Yuiko Sato

Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization

The mobilization of hematopoietic stem cells does not require osteoclasts, which may even have an inhibitory effect.

Osteoclast stimulatory transmembrane protein and dendritic cell–specific transmembrane protein cooperatively modulate cell–cell fusion to form osteoclasts and foreign body giant cells

Cell–cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC‐STAMP), and analyzed its function. OC‐STAMP–deficient mice exhibited a complete lack of cell–cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage‐lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC‐STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC‐STAMP–overexpressing transgenic mice with OC‐STAMP–deficient mice restored inhibited osteoclast and FBGC cell–cell fusion seen in OC‐STAMP–deficient mice. Thus, fusogenic mechanisms in macrophage‐lineage cells are regulated via OC‐STAMP and DC‐STAMP.

MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.

Cell fusion in osteoclasts plays a critical role in controlling bone mass and osteoblastic activity

The balance between osteoclast and osteoblast activity is central for maintaining the integrity of bone homeostasis. Here we show that mice lacking dendritic cell specific transmembrane protein (DC-STAMP), an essential molecule for osteoclast cell-cell fusion, exhibited impaired bone resorption and upregulation of bone formation by osteoblasts, which do not express DC-STAMP, which led to increased bone mass. On the contrary, DC-STAMP over-expressing transgenic (DC-STAMP-Tg) mice under the control of an actin promoter showed significantly accelerated cell-cell fusion of osteoclasts and bone resorption, with decreased osteoblastic activity and bone mass. Bone resorption and formation are known to be regulated in a coupled manner, whereas DC-STAMP regulates bone homeostasis in an un-coupled manner. Thus our results indicate that inhibition of a single molecule provides both decreased osteoclast activity and increased bone formation by osteoblasts, thereby increasing bone mass in an un-coupled and a tissue specific manner.

Aldehyde-stress resulting from Aldh2 mutation promotes osteoporosis due to impaired osteoblastogenesis

Osteoporosis is a complex disease with various causes, such as estrogen loss, genetics, and aging. Here we show that a dominant‐negative form of aldehyde dehydrogenase 2 (ALDH2) protein, ALDH2*2, which is produced by a single nucleotide polymorphism (rs671), promotes osteoporosis due to impaired osteoblastogenesis. Aldh2 plays a role in alcohol‐detoxification by acetaldehyde‐detoxification; however, transgenic mice expressing Aldh2*2 (Aldh2*2 Tg) exhibited severe osteoporosis with increased levels of blood acetaldehyde without alcohol consumption, indicating that Aldh2 regulates physiological bone homeostasis. Wild‐type osteoblast differentiation was severely inhibited by exogenous acetaldehyde, and osteoblastic markers such as osteocalcin, runx2, and osterix expression, or phosphorylation of Smad1,5,8 induced by bone morphogenetic protein 2 (BMP2) was strongly altered by acetaldehyde. Acetaldehyde treatment also inhibits proliferation and induces apoptosis in osteoblasts. The Aldh2*2 transgene or acetaldehyde treatment induced accumulation of the lipid‐oxidant 4‐hydroxy‐2‐nonenal (4HNE) and expression of peroxisome proliferator‐activated receptor gamma (PPARγ), a transcription factor that promotes adipogenesis and inhibits osteoblastogenesis. Antioxidant treatment inhibited acetaldehyde‐induced proliferation‐loss, apoptosis, and PPARγ expression and restored osteoblastogenesis inhibited by acetaldehyde. Treatment with a PPARγ inhibitor also restored acetaldehyde‐mediated osteoblastogenesis inhibition. These results provide new insight into regulation of osteoporosis in a subset of individuals with ALDH2*2 and in alcoholic patients and suggest a novel strategy to promote bone formation in such osteopenic diseases.

An Essential Role for STAT6-STAT1 Protein Signaling in Promoting Macrophage Cell-Cell Fusion

Background: The signaling leading to macrophage fusion remains largely unknown. Results: STAT6 deficiency completely inhibited macrophage fusion, although STAT1 deficiency or OC-STAMP/DC-STAMP co-expression was sufficient to promote macrophage fusion. Conclusion: The STAT6-STAT1-OC-STAMP/DC-STAMP axis is required for macrophage fusion. Significance: The STAT6-STAT1-OC-STAMP/DC-STAMP axis is a novel pathway leading to macrophage fusion. Macrophage lineage cells such as osteoclasts and foreign body giant cells (FBGCs) form multinuclear cells by cell-cell fusion of mononuclear cells. Recently, we reported that two seven-transmembrane molecules, osteoclast stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP), were essential for osteoclast and FBGC cell-cell fusion in vivo and in vitro. However, signaling required to regulate FBGC fusion remained largely unknown. Here, we show that signal transducer and activator of transcription 1 (STAT1) deficiency in macrophages enhanced cell-cell fusion and elevated DC-STAMP expression in FBGCs. By contrast, lack of STAT6 increased STAT1 activation, significantly inhibiting cell-cell fusion and decreasing OC-STAMP and DC-STAMP expression in IL-4-induced FBGCs. Furthermore, either STAT1 loss or co-expression of OC-STAMP/DC-STAMP was sufficient to induce cell-cell fusion of FBGCs without IL-4. We conclude that the STAT6-STAT1 axis regulates OC-STAMP and DC-STAMP expression and governs fusogenic mechanisms in FBGCs.

Smad2/3 Proteins Are Required for Immobilization-induced Skeletal Muscle Atrophy

Skeletal muscle atrophy promotes muscle weakness, limiting activities of daily living. However, mechanisms underlying atrophy remain unclear. Here, we show that skeletal muscle immobilization elevates Smad2/3 protein but not mRNA levels in muscle, promoting atrophy. Furthermore, we demonstrate that myostatin, which negatively regulates muscle hypertrophy, is dispensable for denervation-induced muscle atrophy and Smad2/3 protein accumulation. Moreover, muscle-specific Smad2/3-deficient mice exhibited significant resistance to denervation-induced muscle atrophy. In addition, expression of the atrogenes Atrogin-1 and MuRF1, which underlie muscle atrophy, did not increase in muscles of Smad2/3-deficient mice following denervation. We also demonstrate that serum starvation promotes Smad2/3 protein accumulation in C2C12 myogenic cells, an in vitro muscle atrophy model, an effect inhibited by IGF1 treatment. In vivo, we observed IGF1 receptor deactivation in immobilized muscle, even in the presence of normal levels of circulating IGF1. Denervation-induced muscle atrophy was accompanied by reduced glucose intake and elevated levels of branched-chain amino acids, effects that were Smad2/3-dependent. Thus, muscle immobilization attenuates IGF1 signals at the receptor rather than the ligand level, leading to Smad2/3 protein accumulation, muscle atrophy, and accompanying metabolic changes.

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation

Background: Factors that govern peripheral neuropathy associated with Schwann cell dysfunction are not fully understood. Results: Under hyperglycemic conditions, Schwann cells de-differentiate into immature cells via sorbitol accumulation and Igf1 down-regulation. Conclusion: Schwann cell de-differentiation promotes neuropathy development under hyperglycemic conditions. Significance: These findings reveal new mechanisms underlying neuropathy seen in diabetes mellitus via Schwann cell de-differentiation leading to de-myelination. Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition.

Methotrexate inhibits osteoclastogenesis by decreasing RANKL‑induced calcium influx into osteoclast progenitors

The increasing number of osteoporosis patients is a pressing issue worldwide. Osteoporosis frequently causes fragility fractures, limiting activities of daily life and increasing mortality. Many osteoporosis patients take numerous medicines due to other health issues; thus, it would be preferable if a single medicine could ameliorate osteoporosis and other conditions. Here, we screened 96 randomly selected drugs targeting various diseases for their ability to inhibit differentiation of osteoclasts, which play a pivotal role in development of osteoporosis, and identified methotrexate (MTX), as a potential inhibitor. MTX is currently used to treat sarcomas or leukemic malignancies or auto-inflammatory diseases such as rheumatoid arthritis (RA) through its anti-proliferative and immunosuppressive activities; however, a direct effect on osteoclast differentiation has not been shown. Here, we report that osteoclast formation and expression of osteoclastic genes such as NFATc1 and DC-STAMP, which are induced by the cytokine RANKL, are significantly inhibited by MTX. We found that RANKL-dependent calcium (Ca) influx into osteoclast progenitors was significantly inhibited by MTX. RA patients often develop osteoporosis, and osteoclasts are reportedly required for joint destruction; thus, MTX treatment could have a beneficial effect on RA patients exhibiting high osteoclast activity by preventing both osteoporosis and joint destruction.

PDGFBB promotes PDGFRα-positive cell migration into artificial bone in vivo

Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFβ) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.