Differentiation Of Osteoclast Progenitors Research Articles

Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption

ABSTRACT Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for treating bone loss diseases because excessive osteoclastic bone resorption leads to the development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone-resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti-osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases.

Overexpression of miRNA-22-3p attenuates osteoporosis by targeting MAPK14.

Osteoporosis (OP) results from an imbalance between bone formation, which is regulated by osteoblasts, and bone resorption, which is mediated by osteoclasts. MicroRNA-22-3p (miR-22-3p) expression is decreased during the process of osteoclast differentiation and p38α mitogen-activated protein kinase (MAPK)14 promotes the proliferation and differentiation of osteoclast progenitors. However, whether miR-22-3p could target MAPK14 to regulate the progression of OP remains unknown, which was the aim of the present study. CD14+ PBMCs were used for the establishment of osteoclastic differentiation in vitro. In the present study, reverse transcription quantitative PCR was used to determine the mRNA expression of MAPK14, tartrate resistant acid phosphatase (TRAP), nuclear factor of activated T-cells (NFATC1) and cathepsin K (CTSK). Western blotting was applied to determine the protein expression of MAPK14, TRAP, NFATC1, CTSK, p-p65 and p65. Dual luciferase reporter assay was applied to confirm the relation between miR-22-3p and MAPK14. Cell Counting Kit-8 assay and flow cytometry assays were used to determine the cell proliferation and cell apoptosis, respectively. The results demonstrated that miR-22-3p expression was lower while MAPK14 expression was higher in the serum from patients with OP compared with healthy volunteers. Furthermore, miR-22-3p expression was negatively correlated with MAPK14 expression in patients with OP. In addition, miR-22-3p expression was decreased and MAPK14 expression was increased during the progression of CD14+peripheral blood mononuclear cells (PBMCs) osteoclastic differentiation in a time-dependent manner. Furthermore, miR-22-3p inhibited the proliferation and differentiation and promoted the apoptosis of CD14+PBMCs by targeting MAPK14. In summary, the findings from the present study suggested that miR-22-3p may serve a potential therapeutic role in patients with OP.

RANKL, Ephrin-Eph and Wnt10b are key intercellular communication molecules regulating bone remodeling in autologous transplanted goldfish scales

This study aimed to investigate the precise data of gene expression, functions, and chronological relationships amongst communication molecules involved in the bone remodeling process with an in vivo model using autologous transplanted scales of goldfish. Autotransplantation of methanol-fixed cell-free scales triggers scale resorption and regeneration, as well as helps elucidate the process of bone remodeling. We investigated osteoclastic markers, osteoblastic markers, and gene expressions of communicating molecules (RANKL, ephrinB2, EphB4, EphA4, Wnt10b) by qPCR, in situ hybridization for Wnt10b, and immunohistochemistry for EphrinB2 and EphA4 proteins to elucidate the bone remodeling process. Furthermore, functional inhibition experiments for the signaling of ephrinB2/Eph, ephrin/EphA4, and Wnt10b using specific antibodies, revealed that these proteins are involved in key signaling pathways promoting normal bone remodeling. Our data suggests that the remodeling process comprises of two successive phases. In the first absorption phase, differentiation of osteoclast progenitors by RANKL is followed by the bone absorption by mature, active osteoclasts, with the simultaneous induction of osteoblast progenitors by multinucleated osteoclast-derived Wnt10b, and proliferation of osteoblast precursors by ehprinB2/EphB4 signaling. Subsequently, during the second formation phase, termination of bone resorption by synergistic cooperation occurs, with downregulation of RANKL expression in activated osteoblasts and Ephrin/EphA4-mediated mutual inhibition between neighboring multinucleated osteoclasts, along with simultaneous activation of osteoblasts via forward and reverse EphrinB2/EphB4 signaling between neighboring osteoblasts. In addition, the present study shows that autologous transplantation of methanol-fixed cell-free scale is an ideal in vivo model to study bone remodeling.

P38\u03b1 MAPK regulates proliferation and differentiation of osteoclast progenitors and bone remodeling in an aging-dependent manner

Bone mass is determined by the balance between bone formation, carried out by mesenchymal stem cell-derived osteoblasts, and bone resorption, carried out by monocyte-derived osteoclasts. Here we investigated the potential roles of p38 MAPKs, which are activated by growth factors and cytokines including RANKL and BMPs, in osteoclastogenesis and bone resorption by ablating p38α MAPK in LysM+monocytes. p38α deficiency promoted monocyte proliferation but regulated monocyte osteoclastic differentiation in a cell-density dependent manner, with proliferating p38α−/− cultures showing increased differentiation. While young mutant mice showed minor increase in bone mass, 6-month-old mutant mice developed osteoporosis, associated with an increase in osteoclastogenesis and bone resorption and an increase in the pool of monocytes. Moreover, monocyte-specific p38α ablation resulted in a decrease in bone formation and the number of bone marrow mesenchymal stem/stromal cells, likely due to decreased expression of PDGF-AA and BMP2. The expression of PDGF-AA and BMP2 was positively regulated by the p38 MAPK-Creb axis in osteoclasts, with the promoters of PDGF-AA and BMP2 having Creb binding sites. These findings uncovered the molecular mechanisms by which p38α MAPK regulates osteoclastogenesis and coordinates osteoclastogenesis and osteoblastogenesis.

Differentiation of osteoblast and osteoclast precursors on pure and silicon-substituted synthesized hydroxyapatites

Calcium phosphate-based materials should show excellent bone-bonding and cell-mediated resorption characteristics at the same time, in order to be employed for bone replacement. In this perspective, pure (HAp) and silicon-substituted hydroxyapatite (Si-HAp, 1.4% wt) porous cylinders were prepared starting from synthesized powders and polyethylene spheres used as porogens, and investigated as supports for osteoblast and osteoclast progenitor differentiation. A systematic and detailed biological characterization is reported, in terms of cell adhesion, viability, proliferation, differentiation and bioresorption, aimed at proposing a complete and reliable picture of bone cell in vitro behavior, comprehensive of both the osteogenesis and the bone resorption processes. In order to achieve this purpose, cytocompatibility, differentiation and gene expression by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were carried out using parietal bone-derived pre-osteoblasts obtained from neonatal mice and the bioresorption capability was assessed by seeding human peripheral blood monocytes, as osteoclast precursors. It resulted that both pure and Si-substituted HAps were able to promote differentiation of precursor cells in mature osteoblasts and osteoclasts. In particular, the Si-HAps enhanced the pre-osteoblast proliferation and showed higher osteoclast-mediated bioresorption capability, as supported by the presence of larger and more numerous resorption lacunae, whereas HAps promoted a more robust cell differentiation in terms of both osteocalcin gene expression by qRT-PCR and cell morphological evaluation by SEM analysis.

Botulinum Toxin: A Novel Adjuvant for Giant Cell Tumor

To the Editor Giant cell tumor (GCT) is a locally aggressive, benign bone lesion that shows extensive bone destruction that often leads to pain and pathologic fracture. It usually occurs in the epiphysis and metaphysis of bone near the joint. The destruction of subchondral bone impairs joint function. In rare instances, the tumor can spread to the lungs or can present as multifocal disease. On histologic examination, the tumor shows a mixture of cells consisting of osteoclastlike multinucleated giant cells, mononuclear cells, and stromal cells. This unique composition of different cell types has been the subject of the study of osteoclast biology. The preferred treatment options include surgical treatment such as curettage, in conjunction with chemical or physical adjuvant therapy, or extensive resection followed by major reconstructive surgery. The outcome after surgical resection has been affected negatively by tumor recurrence and additional bone destruction. Many topical adjuvant therapies such as phenol, hydrogen peroxide, or liquid nitrogen have been used in an attempt to decrease local recurrence by inducing physical or chemical damage to the remaining GCT cells after curettage, but a specific adjuvant therapy that directly targets osteoclasts and GCT cells is needed. In GCT, the mesenchymal stromal cells are the neoplastic cells and induce recruitment and formation of osteoclasts. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic preosteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). RANKL is an osteoclastogenesis factor released by osteoblasts, stromal cells, and activated T cells. RANK is a receptor that is present on the cell membrane of osteoclasts, monocytes, and osteoblasts. Studies have shown the role ofRANKL inGCT as evidenced by the inhibitory effect of osteoprotegerin (a decoy receptor of RANKL) on RANKL-mediated tumoral osteoclastogenesis. Cell adhesion of osteoblasts and osteoclastic precursors of hematopoietic origin is a prerequisite for osteoclast maturation. Several studies have demonstrated that interaction of RANKL on osteoblast and RANK on osteoclast precursor provides an essential signal to osteoclast precursors for their maturation into resorbing cells. b1 integrin/focal adhesion kinase (FAK)-mediated signaling is involved in intercellular adhesion molecule 1 (ICAM-1) and RANKL induction of osteoblasts and affects cellular adhesion between osteoblasts and osteoclast precursors through the ICAM-1 andRANKL/RANKpathways and leads to differentiation of osteoclast progenitors to osteoclasts. Therefore, b1 integrin/FAK-mediated signaling is involved in ICAM-1 and RANKL induction of osteoblasts. Small GTPaseRho signaling is involved in b1 integrin-mediated upregulation of ICAM-1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation. Clostridium botulinum, producing two classes of enzymes that have very specific protein targets, the neurotoxin A-G and the ADP-ribosyltransferases C2, C3 bot 1, and C3 bot 2. C2 and C3 bot are part of a larger family of ADP-ribosylating toxins, including diphtheria toxin and cholera toxin, which cleave Published online December 29, 2007. Address correspondence and reprint requests to: Hamid Namazi; E-mail: namazih@sums.ac.ir

Inhibitory activities of plastoquinones and chromene derivative from a brown alga Sagassum micracanthum on bone resorption.

We evaluated inhibitory effects of plastoquinones (1, 2) and chromene derivative (3) from the methanol extract of Sargassum micracanthum on the differentiation of osteoclast progenitors into osteoclast-like cells (OCLs), bone-resorbing activity and the survival of OCLs. When OCL formation was investigated using osteoclast progenitor cells obtained from macrophage-colony stimulating factor (M-CSF)-treated mouse bone marrow, 1-3 inhibited dose-dependently OCL formation at the concentrations of 3-10 muM, the order of inhibitory potency being 1>3>2. In addition, they suppressed dose-dependently the pit formation induced by OCL on dentine slices, the order of inhibitory potency being 1>3>2. The survival of OCLs was inhibited by about 35, 45 and 60% in the presence of 6 muM of 2, 3 and 1, respectively. These results suggest that the inhibitory effects of the three compounds on the differentiation, pit formation and the survival of OCLs might contribute to the suppression of bone resorption.

Mechanisms involved in bone resorption.

Osteoclasts, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. These osteoclasts are derived from hemopoietic progenitors of the monocyte-macrophage lineage. Osteoblasts or bone marrow-derived stromal cells are involved in osteoclastogenesis through a mechanism involving cell-to-cell contact with osteoclast progenitors. Experiments on the osteopetrotic op/op mouse model have established that a product of osteoblasts, macrophage colony-stimulating factor (M-CSF), regulates differentiation of osteoclast progenitors into osteoclasts. Recent discovery of osteoclast differentiation factor (ODF)/receptor activator of NF-kappa B ligand (RANKL) allowed elucidation of the precise mechanism by which osteoblasts regulate osteoclastic bone resorption. Treatment of osteoblasts with bone-resorbing factors up-regulated expression of RANKL mRNA. In contrast, TNF alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the RANKL system. IL-1 also directly acts on mature osteoclasts as a potentiator of osteoclast activation. In addition, TGF-beta super family members, such as bone morphogenetic proteins (BMPs) strikingly enhanced osteoclast differentiation from their progenitors and survival of mature osteoclasts induced by RANKL. These results suggest that BMP-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and function.

Colony-stimulating factor-1 stimulates the fusion process in osteoclasts.

Colony-stimulating factor-1 (CSF-1), also called macrophage colony-stimulating factor, is required for growth, differentiation, activation, and survival of cells of the mononuclear phagocytic system. This cytokine has been shown to be essential for osteoclast development as well as for inducing both proliferation and differentiation of osteoclast progenitors. It also sustains survival of mature osteoclasts and stimulates spreading and migration of these cells. In the present in vitro study, the formation of large tartrate-resistant acid phosphatase (TRAP)-positive cells with a high number of nuclei was observed when osteoclasts isolated from rat long bones were incubated with CSF-1. These large cells, cultured on plastic, bind calcitonin and form F-actin along the edges of the cells. Fusion to such large TRAP-positive multinucleated cells in the presence of CSF-1 and the formation of pits were also observed on dentine slices. Quantitative data obtained from cultures on plastic demonstrated that the number of osteoclasts slightly increased in the course of 72 h in the presence of 250 pM CSF-1, whereas it decreased rapidly after 24 h in the absence of CSF-1, which confirms that this cytokine is required for the survival of osteoclasts. The number of nuclei per osteoclast was maximal after 16 h of incubation with CSF-1, namely twice the value found in the absence of CSF-1. The maximal effect of the cytokine on the fusion process was observed at a concentration of 250 pM. A calculation of the medians of the average frequency of nuclei distribution per osteoclast resulted in four nuclei per osteoclast in the absence and six in the presence of CSF-1. Genistein and herbimycin A, inhibitors of tyrosine kinases, inhibited the fusion induced by CSF-1. The data suggest that CSF-1 induces osteoclast fusion and that tyrosine kinase(s) are involved in this process. The fusion process may continue throughout the entire life of an osteoclast.

Effect of stem cell factor, interleukin-6, nitric oxide and transforming growth factor-beta on the osteoclast differentiation induced by 1 alpha,25-(OH)2D3 in primary murine bone marrow cultures.

Osteotropic hormones and cytokines are involved in the differentiation of osteoclast progenitors from haematopoietic stem cells to multinucleated osteoclasts which mediate bone resorption. Stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta are implicated in the regulation of bone resorption by osteoclast. We test whether stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta affect the generation of osteoclast-like multi-nucleated cells induced by 1 alpha,25-(OH)2D3. 1 alpha,25-(OH)2D3 increase the generation of osteoclast-like cells retaining osteoclast characteristics including multinuclearity and positive staining for tartrate-resistant acid phosphatase. Combined treatment of stem cell factor with interleukin-6 synergistically potentiates the ability of 1 alpha,25-(OH)2D3 to generate tartrate-resistant acid phosphatase-positive multinucleated cells. However, either stem cell factor or interleukin-6 alone does not induce the generation of tartrate-resistant acid phosphatase-positive multinucleated cells. Transforming growth factor-beta produces a biphasic effect on osteoclast generation induced by 1 alpha,25-(OH)2D3. Transforming growth factor-beta stimulates osteoclast generation at low concentration (0.1 ng/ml) whereas it suppresses the formation of osteoclast-like cell at higher concentration (1 ng/ml). Sodium nitroprusside, a donor of nitric oxide, almost completely inhibits the generation of 1 alpha,25-(OH)2D3-induced osteoclast at high concentration (100 microM), but it significantly enhances the osteoclast generation at low concentrations (3 microM). These results suggest that stem cell factor, interleukin-6, transforming growth factor-beta, and nitric oxide interact with 1 alpha,25-(OH)2D3 to modulate the differentiation of hematopoietic precursors toward committed osteoclast precursors.

The Cell-surface Form of Colony-stimulating Factor-1 Is Regulated by Osteotropic Agents and Supports Formation of Multinucleated Osteoclast-like Cells

Colony-stimulating factor-1 (CSF-1) is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. CSF-1 is synthesized as a soluble or cell-surface protein. It is unclear, however, whether human osteoblasts express both molecular forms of CSF-1, and whether these isoforms can independently mediate osteoclastogenesis. In the present study, using a combination of quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western immunoblot analysis, we have demonstrated that human osteoblast-like cells as well as primary human osteoblasts express the cell-surface form of CSF-1 both constitutively and in response to parathyroid hormone and tumor necrosis factor. Furthermore, using an in vitro co-culture system, we have shown that cell-surface CSF-1 alone is sufficient to support osteoclast formation. These findings may be especially significant in view of evidence that direct cell-to-cell contact is critical for osteoclast formation, and suggest that differential regulation of expression of the CSF-1 isoforms may influence osteoclast function modulated by osteotropic hormones.

Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.

Bone biochemistry and physiology from the perspectives of the vitamin D endocrine system.

Vitamin D plays an indispensable role in the dual processes of bone formation (mediated by osteoblasts) and bone resorption (mediated by osteoclasts). More recently, researchers have confirmed the existence of a vitamin D endocrine system, which is responsible for describing the "sphere of biological influence" of vitamin D3. In that system, the kidney serves as the endocrine gland that produces 1,25-dihydroxyvitamin D3. This hormonally active form of vitamin D3 generates many, if not all, of the biologic responses attributed to the parent vitamin D3, including its role in bone formation and bone resorption. In addition, 1,25-dihydroxyvitamin D3 is able to generate biologic responses via both genomic and nongenomic pathways. The classic nuclear receptor for 1,25-dihydroxyvitamin D3 is present in more than 30 target tissues. This paper reviews evidence for the critical role of 1,25-dihydroxyvitamin D3 in cell differentiation, particularly of hematopoietic cells, as well as in the generation of the bone resorptive cell--the osteoclast. In the past year, much evidence has been accumulated supporting the claim that 1,25-dihydroxyvitamin D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors are believed to be derived from the monocyte-macrophage lineage. However, the generation of new osteoclasts is modulated by osteoblastic stromal cells, which are one of the target cells for the nuclear actions of 1,25-dihydroxyvitamin D3.

Inductive effect of human recombinant IL-1 on differentiation of a macrophage-like tumor cell line.

We used the macrophage-like tumor cell line P388D1 to test whether interleukin-1 (IL-1) stimulate differentiation of osteoclast-like progenitors. Recombinant human interleukin-1 (rhIL-1) alpha inhit ited cell growth in a dose-dependent fashion. Incubation of the cells with rhIL-1 alpha resulted in adherence stimulation of non-specific esterase activity, and increased Fc receptor expression. These results suggest th possibility that IL-1 may be involved in the differentiation of osteoclast progenitors and thus may be an irr portant local factor in the mechanism of bone resorption.

Impaired recruitment and differentiation of osteoclast progenitors by osteocalcin-deplete bone implants

This is a report of an experimental system to study differentiation of bone-resorbing osteoclasts and demonstrates that osteocalcin, an extracellular bone-specific component, is necessary for the recruitment of osteoclast progenitor cells. The subcutaneous implantation of devitalized bone particles (BPs) elicits the recruitment and differentiation of osteoclasts that resorb the BPs. In a previous study, we showed by histomorphometric analysis that BPs that were deficient in osteocalcin were resorbed only 60% as well as normal BPs. In this study, the mechanism of this difference was investigated by measurements of recruitment, differentiation and activity of bone resorbing cells by normal and osteocalcin-deficient BP. Mononuclear cells were attracted to control BPs soon after implantation. In dramatic constrast, cellularity was depressed around osteocalcin-deficient BPs with very few mononuclear cells within the implant on day 5 (35% of control cellularity). In implants of normal BPs, tartrate-resistant acid phosphatase-positive multi-nucleated cells were evident by day 5; very few appeared in implants of osteocalcin-deplete BPs even by day 12. The amount of tartrate-resistant acid phosphatase activity in homogenates of the osteocalcin-deficient bone particle specimens not only lagged behind controls but never reached the maximum activity of control BP specimens. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and/or activation of cells for bone resorption.

Colony-stimulating factors regulate the development of multinucleated osteoclasts from recently replicated cells in vitro.

Osteoclasts mediate the process of bone resorption. However, little is known about the mechanisms that regulate the formation of either osteoclasts or osteoclast precursors. In contrast, colony-stimulating factors (CSFs) are well-known to regulate the formation of myeloid cells and their precursors. Because osteoclasts and myeloid cells may originate from a common stem cell, we examined the effects of two CSFs, granulocyte-macrophage CSF (GM-CSF) and interleukin 3 (IL-3), on bone resorption, osteoclast formation, and the incorporation of recently replicated nuclei into the osteoclasts of mouse bone cultures. CSFs had little effect on the formation rate of osteoclasts or their resorptive activity but significantly decreased the percentage of recently replicated osteoclast progenitor cell nuclei present in the osteoclasts of bones treated with parathyroid hormone. GM-CSF also increased the number of myeloid cells in the marrow space of the cultures and the percentage of these cells derived from recently replicated progenitors. These results demonstrate that GM-CSF and IL-3 can regulate the development of osteoclasts from recently replicated precursor cells in cultured fetal mouse long bones. However, the mechanisms by which CSFs influence osteoclast formation are difficult to determine from these studies because markers for the osteoclast progenitor and precursor do not exist. These data also provide evidence that the differentiation of osteoclast progenitors is regulated by different factors at different points in their ontogeny.

Synthesis of colony-stimulating factor (CSF) and differentiation-inducing factor (D-factor) by osteoblastic cells, clone MC3T3-E1

The role of osteoblasts in inducing the proliferation and differentiation of bone marrow cells was examined. Conditioned medium obtained from mouse osteoblastic cell (MC3T3-E1) cultures stimulated colony formation of mouse bone marrow cells (CSF) and differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells (D-factor). The CSF activity increased time dependently in parallel with the increase of alkaline phosphatase activity during the culturing of the MC3T3-E1 cells. The activity of the D-factor attained a maximum on days 12 – 15 and decreased thereafter. Both the CSF and the D-factor were eluted in a range of 25,000 to 67,000 daltons on gel filtration. The fraction containing both factors exhibited bone-resorbing activity. These results suggest that osteoblasts are involved in bone resorption at least in part by enhancing the proliferation and differentiation of osteoclast progenitors.