Functional analysis of Bombyx mori Decapentaplegic gene for bone differentiation in a mammalian cell

Abstract

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF-b) superfamily and are involved in osteoblastic differentiation. The largest TGF-b superfamily subgroup shares genetic homology with human BMPs (hBMPs) and silkworm decapentaplegic (dpp). In addition, hBMPs are functionally interchangeable with Drosophila dpp . Bombyx mori dpp may induce bone formation in mammalian cells. To test this hypothesis, we synthesized the 1,285-base pairs cDNA of full-length B. mori dpp using total RNAs obtained from the fat body of 3-day-old of the 5 th instar larvae and cloned the cDNA into the pCEP4 mammalian expression vector. Next, B. mori dpp was expressed in C3H10T1/2 cells. The target cells transfected with the pCEP4-Bm dpp plasmid showed biological functions similar to those of osteogenic differentiation induction growth factors such as hBMPs. We determined the relative mRNA expression rates of Runt-related transcription factor 2 (RUNX2), osterix, osteocalcin, and alkaline phosphatase (ALP) to validate the osteoblast-specific differentiation effects of B. mori dpp by performing quantitative real-time RT-PCR. Interestingly, mRNA expression levels of the 3 marker genes except RUNX2, in cells expressing B. mori dpp were much higher than those in control cells and C3H10T1/2 cells transfected with pCEP4. These results suggested that B. mori dpp signaling regulates osterix expression during osteogenic differentiation via RUNX2-independent mechanisms.

Expression analysis of decapentaplegic gene in the silkworm, Bombyx mori

“…mori , is highly conserved, and has been found in human, cow, mouse, chicken and fruit fly (Park et al . ). In particular, B .…”

“…Furthermore, it was able to convert the differentiation pathway of these stem cells to form osteoblast lineage cells (Park et al . ). Another study showed that silkworm pupa significantly contributes to the prevention or treatment of bone loss induced by ovariectomization in rats (Ryu et al .…”

“…mori dpp and increased levels of osteogenic‐specific genes has been reported in mouse pluripotent stem cells during osteogenic differentiation (Park et al . ).…”

“…The dpp gene, which has been found in B. mori, is highly conserved, and has been found in human, cow, mouse, chicken and fruit fly (Park et al 2013). In particular, B. mori dpp shares homology with hBMPs and Drosophila dpp.…”

“…Although the functional significance of dpp in B. mori has not been studied in detail, we have shown that dpp protein of this species is produced efficiently in mouse pluripotent stem cells transfected with recombinant pCEP4-Bmdpp vector. Furthermore, it was able to convert the differentiation pathway of these stem cells to form osteoblast lineage cells (Park et al 2013). Another study showed that silkworm pupa significantly contributes to the prevention or treatment of bone loss induced by ovariectomization in rats (Ryu et al 2012).…”