Bone morphogenetic protein (BMP)-2 plays an important role in morphogenesis in both vertebrates and invertebrates. BMP-2 is one of the most powerful bioactive substances known to induce the osteogenic differentiation of mesenchymal cells. We examined the structural and functional conservation of Pinctada fucata BMP-2 in inducing osteogenesis in the murine mesenchymal stem cells, C3H10T1/2. Exposure of C3H10T1/2 cells to the recombinant mature fragment of Pinctada fucata BMP-2 resulted in osteoblastic differentiation. The sequence, SVPKPCCVPTELSSL, within the C-terminal portion of Pinctada fucata BMP-2, is homologous to the knuckle epitope of human BMP-2. This synthetic polypeptide was able to induce differentiation of C3H10T1/2 along the osteoblastic lineage, as confirmed by an increase in alkaline phosphatase activity, and the accumulation of calcium, as determined by von Kossa staining. Furthermore, using immunohistochemical staining, we observed an increased expression of collagen type I, osteopontin, and osteocalcin, which are known markers of osteogenesis. These results show that BMP-2 is conserved, not only in terms of its homology at the amino acid sequence, but also in terms of driving the formation of hard tissues in vertebrates and invertebrates. 1. Introduction The transforming growth factor-beta (TGF-β) supergene family plays various roles in the regulation of cell growth, cell specialization, and morphogenesis [1, 2]. More than 40 genes have been identified in the mammalian TGF-β family [3], including the bone morphogenetic proteins (BMPs). BMPs are well-known regulators of bone and cartilage formation in vertebrates. The BMPs and their homologs are also involved in the regulation of morphogenesis in the shells of mollusks [4–7]. Hard tissue in invertebrates is equivalent to bone of vertebrates in terms of the organization of accumulated minerals. Although it is to be expected that BMP-2 or -4 participates in the formation of hard tissues such as the shells of mollusks, direct proof has not been reported. Previously, to examine the role of BMP-2 in morphogenesis and hard tissues formation of Pinctada fucata, we cloned the Pinctada fucata BMP-2 (PfBMP-2) gene using DNA obtained by PCR-based genome amplification as the probe [8]. The PfBMP-2 gene was expressed strongly in the inner part of the mantle tissue, corresponding to the nacreous aragonite shell layer. This suggested that BMP-2 has a key role in the nacreous layer formation. Recently, indirect proof that PfBMP-2 participates in the hard tissue formation of P. fucata was reported [9].
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