2023, Vol. 27 ›› Issue (31): 4990-4995

Effect of inhibitor of Wnt production 2 on osteoclast differentiation

Liao Zirui, Chen Jianquan   

Abstract: BACKGROUND: Abnormal activation of osteoclast-mediated bone resorption leads to the loss of bone mass, damage and deterioration of bone microarchitecture, which is an important cause of osteoporosis. Wnt/β-catenin signaling pathway may be a potential target and tool for the prevention and treatment of osteoporosis. Overactivation of Wnt/β-catenin signaling pathway inhibits osteoclast differentiation. However, the physiological role of Wnt/β-catenin signaling pathway in osteoclast formation remains to be clarified.
OBJECTIVE: To investigate the effect of inhibitor of Wnt production 2 (IWP-2) on proliferation and differentiation of bone marrow-derived macrophages through inhibiting the Wnt/β-catenin signaling pathway.
METHODS: (1) Bone marrow-derived macrophages were extracted. Cell counting kit-8 assay was performed to assess the viability of bone marrow-derived macrophages after being treated with gradient concentrations from 0 to 40 μmol/L IWP-2 for 24, 72, or 120 hours. (2) Bone marrow-derived macrophages induced by 100 μg/L receptor activator of nuclear factor kappa B ligand (RANKL) were treated with 5 or 10 μmol/L IWP-2 and equivalent solvent, and precipitated proteins were collected at 0, 1, 3 and 5 days. The activity of Wnt/β-catenin signaling pathway and the inhibitory effect of IWP-2 on it during osteoclast differentiation were detected by western blot assay. (3) Bone marrow-derived macrophages induced by 100 μg/L RANKL were treated with non-toxic IWP-2 (0, 5, or 10 μmol/L) for 6 days. The effects of IWP-2 on osteoclast differentiation were evaluated by tartrate-resistant acid phosphatase staining. The mRNA expression of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were detected by real-time fluorescence quantitative PCR.
RESULTS AND CONCLUSION: (1) After treatment with different concentrations of IWP-2 for 24 hours, 10 μmol/L IWP-2 promoted the proliferation of bone marrow-derived macrophages. After treatment with different concentrations of IWP-2 for 72 and 120 hours, the viability of bone marrow-derived macrophages was not significantly affected by IWP-2 at a concentration of 10 μmol/L or less, while high concentrations of IWP-2 (20, 30, and 40 μmol/L) inhibited the proliferation of bone marrow-derived macrophages. (2) After 1, 3, and 5 days of RANKL stimulation, the protein expression of active β-catenin in the control group increased gradually, while that in the 5 and 10 μmol/L IWP-2 treatment group was significantly inhibited. (3) Many large and irregular TRAP-positive multinucleated mature osteoclasts were observed in the control group, while the number and size of the cells decreased in a dose-dependent manner in the IWP-2 treatment groups, suggesting that IWP-2 could inhibit osteoclast differentiation. (4) The expression levels of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were all down-regulated in a dose-dependent manner after IWP-2 treatment. (5) To conclude, IWP-2 can inhibit osteoclast differentiation in vitro and may play a role in the prevention and treatment of osteoporosis by directly or indirectly inhibiting the expression of osteoclast-elated genes.

Key words: osteoporosis, Wnt/β-catenin signaling pathway, IWP-2, bone marrow-derived macrophage, proliferation, differentiation