Takeshi Miyamoto

DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells

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.

Mesenchymal stem cells in perichondrium express activated leukocyte cell adhesion molecule and participate in bone marrow formation

Perichondrium in fetal limb is composed of undifferentiated mesenchymal cells. However, the multipotency of cells in this region and the role of perichondrium in bone marrow formation are not well understood. In this report, we purified and characterized perichondrial cells using a monoclonal antibody against activated leukocyte cell adhesion molecule (ALCAM) and investigated the role of perichondrial cells in hematopoietic bone marrow formation. ALCAM is expressed on hematopoietic cells, endothelial cells, bone marrow stromal cells, and mesenchymal stem cells and mediates homophilic (ALCAM–ALCAM)/heterophilic (ALCAM-CD6) cell adhesion. Here we show by immunohistochemical staining that ALCAM is expressed in perichondrium. ALCAM+ perichondrial cells isolated by FACS® exhibit the characteristics of mesenchymal stem cells. ALCAM+ cells can differentiate into osteoblasts, adipocytes, chondrocytes, and stromal cells, which can support osteoclastogenesis, hematopoiesis, and angiogenesis. Furthermore, the addition of ALCAM-Fc or CD6-Fc to the metatarsal culture, the invasion of the blood vessels to a cartilage was inhibited. Our findings indicate that ALCAM+ perichondrial cells participate in vascular invasion by recruiting osteoclasts and vessels. These findings suggest that perichondrium might serve as a stem cell reservoir and play an important role in the early development of a bone and bone marrow.

Angiopoietin-related growth factor antagonizes obesity and insulin resistance

Angiopoietin-related growth factor (AGF), a member of the angiopoietin-like protein (Angptl) family, is secreted predominantly from the liver into the systemic circulation. Here, we show that most (>80%) of the AGF-deficient mice die at about embryonic day 13, whereas the surviving AGF-deficient mice develop marked obesity, lipid accumulation in skeletal muscle and liver, and insulin resistance accompanied by reduced energy expenditure relative to controls. In parallel, mice with targeted activation of AGF show leanness and increased insulin sensitivity resulting from increased energy expenditure. They are also protected from high-fat diet–induced obesity, insulin resistance and nonadipose tissue steatosis. Hepatic overexpression of AGF by adenoviral transduction, which leads to an approximately 2.5-fold increase in serum AGF concentrations, results in a significant (P < 0.01) body weight loss and increases insulin sensitivity in mice fed a high-fat diet. This study establishes AGF as a new hepatocyte-derived circulating factor that counteracts obesity and related insulin resistance.

Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification

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.

Induction of DC-STAMP by alternative activation and downstream signaling mechanisms.

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.

Role of DC-STAMP in cellular fusion of osteoclasts and macrophage giant cells

Osteoclasts are the only cells that can resorb bone matrix physiologically and maintain the bone content. Osteoclasts are derived from macrophage/monocyte lineage cells; stimulation by macrophage colony stimulating factor and receptor activator of NFκB ligand induces osteoclastogenesis. During osteoclastogenesis, preosteoclasts fuse to form multinuclear mature osteoclasts. Cellular fusion is a unique phenomenon and enables fertilization, myotube formation, and efficient bone resorption in vertebrates. To date, several molecules have been reported to be fusion related in osteoclasts, namely CD44, CD47, ADAM12, MCP-1, and CD9, although the molecules which regulate osteoclast cellular fusion remain unclear. Here, we show that the seven-transmembrane-region receptor dendritic cell-specific transmembrane protein (DC-STAMP) is required for cell–cell fusion of osteoclasts and foreign body giant cells.

A Functional Polymorphism in THBS2 that Affects Alternative Splicing and MMP Binding Is Associated with Lumbar-Disc Herniation

Lumbar-disc herniation (LDH), one of the most common musculoskeletal diseases, has strong genetic determinants. Recently, several genes that encode extracellular matrix (ECM) proteins in the intervertebral disc have been reported to associate with LDH. Thrombospondins (THBSs) 1 and 2 are good candidates for the LDH susceptibility gene: They are intervertebral disc ECM proteins that regulate the effective levels of matrix metalloproteinases (MMPs) 2 and 9, which are key effectors of ECM remodeling. Here, we report that THBS2 is associated with LDH in Japanese populations. An intronic SNP in THBS2 (IVS10-8C --> T; rs9406328) showed significant association (p = 0.0000028) with LDH in two independent Japanese populations. This SNP, located in a polypyrimidine tract upstream of the 3 splice site of intron 10, exerts allelic differences on exon 11 skipping rates in vivo, with the susceptibility allele showing increased skipping. Skipping of exon 11 results in decreased THBS2 interaction with MMP2 and MMP9. Further, a missense SNP in MMP9 (Q279R; rs17576) is also strongly associated with LDH in the Japanese population (p = 0.00049) and shows a combinatorial effect with THBS2 (odds ratio 3.03, 95% confidence interval 1.58-5.77). Thus, a splicing-affecting SNP in THBS2 and a missense SNP in MMP9 are associated with susceptibility to LDH. Our data indicate that regulation of intervertebral disc ECM metabolism by the THBS2-MMP system plays an essential role in the etiology and pathogenesis of LDH.

Tyro 3 Receptor Tyrosine Kinase and its Ligand, Gas6, Stimulate the Function of Osteoclasts

Bone is continuously being formed and resorbed. This process is accomplished by the precise coordination of two cell types: osteoblasts and osteoclasts. Osteoclasts are large, multinucleated cells that are derived from the same hematopoietic precursors as macrophages. However, these bone‐resorbing cells are difficult to study directly because of their relative inaccessibility. The purification of primary osteoclasts from rabbit bones by their adherent nature provides an opportunity for investigating the molecules in osteoclasts. We have examined the expression of receptor tyrosine kinase by polymerase chain reaction (PCR) and found that Tyro 3 was frequently identified from primary osteoclasts in PCR cloning. Immunohistochemistry revealed that Tyro 3 was expressed on the multinucleated osteoclasts which were positive for tartrate‐resistant acid phosphatase (TRAP), but not on mononuclear TRAP‐positive cells. The Tyro 3 ligand, Gas6, induced the phosphorylation of Tyro 3 receptors in osteoclasts in two to five min. Gas6 and protein S directly enhanced the bone resorbing activity of mature osteoclasts. This effect of Gas6 was inhibited by the addition of a tyrosine kinase inhibitor, herbimycin A. However, Gas6 did not affect the differentiation of osteoclasts from bone marrow cells. Gas6 and protein S are dependent on vitamin K, a cofactor for the enzyme responsible for carboxylation of glutamic acid residues. The findings in this study are the first to indicate a new biological activity of Gas6 and protein S as a direct regulator of osteoclastic function; they give an insight into the role of these vitamin K‐dependent ligands in bone resorption in vivo.

FoxO3a regulates hematopoietic homeostasis through a negative feedback pathway in conditions of stress or aging

Stress or aging of tissue-specific stem cells is considered central to the decline of tissue homeostasis in the elderly, although little is known of molecular mechanisms underlying hematopoietic stem cell (HSC) aging and stress resistance. Here, we report that mice lacking the transcription factor forkhead box O3a (FoxO3a) develop neutrophilia associated with inhibition of the up-regulation of negative regulator of cell proliferation, Sprouty-related Ena/VASP homology 1 domain-containing proteins 2 (Spred2) and AKT and ERK activation, in HSCs during hematopoietic recovery following myelosuppressive stress conditions. Compared with aged wild-type mice, more severe neutrophilia was also observed in aged Foxo3a-deficient mice. AKT and ERK activation and inhibition of Spred2 were detected in HSCs from aged FoxO3a-deficient mice. Spred2-deficient mice also developed neutrophilia during hematopoietic recovery following myelosuppressive stress, indicating that FoxO3a plays a pivotal role in maintenance, integrity, and stress resistance of HSCs through negative feedback pathways for proliferation. This will provide new insight into the hematopoietic homeostasis in conditions of aging and stress.

Vascular endothelial growth factor-A is a survival factor for nucleus pulposus cells in the intervertebral disc

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.