Sphingosine-1-Phosphate Receptor 2 Controls Podosome Components Induced by RANKL Affecting Osteoclastogenesis and Bone Resorption.

Proinflammatory cytokine production, cell chemotaxis, and osteoclastogenesis can lead to inflammatory bone loss. Previously, we showed that sphingosine-1-phosphate receptor 2 (S1PR2), a G protein coupled receptor, regulates inflammatory cytokine production and osteoclastogenesis. However, the signaling pathways regulated by S1PR2 in modulating inflammatory bone loss have not been elucidated. Herein, we demonstrated that inhibition of S1PR2 by a specific S1PR2 antagonist (JTE013) suppressed phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-B (NF-κB) induced by an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans, and inhibited the release of IL-1ß, IL-6, TNF-α, and S1P in murine bone marrow cells. In addition, shRNA knockdown of S1PR2 or treatment by JTE013 suppressed cell chemotaxis induced by bacteria-stimulated cell culture media. Furthermore, JTE013 suppressed osteoclastogenesis and bone resorption induced by RANKL in murine bone marrow cultures. ShRNA knockdown of S1PR2 or inhibition of S1PR2 by JTE013 suppressed podosome components, including PI3K, Src, Pyk2, integrin ß3, filamentous actin (F-actin), and paxillin levels induced by RANKL in murine bone marrow cells. We conclude that S1PR2 plays an essential role in modulating proinflammatory cytokine production, cell chemotaxis, osteoclastogenesis, and bone resorption. Inhibition of S1PR2 signaling could be a novel therapeutic strategy for bone loss associated with skeletal diseases.

FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

BACKGROUND: Periodontitis is a bacteria-driven inflammatory bone loss disease. Previous studies showed that the oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) stimulated the generation of sphingosine 1 phosphate (S1P). In addition, S1P signaling regulated the migration of osteoclast precursors and affected osteoclastogenesis. Furthermore, treatment with FTY720 (also called fingolimod, a modulator of multiple S1P receptors) alleviated osteoporosis and suppressed arthritis in animals. This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation. METHODS: Murine bone marrow-derived monocytes and macrophages (BMMs) were treated with vehicle ethanol or FTY720, and were either unstimulated or stimulated for 0.5 to 6 h with A. actinomycetemcomitans. The protein levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the media of BMMs were quantified by enzyme-linked immunosorbent assay (ELISA). Protein expressions, including phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, p-extracellular signal-regulated kinase (p-ERK), PI3K, Akt, and ERK were evaluated by Western blot. In addition, murine bone marrow-derived pre-osteoclasts were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for three days. Then the cells were treated with either vehicle or FTY720 and were either unstimulated or stimulated with A. actinomycetemcomitans for 4 to 24 h. Control cells were treated with M-CSF alone with or without bacterial stimulation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of osteoclastogenic factors, including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (Nfatc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), and RANKL were quantified by quantitative real-time polymerase chain reaction (PCR). RESULTS: FTY720 dose-dependently inhibited IL-1ß, IL-6, and TNF-α protein levels induced by A. actinomycetemcomitans in BMMs compared with controls. Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans. Furthermore, FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts with or without bacterial stimulation and reduced the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar, but not RANKL in bone marrow-derived pre-osteoclasts. CONCLUSION: FTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.

Topical oxygen emulsion: a novel wound therapy.

OBJECTIVE: To investigate the use of a topical oxygen emulsion (TOE), consisting of a supersaturated oxygen suspension using perfluorocarbon components, on second-degree burns and partial-thickness wounds. DESIGN: Oxygen is a required substance for various aspects of wound repair, and increased oxygen tension in a wound has been shown to stimulate phagocytosis and to reduce the incidence of wound infection. Second-degree burns and partial-thickness wounds were created on the backs of specific pathogen-free pigs. Wounds were then randomly assigned to 1 of the following treatment groups: TOE, TOE vehicle, or air-exposed control. MAIN OUTCOME MEASURE: Wounds were assessed for complete epithelialization using a salt-split technique. RESULTS: The TOE was able to significantly (P = .001) enhance the rate of epithelialization compared with both vehicle and untreated control. These data suggest that topical oxygen may be beneficial for acute and burn wounds. CONCLUSIONS: The results obtained from this double-blind, control, in vivo study demonstrate that TOE can significantly enhance the rate of epithelialization of partial-thickness excisional wounds and second-degree burns. These findings could have considerable clinical implications for patients with surgical and burn wounds by providing functional skin at an earlier date to act as a barrier against environmental factors, such as bacteria invasion. Other types of wounds may also benefit from this therapy (eg, chronic wounds and surgical incisions). Additional studies, including clinical studies, are warranted.