Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/β-Catenin Signaling Pathway, Leading to Bone Formation

The Pacific oyster, <i>Crassostrea gigas</i>, is well-known as a nutritious food. Recently, we revealed that fermented extract of <i>C. gigas</i> (FO) inhibited ovariectomy-induced osteoporosis, resulting from suppression of osteoclastogenesis. However, since the beneficial effect of FO on osteogenesis is poorly understood, it was examined in mouse preosteoblast MC3T3-E1 cells, human osteosarcoma MG-63 osteoblast-like cells, and zebrafish larvae in this study. We found that FO increased mitochondrial activity from days 1 to 7; however, total cell number of MC3T3-E1 cells gradually decreased without any change in cell viability, which suggests that FO stimulates the differentiation of MC3T3-E1 cells. FO also promoted the expression of osteoblast marker genes, including <i>runt-related transcription factor 2 (mRUNX2)</i>, <i>alkaline phosphatase (mALP)</i>, <i>collagen type I &#945;1 (mCol1&#945;1)</i>, <i>osteocalcin (mOCN)</i>, <i>osterix (mOSX)</i>, <i>bone morphogenetic protein 2 (mBMP2)</i>, and <i>mBMP4</i> in MC3T3-E1 cells accompanied by a significant increase in ALP activity. FO also increased nuclear translocation of RUNX2 and OSX transcription factors, ALP activity, and calcification in vitro along with the upregulated expression of osteoblast-specific marker proteins such as RUNX2, ALP, Col1&#945;1, OCN, OSX, and BMP4. Additionally, FO enhanced bone mineralization (calcein intensity) in zebrafish larvae at 9 days post-fertilization comparable to that in the &#946;-glycerophosphate (GP)-treated group. All the tested osteoblast marker genes, including <i>zRUNX2a</i>, <i>zRUNX2b</i>, <i>zALP</i>, <i>zCol1a1</i>, <i>zOCN</i>, <i>zBMP2</i>, and <i>zBMP4</i>, were also remarkably upregulated in the zebrafish larvae in response to FO. It also promoted tail fin regeneration in adult zebrafish as same as the GP-treated groups. Furthermore, not only FO positively regulate &#946;-catenin expression and Wnt/&#946;-catenin luciferase activity, but pretreatment with a Wnt/&#946;-catenin inhibitor (FH535) also significantly decreased FO-mediated bone mineralization in zebrafish larvae, which indicates that FO-induced osteogenesis depends on the Wnt/&#946;-catenin pathway. Altogether, the current study suggests that the supplemental intake of FO has a beneficial effect on osteogenesis.