Human Preosteoclastic Cells Research Articles

Complex effect of hydroxyapatite nanoparticles on the differentiation and functional activity of human pre-osteoclastic cells.

Nanosized hydroxyapatite (HA) is a promising material in clinical applications targeting the bone tissue. NanoHA is able to modulate bone cellular events, which accounts for its potential utility, but also raises safety concerns regarding the maintenance of the bone homeostasis. This work analyses the effects of HA nanoparticles (HAnp) on osteoclastic differentiation and activity, an issue that has been barely addressed. Rod-like HAnp, produced by a hydrothermal precipitation method, were tested on peripheral blood mononuclear cells (PBMC), which contains the CD14+ osteoclastic precursors, in unstimulated or osteoclastogenic-induced conditions. HAnp were added at three time-points during the osteoclastic differentiation pathway, and cell response was evaluated for osteoclastic related parameters. Results showed that HAnp modulated the differentiation and function of osteoclastic cells in a dose- and time-dependent manner. In addition, the effects were dependent on the stage of osteoclastic differentiation. In unstimulated PBMC, HAnp significantly increased osteoclastogenesis, leading to the formation of mature osteoclasts, as evident by the significant increase of TRAP activity, number of TRAP-positive multinucleated cells, osteoclastic gene expression and resorbing ability. However, in a population of mature osteoclasts (formed in osteoclastogenic-induced PBMC cultures), HAnp caused a dose-dependent decrease on the osteoclastic-related parameters. These results highlight the complex effects of HAnp in osteoclastic differentiation and activity, and suggest the possibility of HAnp to modulate/disrupt osteoclastic behavior, with eventual imbalances in the bone metabolism. This should be carefully considered in bone-related and other established and prospective biomedical applications of HAnp.

Collinin Reduces Porphyromonas gingivalis Growth and Collagenase Activity and Inhibits the Lipopolysaccharide‐Induced Macrophage Inflammatory Response and Osteoclast Differentiation and Function

Collinin is a secondary plant metabolite belonging to the class of geranyloxycoumarins. We explored the potential beneficial impact of collinin on periodontal health by investigating its effect on Porphyromonas gingivalis (P. gingivalis), lipopolysaccharide (LPS)-induced inflammatory response of macrophages, and osteoclastogenesis. Collinin was synthesized from pyrogallol and propiolic acid. A microdilution assay was used to determine antibacterial activity of collinin. The effect of collinin on collagenase activity of P. gingivalis was determined using fluorescent collagen. Macrophages were treated with collinin before being stimulated with LPS. The secretion of interleukin-6, chemokine (C-C motif) ligand 5, and prostaglandin E2 was assessed by enzyme-linked immunosorbent assays (ELISA). The inhibitory effect of collinin on differentiation of human preosteoclastic cells was assessed by tartrate-resistant acid phosphatase staining, whereas the secretion of matrix metalloproteinase-9 (MMP-9) was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen fragments. Collinin inhibited the growth of P. gingivalis. The effect was more pronounced under iron-restricted conditions. Collinin dose dependently inhibited the degradation of type I collagen by P. gingivalis. It was also a potent inhibitor of the LPS-induced inflammatory response in macrophages and completely inhibited receptor activator of nuclear factor κB ligand-dependent osteoclast differentiation and MMP-9 secretion. Last, collinin affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. Although clinical trials are required, data from these in vitro analyses support the potential of collinin as a therapeutic agent for treating inflammatory periodontitis associated with bone breakdown.

A-Type Cranberry Proanthocyanidins Inhibit the RANKL-Dependent Differentiation and Function of Human Osteoclasts

This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs) on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining, while the secretion of interleukin-8 (IL-8) and matrix metalloproteinases (MMPs) was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold) and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml) affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

Osteoclastic differentiation of mouse and human monocytes in a plasma clot/biphasic calcium phosphate microparticles composite

We recently demonstrated that blood clotted around biphasic calcium phosphate (BCP) microparticles constituted a composite biomaterial that could be used for bone defect filling. In addition, we showed that mononuclear cells, i.e. monocytes and lymphocytes, play a central role in the osteogenic effect of this biomaterial. Hypothesizing that osteoclast progenitors could participate to the pro-osteogenic effect of mononuclear cells we observed previously, we focus on this population through the study of mouse monocyte/macrophage cells (RAW264.7 cell line), as well as human pre-osteoclastic cells derived from mononuclear hematopoietic progenitor cells (monocytes-enriched fraction from peripheral blood). Using monocyte-derived osteoclast progenitors cultured within plasma clot/BCP microparticles composite, we aimed in the present report at the elucidation of transcriptional profiles of genes related to osteoclastogenesis and to bone remodelling. For both human and mouse monocytes, real-time PCR experiments demonstrated that plasma clot/BCP scaffold potentiated the expression of marker genes of the osteoclast differentiation such as Nfactc1, Jdp2, Fra2, Tracp and Ctsk. By contrast, Mmp9 was induced in mouse but not in human cells, and Ctr expression was down regulated for both species. In addition, for both mouse and human precursors, osteoclastic differentiation was associated with a strong stimulation of VegfC and Sdf1 genes expression. At last, using field-emission scanning electron microscopy analysis, we observed the interactions between human monocytes and BCP microparticles. As a whole, we demonstrated that plasma clot/BCP microparticles composite provided monocytes with a suitable microenvironment allowing their osteoclastic differentiation, together with the production of pro-angiogenic and chemoattractant factors.

Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement

Chitosan associated to various scaffolds has been shown to promote growth and mineral rich matrix deposition by osteoblasts in vitro, whereas its influence on osteoclast differentiation, which plays also a central role in bone remodeling, has never been described. The purpose of this study was to investigate the differentiation and activity of human preosteoclastic cells on calcium phosphate cement containing 2% chitosan (Cementek®/chitosan) compared to the Cementek® alone. Human primary osteoclast precursors were cultured directly on both biomaterials in the presence of rhM-CSF and rhRANK-L for 7 days. Using LIVE/DEAD fluorescent assay, tartrate-resistant acid phosphatase staining, scanning electron microscopy and quantitative RT-PCR, we demonstrated that incorporation of chitosan to Cementek® does not affect the proliferation and adhesion of preosteoclasts but inhibits the formation of TRACP positive cells and prevents the osteoclastic resorption of the composite biomaterial compared to Cementek® alone. This inhibitory effect of chitosan on osteoclast resorption activity should have important implications on bone formation and bone remodeling after in vivo implantation. Indeed, based on the positive results obtained in vivo by several investigators, one can suggest that this property of chitosan can be beneficial for bone regeneration.

HERG K+ Channels Activation during β1Integrin-mediated Adhesion to Fibronectin Induces an Up-regulation of αvβ3 Integrin in the Preosteoclastic Leukemia Cell Line FLG 29.1

Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.

In vitro expression of proalpha1(I) collagen mRNA by human pre-osteoclastic cells.

In vitro studies have demonstrated that the extracellular matrix modulates the cell phenotype. In the present study we have investigated in vitro proalpha1(I) collagen mRNA expression in a human pre-osteoclastic cell line (FLG 29.1 cells) in basal condition and after various stimuli. In addition, in order to evaluate the effect of cell-cell interactions on collagen type I mRNA expression, we have cultured the human pre-osteoclastic cells FLG 29.1 with either the human osteoblast-like cell line Saos-2 or the bovine bone endothelial cell line BBE. We showed that the FLG 29.1 cells express proal (I) collagen mRNA, whose expression is modulated by phorbol esters (TPA). Co-culturing FLG 29.1 cells with either Saos-2 or BBE cells induced decrease of proalpha1(I) collagen mRNA expression.

Fluid shear stress increases interleukin-11 expression in human osteoblast-like cells: its role in osteoclast induction.

It is unclear how mechanical stress influences bone cells. Mechanical stress causes fluid shear stress (FSS) in the bone. Osteoblast lineage cells are thought to sense FSS and regulate bone remodeling. We therefore investigated the effects of FSS on human osteoblast-like osteosarcoma cells: SaOS-2 cells in vitro. The conditioned medium of the SaOS-2 cells after 24 h of FSS (24 h-FSS CM) showed such osteoclastic phenotype inductions as significantly increasing the number of tartrate-resistant acid phosphatase (TRAP) positive multinuclear cells in rat bone marrow cells and TRAP-positive cells in human preosteoclastic cells: FLG 29.1 cells. An enzyme-linked immunosorbent assay showed interleukin-11 (IL-11) protein to increase 7-fold in the 24 h-FSS CM. A Northern analysis showed that IL-11 mRNA increased 4-fold in the SaOS-2 cells after 6 h-FSS; however, no IL-6 mRNA expression was detected. Furthermore, the anti-human IL-11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h-FSS CM. The IL-11 mRNA up-regulation in SaOS-2 cells by the 6 h-FSS was not inhibited by the anti-human transforming growth factor-beta1 antibody, but it was significantly inhibited by indomethacin. An enzymeimmunoassay showed prostaglandin E2 to increase 7-fold in the 1 h-FSS CM. These findings thus suggest that FSS induces osteoblasts to produce IL-11 (mediated by prostaglandins) and thus stimulates bone remodeling.

Expression of Src family kinases and their putative substrates in the human preosteoclastic cell line FLG 29.1.

Several lines of evidence suggest that the c-Src tyrosine kinase has a specific role in bone-resorbing osteoclasts. To investigate this further, we examined the expression of c-Src, its kinase family members, and their putative substrates in the human leukemia cell line FLG 29.1. Western blot analysis with specific antibodies against Src family members showed expression of Src, Fyn, and Lyn, lower levels of Yes and Hck, and the absence of Lck tyrosine kinase. During a 3-day treatment with phorbol 12-myristate, 13-acetate (PMA), which induces differentiation of FLG 29.1 cells toward an osteoclast-like phenotype, the levels of Src and Fyn increased and the levels of Lyn decreased. In a similar leukemia cell line, HL-60, Src protein was not constitutively expressed and not induced by PMA treatment, which leads to monocytic differentiation. PMA treatment of FLG 29.1 cells induced a strong increase in the expression of p120 Cbl and Pyk2 kinase, which are putative Src substrates. Pyk2 phosphorylation increased upon adherence of FLG 29.1 cells to fibronectin and to ST2 stromal cells. The expression of other Src substrates and interacting proteins, such as p120 Cas, p130 Cas, vinculin, Fak kinase, and the p85 phosphatidylinositol 3-kinase subunit either did not change or slightly increased during PMA treatment. The elevated total protein tyrosine phosphorylation in PMA-treated FLG 29.1 cells was abolished by herbimycin A, a Src inhibitor. These data are consistent with the proposed role of Src in the osteoclastic function and support the use of FLG 29.1 cells as a model to study Src substrates in the cells of the osteoclastic lineage.

Membrane binding sites and non-genomic effects of estrogen in cultured human pre-osteoclastic cells.

Besides functional estrogen receptors, the presence of signalling cell surface binding sites for 17beta-estradiol (17betaE2) has been reported in osteoblast- and osteoclast-like cells, suggesting that 17betaE2 may influence bone remodelling by a dual mechanism of action: to affect gene expression mediated by the nuclear activity of the steroid-receptor complex, and to initiate rapid responses triggered by a signal-generating receptor on the cell surface. Recently, we demonstrated that the human pre-osteoclastic cell line FLG 29.1 bears functional estrogen receptors. In this study we examined FLG 29.1 cells for the presence of cell surface binding sites for 17betaE2, and whether 17betaE2 could elicit cell signalling. Using a cell-impermeant and fluorescent estrogen conjugate, 17beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17betaE2. Stimulation of FLG 29.1 cells with low (1 nM) and high (1 microM) doses of 17betaE2 induced a prompt and significant (P < 0.05) increase of cellular pH, as measured in single cells using an image analysis system. In addition, both cAMP and cGMP were significantly increased by 17betaE2 with a dose-dependent response. Finally, a rapid increase of intracellular calcium ion concentration [Ca2+] was also induced by 1 nM 17betaE2, as measured in single cells using an image analysis system. Our findings strongly suggest a non-genomic action of 17betaE2 on osteoclast precursors.

Characterization and function of the receptor for IGF-I in human preosteoclastic cells.

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cell's migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.

Catecholamines modulate growth and differentiation of human preosteoclastic cells.

Using a clonal cell line of human osteoclast precursors (FLG 29.1 cells), that after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) show many functional characteristics of osteoclasts, we demonstrated that catecholamines act as inducers of osteoclast maturation in vitro and as stimulators of osteoclast activity via the binding to beta 2 adrenergic receptors. Scatchard analysis of 125I-labelled iodocyanopindolol to untreated (undifferentiated) or TPA-treated (differentiated) FLG 29.1 cells revealed the presence of a single high-affinity site with a Kd value around 24 pM and 8 pM respectively and with superimposable binding capacity (1.18 fmol/mg protein). Catecholamines increased in a dose-dependent fashion the intracellular cyclic AMP (cAMP) accumulation in both undifferentiated and TPA-treated FLG 29.1 cells. Pretreatment of untreated and TPA-treated FLG 29.1 cells with propranolol inhibited the catecholamine effect on cAMP accumulation, while pretreatment with clonidine had no effect. Catecholamines also reduced cell proliferation, increased tartrate-resistant acid phosphatase (TRAcP) activity, interleukin 6 (IL-6) production, multi-nuclearity and response to salmon calcitonin (sCT) in undifferentiated FLG 29.1 cells. In differentiated FLG 29.1 cells only IL-6 release was induced by catecholamine treatment. These findings support a potential role for catecholamines in modulating osteoclast differentiation and mature osteoclast activity.

Functional Estrogen Receptors in a Human Preosteoclastic Cell Line

Abstract The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.

Evidence for Bioeffects of LY 139478 on the Human Preosteoclastic Cell Line FLG 29.1

LY 139478, the hydrochloride salt of LY 117018, is a member of the nonsteroidal antiestrogens, benzothiophene derivatives, described to be full estrogen agonists in bone acting via an estrogen receptor-mediated mechanism. However, the cellular actions of these compounds on bone remodelling need to be established. To investigate the "in vitro" properties of LY 139478 on osteoclast precursors, the human pre-osteoclastic cell line FLG 29.1 was examined for evidence of bioeffects of this compound. Binding studies with tritiated 17β-estradiol (17βE2) demonstrated that the relative potency of LY 139478 in inhibiting estrogen binding to its receptor was equal to that of 17βE2.Significant (p<0.05) dose-dependent inhibition of cell growth was induced by LY 139478 at 10 nM, 100 nM and 1 μM. Calcitonin-induced cAMP accumicrolation was significantly increased by low (1 pM) and high (1 μM) doses of both 17βE2 and the compound with a dose-dependent response. Differently than estrogen, LY 139478 at high dose significantly reduced IL-6 release by these cells. In addition, pharmacological doses of both 17βE2 and LY 139478 activated apoptotic cell death. These findings show that the benzothiophene-derived LY 139478 acts directly on the human pre-osteoclastic cell line FLG 29.1 as an estrogen agonist.

Functional estrogen receptors in a human preosteoclastic cell line.

The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.

Interactions between ipriflavone and the estrogen receptor

Estrogen replacement therapy is effective in the prevention of postmenopausal osteoporosis, and a direct action of 17-beta-estradiol (17 beta E2) on osteoblastic and osteoclastic cells has been demonstrated. The inhibition of bone resorption by ipriflavone (IP), an isoflavone derivative devoid of estrogenic properties but active in potentiating the effects of estrogen on bone tissue, has been shown in in vitro and in vivo studies and confirmed by clinical data. To investigate the molecular mechanisms that underlie IP effect, we studied the possible interactions of IP and its four main in vivo metabolites (I, II, III, and V) with the estrogen receptor (ER) in the human preosteoclastic cell line FLG 29.1, whose growth and function are modulated by the compound. In parallel experiments, the human breast cancer cell line MCF7 was also analyzed. IP binding sites were demonstrated in the nuclear fraction of FLG 29.1 cells. 17 beta E2 and other steroid compounds failed to displace IP binding to intact FLG 29.1 cells. Similarly, IP and metabolites I, III, and V were not able to displace 17 beta E2 binding to intact MCF7 cells, whereas metabolite II showed an IC50 of 61 nM. 17 beta E2 binding to FLG 29.1 cells was increased after preincubation with metabolites I, III, and V. IP and its metabolites did not induce ER-dependent gene expression in FLG 29.1 and MCF7 cells transfected with a reporter gene and an estrogen response element (ERE).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and Bioeffects of Ipriflavone on a Human Preosteoclastic Cell Line

Ipriflavone, a synthetic isoflavone derivative, reduces bone resorption by inhibiting osteoclasts activity. In order to evaluate the role of Ipriflavone on osteoclast growth and differentiation, we tested Ipriflavone and its four "in vivo" main metabolites (Metabolites I, II, III, and V) on a clonal population of human osteoclast precursor cells (FLG 29.1). Pharmacological doses of Ipriflavone and Metabolite III were able to inhibit cell proliferation and interleukin 6 release. In co-cultures of FLG 29.1 cells and osteoblastic (Saos-2) cells Ipriflavone at 1 μM dose inhibited the adhesion of FLG 29.1 cells to the osteoblastic monolayer and reduced the immunocytochemical reaction of the vitronectin receptor. Binding studies with tritiated Ipriflavone showed the presence of a single specific binding site, with a Kd of about 70 nM and a binding capacity of 8 fmol/10 6 cells. These results demonstrate a direct effect of Ipriflavone and of Metabolite III on the human osteoclast precursor cell line FLG 29.1.