[Wild-type Smad3 gene promotes osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro].
OBJECTIVE
To observe the effect and mechanism of wild-type Smad3 gene on the proliferation and osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cell (MSC).


METHODS
Rat bone-derived MSC were cultured and transfected with the complexes of pcDNA3.0-Myc-Smad3 or pcDNA3.0-Myc-Smad3DeltaC and LipofectAMINE reagent. Immunofluorescence technique was performed to evaluate the c-Myc expression. The cell proliferation was detected by MTT method. In order to observe the osteoblastic characteristics in stable expression MSC, alkaline phosphatase (ALP) mRNA and core binding factor alpha1 (cbfa1) mRNA were investigated by RT-PCR technique, and ALP activity and mineralization were examined by PNP method and alizarin red staining respectively. PD98059 was used to interdict selectively ERK pathway in order to investigate the effect of ERK pathway on Smad3 regulating MSC.


RESULTS
c-Myc protein was expressed in Smad3-MSC and Smad3DeltaC-MSC. The proliferation of Smad3-MSC was slower than that of Smad3DeltaC-MSC or V-MSC. The relative levels of ALP mRNA and cbfa1 mRNA in Smad3-MSC were higher markedly than those in Smad3DeltaC-MSC or V-MSC, as well as ALP activity and mineralization. However, without a significant difference ALP activity and mineralization was after PD98059 treatment (both slightly decreased P > 0.05).


CONCLUSIONS
The wild-type Smad3 gene, which is a crucial component promoting bone formation, can inhibit the proliferation of MSC and promote the osteoblastic differentiation and maturation of MSC independent of ERK pathway.
Wang
Yun-Tao
YT
Department of orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zheng
Qi-Xin
QX
Guo
Xiao-Dong
XD
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010006:Osteoblasts / Q000166:cytology*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; D016271:Proto-Oncogene Proteins c-myc; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D015534:Trans-Activators