Hitoshi Amano

Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos.

Osteoclasts are specialized macrophages that resorb bone. Mice lacking the AP-1 component c-Fos are osteopetrotic because of a lack of osteoclast differentiation and show an increased number of macrophages. The nature of the critical function of c-Fos in osteoclast differentiation is not known. Microarray analysis revealed that Nfatc1, another key regulator of osteoclastogenesis, was down-regulated in Fos-/- osteoclast precursors. Chromatin immunoprecipitation assay showed that c-Fos bound to the Nfatc1 and Acp5 promoters in osteoclasts. In vitro promoter analyses identified nuclear factor of activated T-cells (NFAT)/AP-1 sites in the osteoclast-specific Acp5 and Calcr promoters. Moreover, in Fos-/- precursors gene transfer of an active form of NFAT restored transcription of osteoclast-specific genes in the presence of receptor activator of the NF-κB ligand (RANKL), rescuing bone resorption. In the absence of RANKL, however, Fos-/- precursors were insensitive to NFAT-induced osteoclastogenesis unlike wild-type precursors. These data indicate that lack of Nfatc1 expression is the cause of the differentiation block in Fos-/- osteoclast precursors and that transcriptional induction of Nfatc1 is a major function of c-Fos in osteoclast differentiation.

Importance of membrane- or matrix-associated forms of M-CSF and RANKL/ODF in osteoclastogenesis supported by SaOS-4/3 cells expressing recombinant PTH/PTHrP receptors.

SaOS‐4/3, a subclone of the human osteosarcoma cell line SaOS‐2, established by transfecting the human parathyroid hormone/parathyroid hormone‐related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast formation supported by SaOS‐4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony‐stimulating factor (M‐CSF). Expression of messenger RNAs (mRNAs) for receptor activator of NF‐κB ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane‐associated and secreted forms of M‐CSF by SaOS‐4/3 cells was up‐regulated in response to PTH. SaOS‐4/3 cells constitutively expressed OPG mRNA, expression of which was down‐regulated by PTH. To elucidate the mechanism of PTH‐induced osteoclastogenesis, SaOS‐4/3 cells were spot‐cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture was treated for 6 days with both PTH and M‐CSF, osteoclasts were induced exclusively inside the colony of SaOS‐4/3 cells. Osteoclasts were formed both inside and outside the colony of SaOS‐4/3 cells in coculture treated with a soluble form of RANKL/ODF (sRANKL/sODF) in the presence of M‐CSF. When the spot coculture was treated with sRANKL/sODF, osteoclasts were formed only inside the colony of SaOS‐4/3 cells. Adding M‐CSF alone failed to support osteoclast formation in the spot coculture. PTH‐induced osteoclast formation occurring inside the colony of SaOS‐4/3 cells was not affected by the concentration of M‐CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS‐4/3 cells. These findings suggest that the up‐regulation of RANKL/ODF expression is an essential step for PTH‐induced osteoclastogenesis, and membrane‐ or matrix‐associated forms of both M‐CSF and RANKL/ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells.

TNFα mediates the skeletal effects of thyroid-stimulating hormone

We have shown recently that by acting on the thyroid-stimulating hormone (TSH) receptor (TSHR), TSH negatively regulates osteoclast differentiation. Both heterozygotic and homozygotic TSHR null mice are osteopenic with evidence of enhanced osteoclast differentiation. Here, we report that the accompanying elevation of TNFα, an osteoclastogenic cytokine, causes the increased osteoclast differentiation. This enhancement in TSHR−/− and TSHR+/− mice is abrogated in compound TSHR−/−/TNFα−/− and TSHR+/−/TNFα+/− mice, respectively. In parallel studies, we find that TSH directly inhibits TNFα production, reduces the number of TNFα-producing osteoclast precursors, and attenuates the induction of TNFα expression by IL-1, TNFα, and receptor activator of NF-κB ligand. TSH also suppresses osteoclast formation in murine macrophages and RAW-C3 cells. The suppression is more profound in cells that overexpress the TSHR than those transfected with empty vector. The overexpression of ligand-independent, constitutively active TSHR abrogates osteoclast formation even under basal conditions and in the absence of TSH. Finally, IL-1/TNFα and receptor activator of NF-κB ligand fail to stimulate AP-1 and NF-κB binding to DNA in cells transfected with TSHR or constitutively active TSHR. The results suggest that TNFα is the critical cytokine mediating the downstream antiresorptive effects of TSH on the skeleton.

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.

Regulatory mechanisms and physiological relevance of a voltage-gated H+ channel in murine osteoclasts: phorbol myristate acetate induces cell acidosis and the channel activation.

The voltage‐gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis.

Negative feedback loop in the Bim-caspase-3 axis regulating apoptosis and activity of osteoclasts.

Proapoptotic Bcl‐2 family member Bim plays an essential role in the osteoclast apoptosis and is degraded through ubiquitin/proteasome pathways in a caspase‐3–dependent manner. This negative feedback loop in the Bim–caspase‐3 axis is important for regulating the survival and activity of osteoclasts.

Reduced Expression of Thrombospondins and Craniofacial Dysmorphism in Mice Overexpressing Fra1

Fra1 transgenic (Tg) mice develop osteosclerosis and exhibit altered expression of bone matrix proteins. We found that expression of Thbs1 and Thbs2 was reduced in Fra1 Tg osteoblasts. Fra1 Tg and non‐osteosclerotic Thbs1−/−Thbs2−/− mice share an edge‐to‐edge bite. Therefore, reduced expression of thrombospondins may contribute to craniofacial dysmorphism independently of osteosclerosis.

Regulation of Osteoclast Apoptosis by Bcl-2 Family Protein Bim and Caspase-3

Apopotosis of osteoclasts is regulated by the Bcl-2 family protein Bim. Bim is degraded in the course of osteoclast apoptosis, which is regulated by Caspase-3. Osteoclasts generated from caspase-3 -/- mice exhibited a shorter life span and a higher bone-resorbing activity than those generated from normal littermates. These results suggest the important role of Caspase-3-Bim axis in regulating both apoptosis and activation of osteoclasts.

Elderly women with oral exostoses had higher bone mineral density.

Abstract. We examined the relationship between two opposite phenomena in elderly bone, bone loss (osteoporosis) and excessive bone formation (oral exostosis). We recruited, randomly, 44 female subjects without any conditions known to affect bone metabolism. The subjects were examined for exostosis, and bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. The Z score of the BMD was calculated; this is the deviation from the weight-adjusted average BMD of each age. Subjects with palatal tori (n = 15) had a higher femoral BMD than controls (n = 14) (0.580 ± 0.213 vs −0.271 ± 0.182; P = 0.0054). The subjects with palatal tori (n = 12) also had a higher BMD than controls (n = 12) at the radius (0.417 ± 0.235 vs −0.533 ± 0.294; P = 0.0194). In addition, subjects with mandibular tori (n = 13) had a higher femoral BMD than controls (n = 14) (0.569 ± 0.242 vs −0.271 ± 0.182; P = 0.0097). These results suggest that some common mechanisms are involved in the elevation of skeletal BMD and the occurrence of oral exostoses.

Diclofenac Sodium Inhibits NFκB Transcription in Osteoclasts

A non-steroidal anti-inflammatory drug, diclofenac, acts efficiently against inflammation; however, down-regulation of diclofenac on bone remodeling has raised concerns. The inhibitory mechanisms of diclofenac are poorly understood. We hypothesized that diclofenac down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFκB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrateresistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner. Pit formation assay revealed the abolition of osteoclastic bone resorption; levels of cathepsin K transcripts, an osteoclastic resorption marker, were down-regulated time-dependently. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFκB and phosphorylated NFκB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFκB reduction, which regulates osteoclast differentiation and activation.