Downregulation of miR-21 gene expression by CRE-Ter to modulate osteoclastogenesis: De Novo mechanism

miR-21 expression stimulates osteoclast cells in the context of osteoclastogenesis. A previous report showed that NF kappa B-miR-21 pathway could serve as an innovative alternative to devise therapeutics for healing diabetic ulcers. Furthermore, our study demonstrated that a highly water-soluble curcuminoids-rich extract (CRE-Ter) inhibits osteoclastogenesis through NF kappa B pathway. The interplay between miR-21 and CRE-Ter in osteoclastogenesis has not yet been investigated. In this study, we examined the relation of CRE-Ter and miR-21 gene expression in receptor of the nuclear factor kappa B (NF kappa B) ligand (RANKL) - induced murine monocyte/macrophage RAW 264.7 cells, osteoclast cells, in osteoclastogenesis. Effect of CRE-Ter on generation of intracellular reactive oxygen species (ROS) was estimated by dichlorofluorescein diacetate (DCFH-DA). The results reveal that CRE-Ter reduced expression levels of miR-21 gene in osteoclasts. The inhibitory effects of CRE-Ter on in vitro osteoclastogenesis were evaluated by reduction in tartrate-resistant acid phosphatase (TRAP) content, and by reduction in expression levels of an osteoclast-specific gene, cathepsin K. Treatment of the osteoclast cells with CRE-Ter suppressed RANKL-induced NF kappa B activation including phospho-NF kappa B-p65, and phospho I kappa B alpha proteins. Western blot analysis revealed that NF kappa B inhibitor up-regulated CRE-Ter-promoted expression of phospho-NF kappa B-p65. In addition, CRE-Ter dose-dependently inhibited phospho-Akt expression. CRE-Ter also dose-dependently reduced DNA binding activity of NF kappa B and Akt as revealed by EMSA. ChIP assay revealed binding of NF kappa B-p65 to miR-21 promoters. In conclusion, our results demonstrate that CRE-Ter downregulates miR-21 gene expression in osteoclasts via a de novo mechanism, NF kappa B-Akt-miR-21 pathway.