Anti-NFATC1 monoclonal antibody, clone 8B7

Background: Aspirin has been demonstrated to promote osteoblast-mediated bone formation and inhibit osteoclast (OC)-mediated bone resorption. However, it remains unclear whether aspirin influences other immune cells during bone resorption. Dendritic cells (DCs), the most potent antigen-presenting cells, can also transdifferentiate into active OCs in the presence of receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The effects of aspirin on DC-derived OCs (DDOCs) were investigated in the current study. Methods: Flow cytometry and mixed lymphocyte reaction (MLR) assays were used for DC identification. The proliferative capacity of DCs was determined by BrdU assays. Apoptosis was examined by flow cytometry. The osteoclastic potential of DCs was tested using tartrate-resistant acid phosphatase (TRAP) staining, western blotting, and reverse transcription polymerase chain reaction (RT-PCR). Western blotting was also used to examine signaling pathways. A mandibular bone defect model was established to assess the effect of aspirin on bone resorption. Results: Aspirin had no influence on the surface phenotype, proliferation, or apoptosis of DCs, though aspirin significantly inhibited osteoclast differentiation in RANKL-stimulated DCs. DC osteoclast differentiation was modulated by aspirin via the nuclear factor kappa B (NF-kappa B)/nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) signaling pathway. Aspirin treatment also had favorable therapeutic effects on bone regeneration in the bone defect model, and the number of osteoclasts was decreased. Conclusions: Aspirin inhibited RANKL-induced OC differentiation in DCs via the NF-kappa B pathway, downregulating expression of NFATc1. Aspirin treatment promoted bone regeneration by inhibiting DDOC activation in the early stages of inflammation in a rat mandibular bone defect model.

All cells, including nonexcitable cells, maintain a discrete transmembrane potential (V-mem), and have the capacity to modulate V-mem and respond to their own and neighbors' changes in V-mem. Spatiotemporal variations have been described in developing embryonic tissues and in some cases have been implicated in influencing developmental processes. Yet, how such changes in V-mem are converted into intracellular inputs that in turn regulate developmental gene expression and coordinate patterned tissue formation, has remained elusive. Here we document that the V-mem of limb mesenchyme switches from a hyperpolarized to depolarized state during early chondrocyte differentiation. This change in V-mem increases intracellular Ca2+ signaling through Ca2+ influx, via Ca(V)1.2, 1 of L-type voltage-gated Ca2+ channels (VGCCs). We find that Ca(V)1.2 activity is essential for chondrogenesis in the developing limbs. Pharmacological inhibition by an L-type VGCC specific blocker, or limb-specific deletion of Ca(V)1.2, down-regulates expression of genes essential for chondrocyte differentiation, including Sox9, Col2a1, and Agc1, and thus disturbs proper cartilage formation. The Ca2+-dependent transcription factor NFATc1, which is a known major transducer of intracellular Ca2+ signaling, partly rescues Sox9 expression. These data reveal instructive roles of Ca(V)1.2 in limb development, and more generally expand our understanding of how modulation of membrane potential is used as a mechanism of developmental regulation.