteoclast survival. Our results have shown significant differences in osteoclastic survival between unfractionated bone cells and pure osteoclasts. Furthermore, we have shown that addition of 1.25(OH)2D3 to unfractionated bone cells resulted in a dose-dependent increase in osteoclast survival. Finally, we have shown that a conditioned medium obtained from rat osteoblastic cells cultured with calcitriol was able to increase significantly survival of pure osteoclasts. Taken together, these results strongly suggest that osteoblastic cells present in the bone microenvironment might play a role in the osteoclastic survival by producing soluble factor which modulate osteoclast apoptosis.

Animals↗
PubMed1996

Interaction between effects of parathyroid hormone and bisphosphonate on regulation of osteoclast activity by the osteoblast-like cell line UMR-106.

Treatment of osteoblasts by either parathyroid hormone (PTH) or bisphosphonate can affect their regulation of the bone-resorbing activity of osteoclasts in vitro, leading to increased and decreased resorption, respectively. To address this issue, we have examined the interaction between the effects of PTH and bisphosphonate on the regulation of osteoclast activity by the PTH-responsive osteoblast-like cell line UMR-106. When rat osteoclasts were cocultured with UMR-106 cells on bovine bone slices in the presence of 10(-8) mol/L PTH, the number of resorption pits was increased 4.2-fold, whereas the addition of UMR-106 cells or PTH alone had no effect. Pretreatment of the UMR-106 cells for 5 min with increasing concentrations of either of the bisphosphonates, clodronate, and ibandronate before coculture with osteoclasts in 10(-8) mol/L PTH, caused a dose-dependent reduction in the formation of resorption pits, reaching the maximal inhibition level (60%-75% below the control) at approximately 10(-9) mol/L clodronate and 10(-11) mol/L ibandronate. Addition of conditioned medium (CM) from untreated UMR-106 cells to rat osteoclasts had no effect on pit formation, whereas CM from UMR-106 cells pretreated with ibandronate reduced the osteoclastic bone resorption by approximately 40%. However, this effect was abolished by the subsequent culture of the ibandronate-pretreated UMR-106 cells in 10(-8) mol/L PTH before harvesting the CM, because both this CM and CM from non-pretreated UMR-106 cells cultured in 10(-8) mol/L PTH caused an approximately 75% increase in pit formation when added to rat osteoclasts. In conclusion, osteoclastic bone resorption can be directly affected independently as well as at the same time by the ibandronate-induced osteoclast-inhibiting factor and the PTH-induced osteoclast-stimulating factor. The final level of bone resorption depends on the relative concentration of these two factors.

Animals↗
PubMed1998

Macrophage colony-stimulating factor increases bone resorption by osteoclasts disaggregated from human fetal long bones.

Macrophage colony-stimulating factor (M-CSF) is known to play an important role in human and murine osteoclast formation. Although M-CSF has been shown to inhibit isolated neonatal rat osteoclasts from resorbing bone, its action on the mature human osteoclast has not been described. We now report that M-CSF increases osteoclastic bone resorption in a dose-responsive manner. Bone resorption by mature human fetal osteoclasts, including pit area, depth, and volume, was increased in the presence of M-CSF compared with vehicle. The number of osteoclasts in the cultures was similar after 2 and 18 h in the presence of M-CSF, whereas there was a significant reduction in osteoclast number, whether assessed as the number of tartrate-resistant acid phosphatase (TRAP)-positive or vitronectin receptor-positive cells after 18 h in M-CSF-free cultures. The number of nuclei per osteoclast after 2 or 18 h in M-CSF was also similar and there was no difference in the number of vitronectin receptor-positive mononucleate cells at 2 and 18 h. This suggests that the increased bone resorption is likely to be accounted for by enhanced osteoclast survival in M-CSF compared with controls rather than by formation of new osteoclasts.

Acid Phosphatase↗
PubMed1999

Calcitonin receptor antibodies in the identification of osteoclasts.

Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.

Animals↗
PubMed1998

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.

BACKGROUND: Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast-specific biological functions remain elusive because only a limited number of osteoclast-specific genes have been identified. To circumvent this, we isolated a large number of osteoclast-specific genes by preparing a subtracted cDNA library of high quality. RESULTS: We first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast-specific cDNA library of high complexity by subtracting it with biotin-labelled mRNA, derived from rabbit spleen through the biotin-avidin method. The resulting library included a high proportion of full-length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast-specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple-tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast-specific genes. CONCLUSION: From these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up- or down-regulated genes in general.

Animals↗
PubMed2005

The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption.

Bone remodeling involves the interplay of bone resorption and formation and is accurately controlled to maintain bone mass. Both processes require transcellular Ca(2+) transport, but the molecular mechanisms engaged remain largely elusive. The epithelial Ca(2+) channel TRPV5 is one of the most Ca(2+)-selective transient receptor potential (TRP) channels. In this study, the functional role of TRPV5 in bone was investigated. TRPV5 mRNA was expressed in human and murine bone samples and in osteoclasts along with other genes involved in transcellular Ca(2+) transport, including calbindin-D(9K) and calbindin-D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+)-ATPase 1b. TRPV5 expression in murine osteoclasts was confirmed by immunostaining and showed predominant localization to the ruffled border membrane. However, TRPV5 was absent in osteoblasts. Analyses of femoral bone sections from TRPV5 knockout (TRPV5(-/-)) mice revealed increased osteoclast numbers and osteoclast area, whereas the urinary bone resorption marker deoxypyridinoline was reduced compared with WT (TRPV5(+/+)) mice. In an in vitro bone marrow culture system, the amount of osteoclasts and number of nuclei per osteoclast were significantly elevated in TRPV5(-/-) compared with TRPV5(+/+) mice. However, using a functional resorption pit assay, we found that bone resorption was nearly absent in osteoclast cultures from TRPV5(-/-) mice, supporting the impaired resorption observed in vivo. In conclusion, TRPV5 deficiency leads to an increase in osteoclast size and number, in which Ca(2+) resorption is nonfunctional. This report identifies TRPV5 as an epithelial Ca(2+) channel that is essential for osteoclastic bone resorption and demonstrates the significance of transcellular Ca(2+) transport in osteoclastic function.

Acid Phosphatase↗
PubMed1991

Avian osteoclasts as estrogen target cells.

Although in vivo effects of 17 beta-estradiol (estrogen) on bone turnover have been shown to occur mainly through influences on osteoclast-mediated bone resorption, the mechanism by which estrogen reduces bone resorption is unclear. To approach this question, we have examined authentic osteoclasts for evidence of a direct osteoclast response to estrogen in vitro. Highly purified (greater than (90%) viable avian osteoclasts from birds maintained on a low calcium diet were obtained using an osteoclast-specific monoclonal antibody coupled to magnetic beads. Isolated cells were either analyzed directly for estrogen receptor (ER) levels or cultured to assess the biological effects of estrogen. Northern blot analysis revealed a 5.2-kilobase mRNA that hybridized with a cDNA to human ER mRNA in the osteoclasts. An anti-human ER antibody recognized proteins of 66 kDa and 140 kDa in osteoclast extracts by Western blot analysis. The 66-kDa size is in close agreement with the reported size of the human ER. Nuclear binding of estrogen to intact viable osteoclasts was steroid-specific and saturable, with 5662 +/- 1420 molecules bound per nucleus (mean +/- SEM). In vitro estrogen responses in osteoclasts included a dose-dependent decrease in resorption as well as an increase in nuclear protooncogene mRNA levels. These observations indicate that osteoclasts are capable of directly responding to estrogen in vivo.

Animals↗
PubMed1993

Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse.

The development of osteoclastic cell lines would greatly facilitate analysis of the cellular and molecular biology of bone resorption. Several cell lines have previously been reported to be capable of osteoclastic differentiation. However, such cell lines form at best only occasional excavations, suggesting that osteoclastic differentiation is either incomplete or that osteoclasts represent a very small proportion of the cells present. We have used the recently developed H-2KbtsA58 transgenic mouse, in which the interferon-inducible major mouse histocompatibility complex H-2Kb promoter drives the temperature-sensitive (ts) immortalizing gene of simian virus 40 (tsA58), to develop cell lines from bone marrow with high efficiency. Bone marrow cells were incubated with gamma interferon at 33 degrees C, then cloned, and expanded. The cell lines were characterized at 39.5 degrees C in the absence of gamma interferon. First, stromal cell lines were established that induced osteclast formation (resorption of bone slices) when cocultured with hemopoietic spleen cells. Some of the stromal cell lines so generated were able to resorb approximately 30 mm2/cm2 of bone surface. We then established cell lines of hemopoietic origin, several of which possess osteoclastic potential. When these osteoclast-precursor cell lines were cocultured with stromal cell lines, extensive bone resorption was observed. Osteoclast formation did not occur if the precursor cell lines were incubated on bone slices without stromal cells; osteoclast formation was also dependent upon the presence of 1 alpha,25-dihydroxyvitamin D3. These cell lines represent a model for osteoclast formation and a valuable resource for identification of the mechanisms and factors that regulate osteoclast differentiation and function.

Animals↗
PubMed1995

Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms.

Nitric oxide (NO) produces rapid osteoclast detachment and contraction in vitro, and this effect is accompanied by a profound inhibition of bone resorption. Work by others has confirmed these findings in vivo: inhibition of NO synthase [NOS; L-arginine, NADPH: oxygen oxidoreductase (NO-forming), EC 1.14.13.39] in normal rats is followed by increased bone resorption reflected by a marked loss in bone mineral density. In our present study, immunocytochemistry and Northern blotting show the presence of the constitutive calcium-sensitive NOS isoform (cNOS) in normal rat osteoclasts and in the human preosteoclast cell line (FLG 29.1). The inducible NOS isoform (iNOS) was also clearly demonstrable in the rat cells especially after treatment with gamma interferon (IFN-gamma) and bacterial wall products [lipopolysaccharide (LPS)], while a basal level of transcript was detected in the untreated human preosteoclast line. However NADPH-diaphorase activity was intense only in neonatal rat osteoclasts attached to bone, perhaps reflecting either enhancement of cNOS activity by calcium or increased amounts of the inducible isoform in activated osteoclasts in situ compared with isolated neonatal rat osteoclasts. These actively resorb devitalized bone but the untreated cells contain relatively low levels of NOS; they are extremely sensitive to inhibition by NO. The iNOS inhibitor aminoguanidine markedly enhances in vitro resorption by activated NOS-rich chick osteoclasts and by normal rat osteoclasts treated with LPS or IFN-gamma. In contrast, the nonselective NOS inhibitor NG-monomethyl-L-arginine inhibits resorption by untreated neonatal rat osteoclasts. Thus, osteoclast function may require intermittent calcium-stimulated increases in NO production by cNOS against a basal inhibitory background activity of the iNOS isoform. However, bone resorption depends on precursor replication and on the activity of the mature cells, and we found that the NO donor 3-morpholinosydnonimine (SIN-1) (50 microM) profoundly depressed replication in the human preosteoclast line. Taken together, these results strongly suggest that NO maintains a central control of bone resorption in both avian and mammalian species by exerting a powerful tonic restraint of osteoclast numbers and activity. The presence of NOS in human cells implies a similar function in man and that conventional views of calcium homoeostasis and skeletal metabolism will need substantial revision. Since NO also influences behavior of the osteoblast, the bone-forming cell, in vitro, a similar effect in vivo might imply a general influence on bone remodeling.

Adult↗
PubMed2003

Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/- mice by RANKL transgene.

Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2-/- calvaria-derived cell line (CA120-4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120-4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2-/- livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2-/- mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.

Animals↗
PubMed2005

Interleukin-3 and granulocyte-macrophage colony-stimulating factor inhibits tumor necrosis factor (TNF)-alpha-induced osteoclast differentiation by down-regulation of expression of TNF receptors 1 and 2.

Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of monocyte/macrophage lineage, which also give rise to macrophages or dendritic cells. In this study we investigated the mechanism by which interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibit tumor necrosis factor (TNF)-alpha-induced osteoclast differentiation in mouse osteoclast precursors. We show here that both IL-3 and GM-CSF potently inhibits TNF-alpha-induced osteoclast differentiation by direct action on osteoclast precursors. The inhibitory effect of IL-3 and GM-CSF on osteoclast differentiation was completely neutralized by anti-IL-3 and anti-GM-CSF antibodies, respectively. In addition, the inhibitory effect of IL-3 and GM-CSF on TNF-alpha-induced osteoclast differentiation was irreversible. In osteoclast precursors, IL-3 and GM-CSF inhibited c-Fms expression post-transcriptionally. Interestingly, IL-3 and GM-CSF down-regulated both mRNA and surface expression of TNF receptor 1 (TNFR1) and TNFR2. Furthermore, cells in the presence of IL-3 and GM-CSF showed high expression of macrophage antigen CD11b, and low expression of dendritic cells antigen CD11c and prolong exposure of osteoclast precursors to GM-CSF increased the CD11c expression compare with IL-3. In summary, we provide the first evidence that IL-3 and GM-CSF block TNF-alpha-induced osteoclast differentiation by down-regulation of mRNA and surface expression of TNFR1 and TNFR2.

Animals↗