藤田 順之

The intervertebral disc (IVD) is composed of two avascular tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). IVDs is the largest avascular tissue in the human body, however, how these tissues are maintained without a blood supply is poorly understood. Here we show that vascular endothelial growth factor-A (VEGF-A) is highly expressed in NP and that VEGF-A plays a role in NP survival. High VEGF-A expression in NP was detected by microarray analysis, and NP was positive for the hypoxic probe pimonidazole and hypoxia-responsive genes. VEGF-A expression in NP was promoted by hypoxic conditions in vitro. NP cells also expressed the membrane-bound VEGF receptor-1 (VEGFR-1), and the number of apoptotic cells in cultured cell model of NP increased following treatment with VEGFR-1-Fc, which traps VEGF-A in NP. These results indicate that NP is a hypoxic tissue, and that VEGF-A functions in NP survival in an autocrine/paracrine manner.

Although osteoblasts express the angiogenic protein Angiopoietin 1 (Ang1), the role of Ang1 in bone formation remains largely unknown. Here we report that Ang1 overexpression in osteoblasts driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen promoter results in increased bone mass in vivo. In Ang1-transgenic mice (Ang1-Tg), bone volume and bone parameters increased significantly compared with wild-type littermates, although the Ang1 receptor, Tie2 was not expressed in osteoblasts. Tie2 is primarily expressed in vascular endothelial cells, and Ang1-Tie2 signaling is reportedly crucial for angiogenesis. We found that the number of vascular endothelial cells was significantly elevated in Ang1-Tg mice compared with that of wild-type littermates, an increase accompanied by increased alkaline-phosphatase activity, a marker of osteoblast activation. The number of osteoclasts in the bone of Ang1-Tg mice did not differ from wild-type littermates. These results indicate that angiogenesis induced by Ang1 expressed in osteoblasts is coupled with osteogenesis.

Bone resorption by osteoclasts stimulates bone formation by osteoblasts. To isolate osteoblastic factors coupled with osteoclast activity, we performed microarray and cluster analysis of 8 tissues including bone, and found that among 10,490 genes, osteomodulin (OMD), an extracellular matrix keratan sulfate proteoglycan, was simultaneously induced with osteoclast-specific markers such as MMP9 and Acp5. OMD expression was detected in osteoblasts and upregulated during osteoblast maturation. OMD expression in osteoblasts was also detected immunohistochemically using a specific antibody against OMD. The immunoreactivity against OMD decreased in op/op mice, which lack functional macrophage colony stimulating factor (M-CSF) and are therefore defective in osteoclast formation, when compared to wild-type littermates. OMD expression in op/op mice was upregulated by M-CSF treatment. Since the M-CSF receptor c-Fms was not expressed in osteoblasts, it is likely that OMD is an osteoblast maturation marker that is induced by osteoclast activity.

Chondrocyte hypertrophy during endochondral ossification is a well-controlled process in which proliferating chondrocytes stop proliferating and differentiate into hypertrophic chondrocytes, which then undergo apoptosis. Chondrocyte hypertrophy induces angiogenesis and mineralization. This step is crucial for the longitudinal growth and development of long bones, but what triggers the process is unknown. Reactive oxygen species (ROS) have been implicated in cellular damage; however, the physiological role of ROS in chondrogenesis is not well characterized. We demonstrate that increasing ROS levels induce chondrocyte hypertrophy. Elevated ROS levels are detected in hypertrophic chondrocytes. In vivo and in vitro treatment with N-acetyl cysteine, which enhances endogenous antioxidant levels and protects cells from oxidative stress, inhibits chondrocyte hypertrophy. In ataxia telangiectasia mutated (Atm)-deficient (Atm(-/-)) mice, ROS levels were elevated in chondrocytes of growth plates, accompanied by a proliferation defect and stimulation of chondrocyte hypertrophy. Decreased proliferation and excessive hypertrophy in Atm(-/-) mice were also rescued by antioxidant treatment. These findings indicate that ROS levels regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes.

UNLABELLED: DC-STAMP is essential for fusion of osteoclasts and foreign body giant cells; however, it is not known whether dc-stamp expression in these two cell types is differentially regulated. Here, we show that dc-stamp expression and cell-cell fusion are regulated in a cell type-specific manner. INTRODUCTION: The transcription factors c-Fos and NFATc1 cooperate to regulate osteoclast differentiation, whereas PU.1 and NF-kappaB are activated in macrophages and osteoclasts or in both cell types. Thus, we asked what role c-Fos, NFATc1, PU.1, and NF-kappaB played in regulating dendritic cell-specific transmembrane protein (dc-stamp) expression and fusion of osteoclasts and macrophage giant cells. MATERIALS AND METHODS: Transcriptional activation by c-Fos and NFATc1 was examined by dc-stamp promoter analysis. Multinuclear cell formation was analyzed in cells from c-Fos-deficient mice or in wildtype cells treated with the NFAT inhibitor FK506. The role of DC-STAMP in cell fusion was examined in vitro in a macrophage giant cell formation assay using DC-STAMP-deficient cells. Recruitment of c-Fos, NFATc1, PU.1, and NF-kappaB to the dc-stamp promoter in osteoclasts and macrophage giant cells was analyzed by chromatin-immunoprecipitation analysis. RESULTS: Both activator protein-1 (AP-1) and NFAT binding sites in the dc-stamp promoter were needed for dc-stamp expression after RANKL stimulation of osteoclasts. dc-stamp expression was induced in osteoclasts and macrophage giant cells, and cells from DC-STAMP-deficient mice failed to form either multinuclear osteoclasts or macrophage giant cells. In contrast, c-Fos is indispensable for dc-stamp expression and cell-cell fusion under conditions favoring in vitro and in vivo induction of osteoclasts but not macrophage giant cells. Consistently, an NFAT inhibitor suppressed multinuclear osteoclast formation but not macrophage giant cell formation. In addition, PU.1 and NF-kappaB binding sites were detected in the dc-stamp promoter, and both PU.1 and NF-kappaB were recruited to the dc-stamp promoter after granulocyte-macrophage colony stimulating factor (GM-CSF) + interleukin (IL)-4 stimulation. CONCLUSIONS: dc-stamp expression is regulated differently in osteoclasts and macrophage giant cells. c-Fos and NFATc1, both of which are essential for osteoclast differentiation, are needed for dc-stamp expression and cell-cell fusion in osteoclasts, but both factors are dispensable for giant cell formation by macrophages. Because PU.1 and NF-kappaB are recruited to the dc-stamp promoter after stimulation with GM-CSF + IL-4, dc-stamp transcription is regulated in a cell type-specific manner.

Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD.

Osteoclasts are bone-resorbing cells that play a pivotal role in bone remodeling. Osteoclasts form large multinuclear giant cells by fusion of mononuclear osteoclasts. How cell fusion is mediated, however, is unclear. We identify the dendritic cell-specific transmembrane protein (DC-STAMP), a putative seven-transmembrane protein, by a DNA subtraction screen between multinuclear osteoclasts and mononuclear macrophages. DC-STAMP is highly expressed in osteoclasts but not in macrophages. DC-STAMP-deficient mice were generated, and osteoclast cell fusion was completely abrogated in homozygotes despite normal expression of osteoclast markers and cytoskeletal structure. As osteoclast multinucleation was restored by retroviral introduction of DC-STAMP, loss of cell fusion was directly attributable to a lack of DC-STAMP. Defects in osteoclast multinucleation reduce bone-resorbing activity, leading to osteopetrosis. Similar to osteoclasts, foreign body giant cell formation by macrophage cell fusion was also completely abrogated in DC-STAMP-deficient mice. We have thus identified an essential regulator of osteoclast and macrophage cell fusion, DC-STAMP, and an essential role of osteoclast multinucleation in bone homeostasis.

We describe a case of regional migratory osteoporosis (RMO) with clinical images clearly illustrating the migratory behavior of this unusual disorder. RMO is a relatively rare disorder that manifests as rapidly developing, self-limiting, reversible osteoporosis typically seen in the lower limbs of middle-aged men. In our case, the lesion was observed migrating not only from the knee to the ankle within the same limb but also within two compartments of the same knee. To our knowledge, this is the first case showing migration of a lesion both within the same joint and to the adjacent joint. We also present computed tomography images showing characteristic spotty bone defects that are rarely described in the literature, along with magnetic resonance imaging scans revealing bone marrow edema in the affected region. Although little is understood about the etiology or treatment of RMO, knowledge of this disorder is mandatory to avoid its misdiagnosis as some other, more aggressive disease, leading to unnecessary treatment.