Site-Specific Fracture Healing: Comparison between Diaphysis and Metaphysis in the Mouse Long Bone.

The process of fracture healing varies depending upon internal and external factors, such as the fracture site, mode of injury, and mechanical environment. This review focuses on site-specific fracture healing, particularly diaphyseal and metaphyseal healing in mouse long bones. Diaphyseal fractures heal by forming the periosteal and medullary callus, whereas metaphyseal fractures heal by forming the medullary callus. Bone healing in ovariectomized mice is accompanied by a decrease in the medullary callus formation both in the diaphysis and metaphysis. Administration of estrogen after fracture significantly recovers the decrease in diaphyseal healing but fails to recover the metaphyseal healing. Thus, the two bones show different osteogenic potentials after fracture in ovariectomized mice. This difference may be attributed to the heterogeneity of the skeletal stem cells (SSCs)/osteoblast progenitors of the two bones. The Hox genes that specify the patterning of the mammalian skeleton during embryogenesis are upregulated during the diaphyseal healing. Hox genes positively regulate the differentiation of osteoblasts from SSCs in vitro. During bone grafting, the SSCs in the donor's bone express Hox with adaptability in the heterologous bone. These novel functions of the Hox genes are discussed herein with reference to the site-specificity of fracture healing.

Heterogeneity and Actin Cytoskeleton in Osteoclast and Macrophage Multinucleation.

Osteoclast signatures are determined by two transcriptional programs, the lineage-determining transcription pathway and the receptor activator of nuclear factor kappa-B ligand (RANKL)-dependent differentiation pathways. During differentiation, mononuclear precursors become multinucleated by cell fusion. Recently, live-cell imaging has revealed a high level of heterogeneity in osteoclast multinucleation. This heterogeneity includes the difference in the differentiation states and the mobility of the fusion precursors, as well as the mode of fusion among the fusion precursors with different numbers of nuclei. In particular, fusion partners often form morphologically distinct actin-based linkages that allow two cells to exchange lipids and proteins before membrane fusion. However, the origin of this heterogeneity remains elusive. On the other hand, osteoclast multinucleation is sensitive to the environmental cues. Such cues promote the reorganization of the actin cytoskeleton, especially the formation and transformation of the podosome, an actin-rich punctate adhesion. This review covers the heterogeneity of osteoclast multinucleation at the pre-fusion stage with reference to the environment-dependent signaling pathway responsible for reorganizing the actin cytoskeleton. Furthermore, we compare osteoclast multinucleation with macrophage fusion, which results in multinucleated giant macrophages.

The Sealing Zone in Osteoclasts: A Self-Organized Structure on the Bone.

Osteoclasts form a specialized cell-matrix adhesion structure, known as the "sealing zone", during bone resorption. The sealing zone is a dynamic actin-rich structure that defines the resorption area of the bone. The detailed dynamics and fine structure of the sealing zone have been elusive. Osteoclasts plated on glass do not form a sealing zone, but generate a separate supra-molecular structure called the "podosome belt". Podosomes are integrin-based adhesion complexes involved in matrix adhesion, cell migration, matrix degradation, and mechanosensing. Invadopodia, podosome-like protrusions in cancer cells, are involved in cell invasion into other tissues by promoting matrix degradation. Both podosomes and invadopodia exhibit actin pattern transitions during maturation. We previously found that Arp2/3-dependent actin flow occurs in all observed assembly patterns of podosomes in osteoclasts on glass. It is known that the actin wave in Dictyostelium cells exhibits a similar pattern transition in its evolution. Because of significant advances in our understanding regarding the mechanism of podosomes/invadopodia formation over the last decade, we revisited the structure and function of the sealing zone in this review, highlighting the possible involvement of self-organized actin waves in the organogenesis of the sealing zone.

Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.

Regulation of Osteoclast Multinucleation by the Actin Cytoskeleton Signaling Network.

Although it is known that osteoclasts are multinucleated cells that are responsible for bone resorption, the mechanism by which their size is regulated is unclear. We previously reported that an actin-rich superstructure, termed the zipper-like structure, specifically appears during the fusion of large osteoclast-like cells (OCLs). Actin cytoskeleton reorganization in osteoclasts is regulated by a signaling network that includes the macrophage colony-stimulating factor (M-CSF) receptor, a proto-oncogene, Src, and small GTPases. Here, we examined the role of actin reorganization in the multinucleation of OCLs differentiated from RAW 264.7 cells using various pharmacological agents. Jasplakinolide, which stabilizes actin stress fibers, induced the development of small OCLs, and the Src inhibitor SU6656 and the dynamin inhibitor dynasore impaired the maintenance of the podosome belt and the zipper-like structure. These inhibitors decreased the formation of large OCLs but increased the number of small OCLs. M-CSF is known to stimulate osteoclast fusion. M-CSF signaling via Src up-regulated Rac1 activity but down-regulated Rho activity. Rac1 and Rho localized to the center of the zipper-like structure. Rho activator II promoted the formation of small OCLs, whereas the Rho inhibitor Y27632 promoted the generation of large OCLs. These results suggest that the status of the actin cytoskeleton signaling network determines the size of OCLs during cell fusion.

The transient appearance of zipper-like actin superstructures during the fusion of osteoclasts.

Multinucleated osteoclasts are responsible for bone resorption. Hypermultinucleated osteoclasts are often observed in some bone-related diseases such as Paget's disease and cherubism. The cellular mechanics controlling the size of osteoclasts is poorly understood. We introduced EGFP-actin into RAW 264.7 cells to monitor actin dynamics during osteoclast differentiation. Before their terminal differentiation into osteoclasts, syncytia displayed two main types of actin assembly, podosome clusters and clusters of zipper-like structures. The zipper-like structures morphologically resembled the adhesion zippers found at the initial stage of cell-cell adhesion in keratinocytes. In the zipper-like structure, Arp3 and cortactin overlapped with the distribution of dense F-actin, whereas integrin ß3, paxillin and vinculin were localized to the periphery of the structure. The structure was negative for WGA-lectin staining and biotin labeling. The zipper-like structure broke down and transformed into a large actin ring, called a podosome belt. Syncytia containing clusters of zipper-like structures had more nuclei than those with podosome clusters. Differentiated osteoclasts with a podosome belt also formed the zipper-like structure at the cell contact site during cell fusion. The breakdown of the cell contact site resulted in the fusion of the podosome belts following plasma membrane fusion. Additionally, osteoclasts in mouse calvariae formed the zipper-like structure in the sealing zone. Therefore, we propose that the zipper-like actin superstructures might be involved in cell-cell interaction to achieve efficient multinucleation of osteoclasts. Understanding of the zipper-like structure might lead to selective therapeutics for bone diseases caused by hypermultinucleated osteoclasts.

Dual functions of cell-autonomous and non-cell-autonomous ADAM10 activity in granulopoiesis.

Previous studies have revealed various extrinsic stimuli and factors involved in the regulation of hematopoiesis. Among these, Notch-mediated signaling has been suggested to be critically involved in this process. Herein, we show that conditional inactivation of ADAM10, a membrane-bound protease with a crucial role in Notch signaling (S2 cleavage), results in myeloproliferative disorder (MPD) highlighted by severe splenomegaly and increased populations of myeloid cells and hematopoietic stem cells. Reciprocal transfer of bone marrow cells between wild-type and ADAM10 mutant mice revealed that ADAM10 activity in both hematopoietic and nonhematopoietic cells is involved in the development of MPD. Notably, we found that MPD caused by lack of ADAM10 in nonhematopoietic cells was mediated by G-CSF, whereas MPD caused by ADAM10-deficient hematopoietic cells was not. Taken together, the present findings reveal previously undescribed nonredundant roles of cell-autonomous and non-cell-autonomous ADAM10 activity in the maintenance of hematopoiesis.

The IRE1α-XBP1 pathway is essential for osteoblast differentiation through promoting transcription of Osterix.

During skeletal development, osteoblasts produce large amounts of extracellular matrix proteins and must therefore increase their secretory machinery to handle the deposition. The accumulation of unfolded protein in the endoplasmic reticulum induces an adoptive mechanism called the unfolded protein response (UPR). We show that one of the most crucial UPR mediators, inositol-requiring protein 1α (IRE1α), and its target transcription factor X-box binding protein 1 (XBP1), are essential for bone morphogenic protein 2-induced osteoblast differentiation. Furthermore, we identify Osterix (Osx, a transcription factor that is indispensible for bone formation) as a target gene of XBP1. The promoter region of the Osx gene encodes two potential binding motifs for XBP1, and we show that XBP1 binds to these regions. Thus, the IRE1α-XBP1 pathway is involved in osteoblast differentiation through promoting Osx transcription.

PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts.

Cytokine signaling via various transcription factors regulates receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from monocyte/macrophage lineage cells involved in propagation and resolution of inflammatory bone destruction. Protein inhibitor of activated STAT3 (PIAS3) was initially identified as a molecule that inhibits DNA binding of STAT3 and regulates many transcription factors through distinct mechanisms. To analyze PIAS3 function in osteoclasts in vivo, we have generated transgenic mice in which PIAS3 is specifically expressed in the osteoclast lineage using the tartrate-resistant acid phosphatase (TRAP) gene promoter. PIAS3 transgenic mice showed an osteopetrotic phenotype due to impairment of osteoclast differentiation. Overexpression of PIAS3 in RAW264.7 cells suppressed RANKL-induced osteoclastogenesis by inhibiting the expression of c-Fos and NFATc1. Interestingly, PIAS3 inhibits the transcriptional activity of microphthalmia-associated transcription factor (MITF) independent of sumoylation. Down-regulation of PIAS3 markedly enhances RANKL-mediated osteoclastogenesis in RAW264.7 cells. Furthermore, overexpression of PIAS3 in mouse primary osteoblast (POB), down-regulates RANKL expression induced by interleukin-6 (IL-6) cytokine family, and inhibits osteoclast formation from bone marrow macrophages (BMMs) in vitro coculture system. Down-regulation of PIAS3 leads to the accelerated expression of RANKL in POB stimulated with IL-6 and soluble IL-6 receptor (sIL-6R). Taken together, our results clearly indicate that PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts.

IL-27 abrogates receptor activator of NF-kappa B ligand-mediated osteoclastogenesis of human granulocyte-macrophage colony-forming unit cells through STAT1-dependent inhibition of c-Fos.

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Ralpha (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kappaB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-kappaB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.

Conversion of ES cells to columnar epithelia by hensin and to squamous epithelia by laminin.

Single-layered epithelia are the first differentiated cell types to develop in the embryo, with columnar and squamous types appearing immediately after blastocyst implantation. Here, we show that mouse embryonic stem cells seeded on hensin or laminin, but not fibronectin or collagen type IV, formed hemispheric epithelial structures whose outermost layer terminally differentiated to an epithelium that resembled the visceral endoderm. Hensin induced columnar epithelia, whereas laminin formed squamous epithelia. At the egg cylinder stage, the distal visceral endoderm is columnar, and these cells begin to migrate anteriorly to create the anterior visceral endoderm, which assumes a squamous shape. Hensin expression coincided with the dynamic appearance and disappearance of columnar cells at the egg cylinder stage of the embryo. These expression patterns, and the fact that hensin null embryos (and those already reported for laminin) die at the onset of egg cylinder formation, support the view that hensin and laminin are required for terminal differentiation of columnar and squamous epithelial phenotypes during early embryogenesis.

Decisive differences in the bone repair processes of the metaphysis and diaphysis in young mice.

Fractures are common traumatic injuries that mainly occur in the metaphyses of long bones such as the proximal humerus, distal radius, and proximal femur. However, most studies of fracture repair processes have focused on the diaphyseal region. In this study, we compared the bone repair processes of the metaphysis and the diaphysis of the mouse tibia. Bone apertures were formed in the tibial metaphysis and diaphysis. At indicated times after surgery, samples were collected, and the healing process was investigated using micro-computed tomography, as well as histological, immunohistochemical, and mRNA expression analyses. In the metaphysis, cartilage formation was not detected on the periosteal side. The bone aperture was filled with newly formed bone produced from bone marrow at day 7. In the case of the diaphysis, cartilage was formed around the aperture at day 4 and sequentially replaced by bone on the periosteal side. The bone aperture was filled with newly formed bone at day 14. In the bone marrow, expression of the osteogenic markers such as alkaline phosphatase, osteocalcin, and type I collagen, appeared earlier with metaphyseal injury than with diaphyseal injury. The mRNA expression of chondrogenesis markers was markedly upregulated in the diaphysis compared with that in the metaphysis on the periosteal side. These results indicate differences in the bone repair processes of the two regions, suggesting functional heterogeneity of the periosteum and bone marrow mesenchymal cells in response to bone fractures.

Unfolding of the myosin head by purealin in glycerol.

Purealin is a small bioactive compound obtained from the marine sponge. The compound modulates various types of ATPase activity of myosin from skeletal muscle, cardiac muscle, and smooth muscle. To elucidate the structural basis of these effects of purealin on myosin ATPases, we examined the effect of purealin on the conformation of skeletal muscle myosin in aqueous solution and in glycerol. Analysis of the circular dichroism spectrum of subfragment 1, a single-headed fragment of myosin, revealed that in 10% glycerol purealin decreased the ß-sheet content of S1, but in aqueous solution it had little effect on the secondary structure of S1. A myosin monomer conforms to two pear-like globular heads attached to a long tail. Electron microscopy observations with rotary shadowing revealed that purealin unfolded each globular head to an extended single strand. The tips of the unfolded strand bound each other and formed a ring in one molecule. These results suggest that binding of purealin affects the critical parameters of myosin folding.

Symmetrical retrograde actin flow in the actin fusion structure is involved in osteoclast fusion.

The aim of this study was to elucidate the role of the zipper-like structure (ZLS), a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3). Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

Extraction of nobiletin from Citrus Unshiu peels by supercritical fluid and its CRE-mediated transcriptional activity.

BACKGROUND: Polymethoxyflavone (PMF) is one of bioactive compounds in Citrus Unshiu and included mainly in the peels rather than the fruits, seeds and leaves. HYPOTHESIS/PURPOSE: Supercritical CO2 extraction is one candidate for selective extraction of polymethoxyflavone and in this study, supercritical CO2 extraction with/without ethanol entrainer from Citrus Unshiu peels was examined at a temperature of 333K and a pressure of 30MPa. METHODS: CRE (cyclic AMP response element)-mediated transcriptional assay was examined by using the extracts from supercritical fluid extraction. RESULTS: The results showed that extracts including nobiletin increased with increasing ethanol concentration in supercritical CO2 and the elapsed extraction time. Extracts at ethanol concentration of 5 mol% showed high CRE-mediated transcription activity. This can be caused by activity of the extract including nobiletin in addition to the other methoxylated flavonoid species such as tangeretin. Extracts at ethanol concentration of 50% showed the highest CRE-mediated transcription activity, which can be attributed to flavonoid glycoside such as hesperidin. From our investigations, flavonoid glycoside can be one of promoters of CRE-mediated transcription activity.

The expression of embryonic globin mRNA in a severely anemic mouse model induced by treatment with nitrogen-containing bisphosphonate.

BACKGROUND: Mammalian erythropoiesis can be divided into two distinct types, primitive and definitive, in which new cells are derived from the yolk sac and hematopoietic stem cells, respectively. Primitive erythropoiesis occurs within a restricted period during embryogenesis. Primitive erythrocytes remain nucleated, and their hemoglobins are different from those in definitive erythrocytes. Embryonic type hemoglobin is expressed in adult animals under genetically abnormal condition, but its later expression has not been reported in genetically normal adult animals, even under anemic conditions. We previously reported that injecting animals with nitrogen-containing bisphosphonate (NBP) decreased erythropoiesis in bone marrow (BM). Here, we induced severe anemia in a mouse model by injecting NBP injection in combination with phenylhydrazine (PHZ), and then we analyzed erythropoiesis and the levels of different types of hemoglobin. METHODS: Splenectomized mice were treated with NBP to inhibit erythropoiesis in BM, and with PHZ to induce hemolytic anemia. We analyzed hematopoietic sites and peripheral blood using morphological and molecular biological methods. RESULTS: Combined treatment of splenectomized mice with NBP and PHZ induced critical anemia compared to treatment with PHZ alone, and numerous nucleated erythrocytes appeared in the peripheral blood. In the BM, immature CD71-positive erythroblasts were increased, and extramedullary erythropoiesis occurred in the liver. Furthermore, embryonic type globin mRNA was detected in both the BM and the liver. In peripheral blood, spots that did not correspond to control hemoglobin were observed in 2D electrophoresis. ChIP analyses showed that KLF1 and KLF2 bind to the promoter regions of ß-like globin. Wine-colored capsuled structures were unexpectedly observed in the abdominal cavity, and active erythropoiesis was also observed in these structures. CONCLUSION: These results indicate that primitive erythropoiesis occurs in adult mice to rescue critical anemia because primitive erythropoiesis does not require macrophages as stroma whereas macrophages play a pivotal role in definitive erythropoiesis even outside the medulla. The cells expressing embryonic hemoglobin in this study were similar to primitive erythrocytes, indicating the possibility that yolk sac-derived primitive erythroid cells may persist into adulthood in mice.

Precursors linked via the zipper-like structure or the filopodium during the secondary fusion of osteoclasts.

We previously reported the transient appearance of an actin superstructure, called the zipper-like structure, during the primary fusion (fusion of mononuclear precursors) and the secondary fusion (fusion of multinucleated cells) of osteoclasts. Here, we focus on the actin-based superstructures that link two precursor cells during the secondary fusion event. In one type of secondary fusion, the osteoclasts transformed the podosome belts into the zipper-like structure at the site of cell contact and the apposed plasma membranes in the zipper-like structure attached to each other via a discontinuous interface. In another type of secondary fusion, the osteoclasts used a filopodium-like protrusion that linked the two cells. Both types of cell fusion required a lag period between the adhesion of the cells and the fusion of cell bodies. Thus, the secondary fusion of osteoclasts uses actin-based superstructures for cell-cell interactions before the definitive fusion of the plasma membranes.

Inhibition of STAT1 accelerates bone fracture healing.

Skeletal fracture healing involves a variety of cellular and molecular events; however, the mechanisms behind these processes are not fully understood. In the current study, we investigated the potential involvement of the signal transducer and activator of transcription 1 (STAT1), a critical regulator for both osteoclastogenesis and osteoblast differentiation, in skeletal fracture healing. We used a fracture model and a cortical defect model in mice, and found that fracture callus remodeling and membranous ossification are highly accelerated in STAT1-deficient mice. Additionally, we found that STAT1 suppresses Osterix transcript levels and Osterix promoter activity in vitro, indicating the suppression of Osterix transcription as one of the mechanisms behind the inhibitory effect of STAT1 on osteoblast differentiation. Furthermore, we found that fludarabine, a potent STAT1 inhibitor, significantly increases bone formation in a heterotopic ossification model. These results reveal previously unknown functions of STAT1 in skeletal homeostasis and may have important clinical implications for the treatment of skeletal bone fracture.

Accelerated cartilage resorption by chondroclasts during bone fracture healing in osteoprotegerin-deficient mice.

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG), a decoy receptor of RANKL, maintain bone mass by regulating the differentiation of osteoclasts, which are bone-resorbing cells. Endochondral bone ossification and bone fracture healing involve cartilage resorption, a less well-understood process that is needed for replacement of cartilage by bone. Here we describe the role of OPG produced by chondrocytes in chondroclastogenesis. Fracture healing in OPG(-/-) mice showed faster union of the fractured bone, faster resorption of the cartilaginous callus, and an increased number of chondroclasts at the chondroosseous junctions compared with that in wild-type littermates. When a cultured pellet of OPG(-/-) chondrocytes was transplanted beneath the kidney capsule, the pellet recruited many chondroclasts. The pellet showed the ability to induce tartrate-resistant acid phosphatase-positive multinucleated cells from RAW 264.7 cells in vitro. Finally, OPG(-/-) chondrocytes (but not wild-type chondrocytes) cultured with spleen cells induced many tartrate-resistant acid phosphatase-positive multinucleated cells. The expression of RANKL and OPG in chondrocytes was regulated by several osteotropic factors including 1,25-dihydroxyvitamin D(3), PTHrP, IL-1alpha, and TNF-alpha. Thus, local OPG produced by chondrocytes probably controls cartilage resorption as a negative regulator for chondrocyte-dependent chondroclastogenesis.

Administration of cyclooxygenase-2 inhibitor reduces joint inflammation but exacerbates osteopenia in IL-1 alpha transgenic mice due to GM-CSF overproduction.

IL-1alpha transgenic (Tg) mice exhibit chronic inflammatory arthritis and subsequent osteopenia, with IL-1-induced GM-CSF playing an important role in the pathogenesis. This study analyzed the mechanisms underlying osteopenia in Tg mice, and the therapeutic effects of the cyclooxygenase-2 inhibitor celecoxib on such osteopenia. Inhibited osteoclast formation was observed in RANKL-treated bone marrow cell (BMC) cultures from Tg mice and coculture of Tg-derived BMCs and wild-type-derived primary osteoblasts (POBs). FACS analysis indicated that this inhibition was attributable to a decreased number of osteoclast precursors within Tg-derived BMCs. Moreover, in coculture of Tg-derived POBs and either Tg- or wild-type-derived BMCs, osteoclast formation was markedly inhibited because Tg-derived POBs produced abundant GM-CSF, known as a potent inhibitor of osteoclast differentiation. Histomorphometric analysis of Tg mice revealed that both bone formation and resorption were decreased, with bone formation decreased more prominently. Interestingly, administration of celecoxib resulted in further deterioration of osteopenia where bone formation was markedly suppressed, whereas bone resorption remained unchanged. These results were explained by our in vitro observation that celecoxib dose-dependently and dramatically decreased osteogenesis by Tg mouse-derived POBs in culture, whereas mRNA expressions of GM-CSF and M-CSF remained unchanged. Consequently, blockade of PGE(2) may exert positive effects on excessively enhanced bone resorption observed in inflammatory bone disease, whereas negative effects may occur mainly through reduced bone formation, when bone resorption is constitutively down-regulated as seen in Tg mice.