Bone Morphogenetic Protein 2/4 in Mollusk, Haliotis diversicolor: Its Expression and Osteoinductive Function In Vitro.

Bone morphogenetic proteins (BMPs), which are members of the superfamily of transforming growth factor-β (TGF-β), are known both in vitro and in vivo for their osteoinduction properties on the osteoblastic cells. Its role in the mollusk shell formation has also been gradually established. Using Haliotis diversicolor as a model, we characterized the HdBMP2/4 gene in the mantle tissue and showed its expression in the outer fold epithelium (particularly at the periostracal groove) the epithelial site which is involved in shell formation, both prismatic and nacreous layers. Shell notching experiments following gene analysis by qPCR revealed the upregulation of the HdBMP2/4 gene up to 3.2-fold than that of the control animals. In vitro treatments of the preosteoblastic cells, MC3T3-E1 with HdBMP2/4 synthetic peptide demonstrated the enhanced effect of many osteogenic genes that are known to regulate bone and shell biomineralization including ALP, Runx2, and OCN with 2-4 fold-change throughout 14 days of culture. In addition, the increased deposition of calcium-based mineral (as assessed by Alizarin red staining) of the treated cells was comparable to the ascorbic acid (Vit C) + glycerophosphate positive control which revealed the enhanced effect of HdBMP2/4 peptide on matrix biomineralization of the preosteoblastic cells. In conclusion, these results indicated the presence of the HdBMP2/4 gene in the mantle tissue at the site involved in shell formation and the effect of the HdBMP2/4 knuckle epitope peptide in osteoinduction in vitro.