Toshihiro Tanioka1, Kohei Maeda1, Rei Takahashi1 and Takeo Isozaki2, 1Division of Pathogenesis and Translational Medicine, Department of Clinical Pharmacy, Showa University School of Pharmacy, Tokyo, Japan, 2Division of Rheumatology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
Background/Purpose: S-nitrosoglutathione (GSNO) reductase (GSNOR) is an enzyme which decomposes GSNO and downregulates protein S-nitrosylation. It has been reported that GSNOR KO mice have enhanced bone formation and resorption resulting higher bone turnover, with a net result of lower bone volume. Bone marrow derived macrophages (BMM) from GSNOR KO mice also enhanced osteoclast differentiation in vitro. However, the role of GSNOR on osteoclast differentiation not been fully elucidated. Here, we examined molecular mechanisms of osteoclast differentiation in BMM isolated from GSNOR KO mice.
Methods: BMM form WT and GSNOR KO mice were cultured in the presence of 50ng/m M-CSF and 50ng/m sRANKL. Osteoclasts were stained by tartrate resistant acid phosphatase (TRAP) and numbers of TRAP-positive multinucleated cells (three or more nuclei) were evaluated. In addition to primary cells, a transient knockdown of mouse GSNOR in the RAW264.7 cells were used. For detection of protein expression, cell lysates were subjected to immunoblotting analysis. Real-time PCR (qPCR) was also carried out to measure mRNA levels of osteoclastic markers such as NFATc1, c-Fos, Cathepsin K relative to β-actin as endogenous control.
Results: BMM from WT mice were allowed to differentiate into mononuclear or multinucleated TRAP-positive osteoclasts in the presence of M-CSF and sRANKL for 4 days. In contrast, BMM form KO mice showed a marked reduction in the number of TRAP-positive multinucleated cells, suggesting that osteoclast differentiation was regulated by GSNOR. To further confirm the role of GSNOR in the osteoclast differentiation, expression of osteoclastic marker molecules, NFATc1 and c-Fos, were assessed. NFATc1 and c-Fos expression was induced after M-CSF/sRANKL stimulation of BMM from WT mice, while expression level of these molecules were impaired in BMM from KO mice. A similar result was observed in GSNOR siRNA-treated cells. These results suggest that GSNOR deficiency is crucial for sRANKL-mediated signaling as a negative regulator of osteoclast differentiation.
Conclusion: GSNOR deficiency in macrophages plays an important role in suppression of osteoclast differentiation. These data indicate that GSNOR inhibitor may considered as a therapeutic drug for inflammation disease such as rheumatoid arthritis.
Disclosures: T. Tanioka, None; K. Maeda, None; R. Takahashi, None; T. Isozaki, None.
