Abstract
Objectives
Nutritional factors influence bone development. We have previously reported that Blueberry (BB) and BB-associated phenolic acids had significant effects on increasing bone mass in rats. We also found BB-associated phenolic acids [hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA)] inhibited osteoclastogenesis in vitro and in ex vivo. Here, we investigated the mechanisms by which HA or 3-3-PPA inhibit osteoclast formation and if HA or 3-3-PPA inhibits bone resorption in vivo.
Methods
Murine osteoclast (macrophage) cell line, RAW 264.7 cells, and hematopoietic osteoclast progenitor cells (isolated from 4 weeks old C57BL6/J mice) were cultured. Cells were treated with HA or 3-3-PPA (0.6, 6, 60, 600 µg/dL) for 4 days in the presence of 50 ng/ml of RANKL (Receptor activator of nuclear factor kappa-Β ligand). In animal studies, dietary HA or 3-3-PPA was provided at 1, 5 or 10 mg/kg/d to female mice for 4 weeks, starting at postnatal day 28.
Results
In RAW 264.7 cell and primary cell cultures, TRAPase staining showed that RANKL-stimulated osteoclast number/well dose-dependently decreased with treatments of HA or 3-3-PPA. HA and 3-3-PPA significantly inhibited RANKL-induced β-catenin and GPR109A (G protein coupled receptor 109A) protein expression. Moreover, RANKL-induced β-catenin and GPR109A mRNA expression were also suppressed by HA and 3-3-PPA. Furthermore, after pre-treatment with HA or 3-3-PPA, RANKL-stimulated increases of Wnt/β-catenin down-stream genes such as c-myc, and osteoclastogenic genes such as NFkB and NFATc1 gene expression were blunted. In mice, we found significantly increased bone mass in dietary HA and 3-3-PPA supplemented groups compared to control group (P < 0.05). Increased bone mass in HA and 3-3-PPA treated mice was accompanied by decreased bone resorption (bone resorption markers and osteoclastogenic gene expression), but increased β-catenin expression in total protein isolated from bone.
Conclusions
These results indicate significant inhibition of osteoclastogenesis and bone resorption by HA and 3-3-PPA, and suggest that HA and 3-3-PPA inhibit bone marrow the hematopoietic cell differentiation program through non-canonical Wnt signaling to protect against increased bone resorption.
Funding Sources
USDA-ARS Project.