Effects of the Lysine Methyltransferase Inhibitor AZ505 on Bone Metabolism

Background@#Protein methylation has important role in regulating diverse cellular responses, including differentiation, by affecting protein activity, stability, and interactions. AZ505 is an inhibitor of the SET and MYND domain-containing protein 2 lysine methylase. In this study, we investigated the effect of AZ505 on osteoblast and osteoclast differentiation in vitro and evaluated the effect of AZ505 in vivo on the long bones in mice. @*Methods@#Osteoblast differentiation was assessed by alkaline phosphatase (ALP) and Alizarin red staining after culturing calvarial preosteoblasts in an osteogenic medium. Osteoclast differentiation was analyzed by tartrate-resistant acid phosphatase (TRAP) staining in bone marrow-derived macrophages cultured with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). For in vivo experiments, mice were intraperitoneally injected with AZ505 and femurs were examined by micro-computed tomography. @*Results@#AZ505 increased ALP and Alizarin red staining in cultured osteoblasts and the expression of osteoblast marker genes, including Runx2 and osteocalcin. AZ505 resulted in decreased TRAP-staining of osteoclasts and expression of c-Fos and nuclear factor of activated T cells transcription factors and osteoclast marker genes, including cathepsin K and dendritic cell-specific transmembrane protein. Unexpectedly, in vivo administration of AZ505 markedly decreased the trabecular bone mass of femurs. In support of this catabolic result, AZ505 strongly upregulated RANKL expression in osteoblasts. @*Conclusions@#The results indicate that AZ505 has a catabolic effect on bone metabolism in vivo despite its anabolic effect in bone cell cultures. The findings indicate that cell culture data should be extrapolated cautiously to in vivo outcomes for studying bone metabolism.

Induction of Anti-Aquaporin 5 Autoantibody Production by Immunization with a Peptide Derived from the Aquaporin of Prevotella melaninogenica Leads to Reduced Salivary Flow in Mice

Sjögren's syndrome (SS) is an autoimmune disease characterized by dryness of the mouth and eyes. The glandular dysfunction in SS involves not only T cell-mediated destruction of the glands but also autoantibodies against the type 3 muscarinic acetylcholine receptor or aquaporin 5 (AQP5) that interfere with the secretion process. Studies on the breakage of tolerance and induction of autoantibodies to these autoantigens could benefit SS patients. To break tolerance, we utilized a PmE-L peptide derived from the AQP5-homologous aquaporin of Prevotella melaninogenica (PmAqp) that contained both a B cell “E” epitope and a T cell epitope. Repeated subcutaneous immunization of C57BL/6 mice with the PmE-L peptide efficiently induced the production of Abs against the “E” epitope of mouse/human AQP5 (AQP5E), and we aimed to characterize the antigen specificity, the sequences of AQP5Especific B cell receptors, and salivary gland phenotypes of these mice. Sera containing anti-AQP5E IgG not only stained mouse Aqp5 expressed in the submandibular glands but also detected PmApq and PmE-L by immunoblotting, suggesting molecular mimicry.Characterization of the AQP5E-specific autoantibodies selected from the screening of phage display Ab libraries and mapping of the B cell receptor repertoires revealed that the AQP5E-specific B cells acquired the ability to bind to the Ag through cumulative somatic hypermutation. Importantly, animals with anti-AQP5E Abs had decreased salivary flow rates without immune cell infiltration into the salivary glands. This model will be useful for investigating the role of anti-AQP5 autoantibodies in glandular dysfunction in SS and testing new therapeutics targeting autoantibody production.