Wild-type Smad3 Gene Enhances the Osteoblastic Differentiation of Rat Bone Marrow-derived Mesenchymal Stem Cells in Vitro

Abstract

This study examined the effect of wild-type Smad3 gene on the osteoblastic differentiation of rat bone marrow derived mesenchymal stem cells in vitro. Bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the complexes of pcDNA3.0-Myc-Smad3 or pcDNA3.0-Myc-Smad3△C and Lipofectamine reagent. Immunofluorescence staining was performed to evaluate the c-Myc signal in MSCs. The cell proliferation was detected by MTT method. To clarify the osteoblastic characteristics in stably transfected MSCs, alkaline phosphatase (ALP) mRNA and core binding factor α1 (Cbfa1) mRNA were investigated by RT-PCR, and ALP activity and mineralization were examined by p-nitrophenolphosphate method and alizarin red staining respectively. PD98059, a specific inhibitor of the ERK signaling pathway, was used to determine the role of ERK in Smad3-MSCs osteoblastic differentiation. c-Myc signal was detected in Smad3-MSCs and Smad3△C-MSCs. The proliferation of Smad3-MSCs was slower than that of Smad3△C-MSCs or V-MSCs.The relative levels of ALP mRNA and Cbfa1 mRNA in Smad3-MSCs, as well as ALP activity and mineralization, were markedly higher than those in Smad3△C-MSCs or V-MSCs. Although ALP activity and mineralization were slightly lower in Smad3-MSCs.treated with PD98059 than in those without PD98059 treatment, no significant difference was found between them (P＞0.05). It is concluded that the wild-type Smad3 gene, which is a crucial component promoting bone formation,can inhibit the proliferation of MSCs and enhance the osteoblastic differentiation of uncommitted MSCs and the maturation of committed MSCs independent of the ERK signaling pathway.