miR-302a-3p regulates RANKL expression in human mandibular osteoblast-like cells.

Receptor activator of nuclear factor kappa-B ligand (RANKL) is important substance during osteoclastogenesis that resulted in alveolar bone loss of periodontitis. MicroRNAs (miRNAs) regulate gene expression in several biological processes including osteoclastogenesis. We investigated the function of microRNA-302a-3p (miR-302a-3p) to regulate receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human mandibular osteoblast-like cells (HMOBs). HMOBs were incubated with prostaglandin E2 (PGE2 ) to mimic inflammation, or with PGE2 and interferon gamma (IFNγ) to mimic homeostasis. MicroRNA (miRNA) profiles related to RANKL expression were demonstrated by PCR array, and miR-302a-3p was identified. Using TargetScanHuman 7.0, a target of miR-302a-3p was predicted. To confirm its function, miR-302a-3p was overexpressed, or silenced, by transfection with miR-302a-3p mimic, or inhibitor, respectively. Level of miR-302a-3p and RANKL mRNA was assessed by qRT-PCR. Soluble RANKL (sRANKL), and membrane-bound RANKL (mRANKL) were measured by ELISA and by Western blot, respectively. When PGE2 stimulated RANKL in HMOBs, miR-302a-3p was lower than baseline level. However, upregulation of miR-302a-3p is observed when IFNγ suppressed RANKL expression in PGE2 -stimulated HMOBs. miR-302a-3p was predicted to target PRKACB mRNA encoding the catalytic subunit in cAMP/PKA pathway. Overexpression of miR-302a-3p could decrease RANKL expression during PGE2 stimulation. In contrast, silencing of miR-302a-3p by its inhibitor increased RANKL expression in PGE2 -IFNγ conditioned HMOBs. miR-302a-3p regulates RANKL expression in HMOBs within PGE2 -IFNγ regulatory network.

Epithelial Cells Secrete Interferon-γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa-B Ligand in Human Mandibular Osteoblast-Like Cells.

BACKGROUND: Prostaglandin (PG)E2 accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa-B ligand (RANKL)-RANK-osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. METHODS: To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast-like cells (HMOBs) were stimulated with PGE2. Effect of recombinant human interferon (IFN)-γ or epithelial-derived IFN-γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)-stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti-IFN-γ antibody before PGE2 stimulation. THP-1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL-driven THP-1 osteoclastic activity. RESULTS: PGE2 significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose-dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN-γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN-γ, concurrently with PGE2 stimulation, reduced RANKL, but not OPG, expression. In contrast, anti-IFN-γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP-1, RANKL released by PGE2-stimulated HMOBs is adequate to drive THP-1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN-γ, or IFN-γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP-1-derived osteoclastic activity. CONCLUSION: Oral epithelial cells interact with HMOBs by releasing IFN-γ to regulate RANKL expression and contribute to osteoclastogenesis.

Effects of 1.23% acidulated phosphate fluoride gel and drinkable yogurt on human enamel erosion, in vitro.

OBJECTIVES: In this in vitro study, the authors sought to determine the effects of 1.23% acidulated phosphate fluoride gel (APF) or drinkable yogurt on human enamel after exposure to an acidic drink. METHOD AND MATERIALS: Sixteen surgically removed, caries-free, human third molars were cut into four portions: mesial, distal, buccal, and lingual. Each portion was distributed into one of four groups, with each specimen embedded in acrylic resin and its enamel center lightly ground and polished. Three groups of specimens were immersed in orange juice for 2 minutes. One group received no other treatment (OR), one group was subsequently immersed in drinkable yogurt for 5 minutes (OR:YO), while another group had 1.23% APF applied for 4 minutes (OR:APF). The final group was immersed in only drinkable yogurt for 5 minutes (YO). Each exposure was performed twice daily for 60 days; between exposures, the samples were stored in artificial saliva. The enamel surfaces were monitored by three criteria: erosion depth, surface hardness, and SEM. RESULTS: Erosion depth increased progressively in all groups. Surface microhardness progressively decreased in all groups except the OR:APF group, where hardness was significantly higher than other groups at 60 days (P < .05). SEM inspection revealed preferential loss of rod crystallites with retention of interrod crystallites in all specimens exposed to orange juice. The enamel exposed to only drinkable yogurt revealed modest and uniform etching. CONCLUSION: Drinkable yogurt alone or posttreatment of enamel after exposure to an acidic drink with either 1.23% APF or drinkable yogurt leads to enamel dissolution and does not reduce enamel erosion, in vitro.