Chalcone T4 Inhibits RANKL-Induced Osteoclastogenesis and Stimulates Osteogenesis In Vitro

Abstract

Chalcones are phenolic compounds produced during the biosynthesis of flavonoids that have numerous biological activities, including anti-inflammatory, antioxidant and anticancer. In this in vitro study, we investigate a newly synthesized chalcone (Chalcone T4) in the context of bone turnover, specifically on the modulation of osteoclast differentiation and activity and osteoblast differentiation. Murine macrophages (RAW 264.7) and pre-osteoblasts (MC3T3-E1) were used as models of osteoclasts and osteoblasts, respectively. Differentiation and activity osteoclasts were induced by RANKL in the presence and absence of non-cytotoxic concentrations of Chalcone T4, added in different periods during osteoclastogenesis. Osteoclast differentiation and activity were assessed by actin ring formation and resorption pit assay, respectively. Expression of osteoclast-specific markers (Nfatc1, Oscar, Acp5, Mmp-9 and Ctsk) was determined by RT-qPCR, and the activation status of relevant intracellular signaling pathways (MAPK, AKT and NF-kB) by Western blot. Osteoblast differentiation and activity was induced by osteogenic culture medium in the presence and absence of the same concentrations of Chalcone T4. Outcomes assessed were the formation of mineralization nodules via alizarin red staining and the expression of osteoblast-related genes (Alp e Runx2) by RT-qPCR. Chalcone T4 reduced RANKL-induced osteoclast differentiation and activity, suppressed Oscar, Acp5 and Mmp-9 expression, and decreased ERK and AKT activation in a dose-dependent manner. Nfact1 expression and NF-kB phosphorylation were not modulated by the compound. Mineralized matrix formation and the expression of Alp and Runx2 by MC3T3-E1 cells were markedly stimulated by Chalcone T4. Collectively, these results demonstrate that Chalcone T4 inhibits in osteoclast differentiation and activity and stimulates osteogenesis, which indicates a promising therapeutic potential in osteolytic diseases.

Keywords: chalcone; intracellular signaling; osteoclastogenesis; osteogenesis.

Figure 1

Chalcone T4 reduces osteoclast differentiation in a dose-dependent manner, even when added up to 96 h after RANKL stimulation. Cells were seeded and stimulated with RANKL (100 ng/mL) and/or treated with different concentrations of Chalcone T4. In (A) The bar graph represents the number of osteoclasts per well after stimulation with RANKL, and treatment with Chalcone at different times ((I): 30 min before stimulation with RANKL, (II): 48 h and (III): 96 h after stimulation). In (B) Images obtained in fluorescence microscopy (4× magnification), representative of the differentiation of osteoclasts induced by RANKL, after treatment or not of cells with Chalcone T4. The bars indicate the mean values and the vertical lines the standard error of the mean (SEM) of three independent experiments, performed in duplicate. p values are depicted in the figure.

Figure 2

Chalcone T4 inhibits osteoclast activity in a dose-dependent manner, in all analyzed periods. Cells were stimulated with RANKL and treated with chalcone as described above. The resorptive activity of osteoclasts was evaluated through the area of the resorption gaps (underlined in red) in the plates coated with synthetic calcium phosphate. (A) (I–III) quantitative result of the area reabsorbed by osteoclasts, in the different periods of chalcone addition. (B) (I–III) representative images of the area of resorption after stimulation of cells with RANKL and treatment with the compound (4× magnification). The bars indicate the mean values and the vertical lines the standard error of the mean (SEM) of three different experiments evaluated in triplicate. Statistically significant differences between groups, as well as p values are indicated in the figure.

Figure 3

Chalcone T4 inhibits gene expression of bone markers in RANKL-stimulated RAW 264.7 cells. Effect of chalcone T4 on gene expression of Oscar (A), Nfatc1 (B), Acp5 (C), Cathepsin k (D) and Mmp-9 (E). Chalcone significantly inhibited the gene expression of Mmp-9, Cathepsin k and Oscar on the third and fifth day after stimulation with RANKL (p < 0.0001), and only in the later period (fifth day) the expression of Acp5. Nfatc1 expression was not altered by Chalcone treatment. Data is presented as fold change to unstimulated (control) cells. The bars indicate the mean results and the vertical lines the standard error (SEM) of three independent experiments performed in duplicate. Statistically significant differences between groups, as well as p values are indicated in the figure.

Figure 4

Chalcone T4 inhibits ERK and AKT activation. The graphs depict the mean and standard deviations of the densitometric analysis for the bands corresponding to ERK (A), AKT (B), p-38 (C) and NF-KB (D), after 10 (I) or 40 min (II) of stimulation with RANKL. Chalcone T4 suppressed ERK and AKT activation after 10 and 40 min, respectively, of stimulation with RANKL. Bars indicate mean results and vertical lines the standard error (SEM) of three duplicate experiments. p values are presented in the figure.

Figure 5

Chalcone T4 increases the formation of mineralized matrix nodules and gene expression of osteoblast markers. MC3TE-E1 were cultivated in osteogenic medium, and after 24 h treated with different concentrations of Chalcone T4. In (A), mean absorbance values at the end of each experimental period (7, 14 and 21 days). (B) Representative image of alizarin red staining. Images were registered on a digital inverted microscope (brightfield) (at a 40× magnification). The arrows highlight the formed calcification nodules. (C) Gene expression of Alp and Runx2 in cells treated with the compound for 7 days. Bars indicate mean results and vertical lines the standard error (SEM) of three duplicate experiments.