Yoshiyuki Hakeda

Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts

In bone development and regeneration, angiogenesis and bone/cartilage resorption are essential processes and are closely associated with each other, suggesting a common mediator for these two biological events. To address this interrelationship, we examined the effect of vascular endothelial growth factor (VEGF), the most critical growth factor for angiogenesis, on osteoclastic bone‐resorbing activity in a culture of highly purified rabbit mature osteoclasts. VEGF caused a dose‐ and time‐dependent increase in the area of bone resorption pits excavated by the isolated osteoclasts, partially by enhancing the survival of the cells. Two distinct VEGF receptors, KDR/Flk‐1 and Flt‐1, were detectable in osteoclasts at the gene and protein levels, and VEGF induced tyrosine phosphorylation of proteins in osteoclasts. Thus, osteoclastic function and angiogenesis are up‐regulated by a common mediator such as VEGF.

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.

Identification of osteopontin in isolated rabbit osteoclasts

Bone remodeling is a complex process coupling bone formation and resorption. Osteoblasts, the bone-forming cells, are known to produce various bone matrix proteins and cytokines; however, little is known about protein factors produced by osteoclasts or bone-resorbing cells. A method utilizing the high affinity of osteoclasts for tissue culture dishes was developed to isolate a large number of pure osteoclasts from rabbit long bones. A cDNA library was then constructed from these isolated osteoclasts, and differential cDNA screening was performed between osteoclasts and spleen cells. Two clones representing osteoclast-specific clones, named OC-1 and OC-2, were isolated. By Northern blot analysis, OC-1 was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts. OC-1 was found to encode osteopontin from its nucleotide sequence, and therefore, osteopontin may have other functions for osteoclastic bone resorption besides osteoclast attachment to bone.

Isolation of mouse and human cDNA clones encoding a protein expressed specifically in osteoblasts and brain tissues.

Using the differential hybridization screening method between osteoblastic and fibroblastic cells, a cDNA clone coding for an osteoblast specific protein, named OSF-1, consisting of 168 amino acid residues including a possible 32 amino acid long leader sequence, was isolated from murine osteoblastic cell line MC3T3-E1. The OSF-1 gene was shown by Northern blotting analysis to be expressed in mouse calvarial osteoblast-enriched cells and in mouse brain tissues, but not in thymus, spleen, kidney, liver, lung, testis or heart. The human counterpart was also found in cDNA libraries from human osteosarcoma cell line MG63 and normal brain tissues. DNA sequence analysis revealed four amino acid sequence differences between the mouse and human, of which only one is located in the mature protein. This extremely high sequence conservation suggests that OSF-1 plays a fundamental role in bone and brain functions.

Endogenous Production of TGF-β Is Essential for Osteoclastogenesis Induced by a Combination of Receptor Activator of NF-κB Ligand and Macrophage-Colony- Stimulating Factor

Differentiation of osteoclasts, the cells primarily responsible for bone resorption, is controlled by a variety of osteotropic hormones and cytokines. Of these factors, receptor activator of NF-κB (RANK) ligand (RANKL) has been recently cloned as an essential inducer of osteoclastogenesis in the presence of M-CSF. Here, we isolated a stroma-free population of monocyte/macrophage (M/Mφ)-like hemopoietic cells from mouse unfractionated bone cells that were capable of differentiating into mature osteoclasts by treatment with soluble RANKL (sRANKL) and M-CSF. However, the efficiency of osteoclast formation was low, suggesting the requirement for additional factors. The isolated M/Mφ-like hemopoietic cells expressed TGF-β and type I and II receptors of TGF-β. Therefore, we examined the effect of TGF-β on osteoclastogenesis. TGF-β with a combination of sRANKL and M-CSF promoted the differentiation of nearly all M/Mφ-like hemopoietic cells into cells of the osteoclast lineage. Neutralizing anti-TGF-β Ab abrogated the osteoclast generation. These TGF-β effects were also observed in cultures of unfractionated bone cells, and anti-TGF-β blocked the stimulatory effect of 1,25-dihydroxyvitamin D3. Translocation of NF-κB into nuclei induced by sRANKL in TGF-β-pretreated M/Mφ-like hemopoietic cells was greater than that in untreated cells, whereas TGF-β did not up-regulate the expression of RANK, the receptor of RANKL. Our findings suggest that TGF-β is an essential autocrine factor for osteoclastogenesis.

A simple method to assess osteoclast-mediated bone resorption using unfractionated bone cells

To determine osteoclastic bone resorption we established a simple assay system in which unfractionated cells obtained from femora of 13-day-old mice were cultured on a dentine slice and the number of osteoclasts and their induced pit area on the slices were measured. When the bone cells (1 x 10(5) cells/dentine slice) were cultured in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or human parathyroid hormone (hPTH) for 4 days, at which time newly-formed osteoclasts were not detected, the pit area was dose-dependently increased, being a 4.3- or 4.1-fold respective increase over the control at a 10(-8) M concentration of hormones. Chick calcitonin (cCT) inhibited the osteoclastic bone resorption induced by either of these hormones. cCT alone also suppressed the bone resorption by the cells (3 x 10(5) cells/dentine slice). These findings indicate that 1,25(OH)2D3 or hPTH may mainly activate pre-existing osteoclasts, resulting in increased bone resorption, and that cCT may suppress this osteoclastic activity. When 1,25(OH)2D3 or hPTH was added to the cells pre-cultured in factor-free medium for 6 days, at which time pre-existing osteoclasts had almost degenerated, new osteoclasts were formed, resulting in an increase in pit formation. Thus this system is a useful method which could more sensitively evaluate the effects of hormones or factors on osteoclast formation and activation than other previous systems.

Regulation of Receptor Activator of NF-κB Ligand-induced Osteoclastogenesis by Endogenous Interferon-β (INF-β) and Suppressors of Cytokine Signaling (SOCS) THE POSSIBLE COUNTERACTING ROLE OF SOCSs IN IFN-β-INHIBITED OSTEOCLAST FORMATION

Bone resorption and the immune system are correlated with each other, and both are controlled by a variety of common cytokines produced in the bone microenvironments. Among these immune mediators, the involvement of type I interferons (IFNs) in osteoclastic bone resorption remains unknown. In this study, we investigated the participation of IFN-β and suppressors of cytokine signaling (SOCS)-1 and -3 in osteoclastogenesis. Addition of exogenous IFN-β to osteoclast progenitors (bone-derived monocytes/macrophages) inhibited their differentiation toward osteoclasts induced by the receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor with/without transforming growth factor-β, where inhibition was associated with down-regulation of the gene expressions of molecules related to osteoclast differentiation. In addition, RANKL induced the expression of IFN-β; furthermore, neutralizing antibody against type I IFNs accelerated the osteoclast formation, indicating type I IFNs as potential intrinsic inhibitors. On the other hand, RANKL also induced the expression of SOCS-1 and -3, suppressors of the IFN signaling. Pretreatment with RANKL for a sufficient time for the induction of SOCSs attenuated phosphorylation of STAT-1 in response to IFN-β in osteoclast progenitors, causing a decrease in the binding activity of nuclear extracts toward the interferon-stimulated response element. mRNA levels of STAT-1, STAT-2, and IFN-stimulated gene factor-3γ, comprising IFN-stimulated gene factor-3, were not altered by RANKL. Thus, although the inhibitory cytokine such as IFN-β is produced in response to RANKL, the inhibition of osteoclastogenesis may be rescued by the induction of signaling suppressors such as SOCSs.

Dexamethasone Enhances Osteoclast Formation Synergistically with Transforming Growth Factor-β by Stimulating the Priming of Osteoclast Progenitors for Differentiation into Osteoclasts

Long-term administration of glucocorticoids (GCs) causes osteoporosis with a rapid and severe bone loss and with a slow and prolonged bone disruption. Although the involvement of GCs in osteoblastic proliferation and differentiation has been studied extensively, their direct action on osteoclasts is still controversial and not conclusive. In this study, we investigated the direct participation of GCs in osteoclastogenesis. Dexamethasone (Dex) at <10–8 m stimulated, but at >10–7 m depressed, receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation synergistically with transforming growth factor-β. The stimulatory action of Dex was restricted to the early phase of osteoclast differentiation and enhanced the priming of osteoclast progenitors (bone marrow-derived monocytes/macrophages) toward differentiation into cells of the osteoclast lineage. The osteoclast differentiation depending on RANKL requires the activation of NF-κB and AP-1, and the DNA binding of these transcription factors to their respective consensus cis-elements was enhanced by Dex, consistent with the stimulation of osteoclastogenesis. However, Dex did not affect the RANKL-induced signaling pathways such as the activation of IκB kinase followed by NF-κB nuclear translocation or the activation of JNK. On the other hand, Dex significantly decreased the endogenous production of interferon-β, and this cytokine depressed the RANKL-elicited DNA binding of NF-κB and AP-1, as well as osteoclast formation. Thus, the down-regulation of inhibitory cytokines such as interferon-β by Dex may allow the osteoclast progenitors to be freed from the suppression of osteoclastogenesis, resulting in an increased number of osteoclasts, as is observed in the early phase of GC-induced osteoporosis.

Direct and indirect actions of fibroblast growth factor-2 on osteoclastic bone resorption in cultures

Fibroblast growth factor 2 (FGF‐2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF‐2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF‐2 at 10−9 M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti‐inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF‐2 (10−8 M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX‐2) than COX‐1, suggesting the mediation of COX‐2 induction. COX‐2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF‐2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF‐2 in cells of osteoclast lineage. To further investigate the direct actions of FGF‐2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF‐2 (≥10−12 M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0‐fold at 10−11 M, and no further stimulation was observed at higher concentrations. However, FGF‐2 at 10−9 M − 10−8 M stimulated resorbed pit formation by unfractionated bone cells up to 9.7‐fold. NS‐398, a specific COX‐2 inhibitor, did not affect the FGF‐2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF‐2 at low concentrations (≥10−12 M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (≥10−9 M) it acts on osteoblastic cells to induce COX‐2 and stimulates bone resorption potently.

Prostaglandin E2 stimulates collagen and non-collagen protein synthesis and prolyl hydroxylase activity in osteoblastic clone MC3T3-E1 cells

We investigated the stimulative effect of prostaglandin E2 (PGE2) on an osteoblastic cell line, clone MC3T3-E1, in serum-free medium. PGE2 elevated collagen and non-collagen protein syntheses in a dose-related fashion up to 2 micrograms/ml, the maximal increases being 2- and 3-fold, respectively, over that in the control. Its stimulative effect was evident as early as 12 h. PGE2 slightly increased DNA content, but its effect was less than that on collagen and non-collagen protein syntheses. Moreover, PGE stimulated an increase in prolyl hydroxylase activity with a maximal effect at 1-2 micrograms/ml, the activity being 15-fold over that of the control. These results strongly indicate that PGE2 directly enhances total protein synthesis including that of collagen in osteoblasts in vitro, suggesting its direct effect on bone formation in vivo as well.