TGF-beta activated Smad signalling leads to a Smad3-mediated down-regulation of DSPP in an odontoblast cell line.
OBJECTIVE
Transforming growth factor-beta (TGF-beta) regulates odontoblast differentiation and stimulates dentine extracellular matrix synthesis. However, until recently, the molecular mechanisms of action of TGF-beta have been unknown. Smad proteins have recently been identified as intracellular signalling mediators of TGF-beta. In this study, we characterise the role of Smad proteins as mediators of TGF-beta in a mouse odontoblast cell line MDPC-23.


METHODS
Transcription of Smads was detected by RT-PCR. The change of intracellular location of Smad proteins treated by TGF-beta1 was evaluated immunocytochemically. Smad function and its role in transcription of dentin sialophosphoprotein (DSPP) were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs.


RESULTS
MDPC-23 cells expressed Smad2, Smad3 and Smad4 mRNA. Endogenous Smad2, Smad3 and Smad4 rapidly translocated from the cytoplasm into the nucleus in response to TGF-beta1. The activity of the TGF-beta-responsive p3TP-Lux reporter construct was stimulated by 12.7-fold with TGF-beta1 treatment. Over-expression of wild-type Smad3 promoted TGF-beta1-induced luciferase activity, whereas dominant negative Smad3 inhibited it. TGF-beta1 also inhibited the activity of DSPP promoter luciferase reporter construct containing the sequence between -791 bp and +54 bp of the mouse DSPP gene. Over-expression of wild-type Smad3 potentiate the inhibitory effect of TGF-beta1 on transcriptional regulation of DSPP, while dominant negative Smad3 decreased the effect. In contrast to Smad3, wild-type Smad2 or its dominant negative mutant had little effect on TGF-beta1 regulation of the promoter activity of DSPP.


CONCLUSIONS
Smad2, Smad3 and Smad4 are present and activated by TGF-beta1 in MDPC-23 cells. The Smad pathway is functional in these cells and Smad3 appears to be involved in down-regulation of DSPP by TGF-beta1. These findings raise the possibility that Smad signalling plays a role in dentinogenesis.
He
Wen-Xi
WX
Department of Operative Dentistry, Qin Du Stomatological Hospital, Xi'an, 710032, PR China. hwenxi@yahoo.com.cn
Niu
Zhong-Ying
ZY
Zhao
Shou-Liang
SL
Jin
Wei-Lin
WL
Gao
Jie
J
Smith
Anthony J
AJ
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D003804:Dentin / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D016326:Extracellular Matrix Proteins; D007124:Immunoenzyme Techniques; D051379:Mice; D009804:Odontoblasts / Q000378:metabolism*; D010750:Phosphoproteins; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012795:Sialoglycoproteins; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins; D051900:Smad3 Protein; D015534:Trans-Activators / Q000502:physiology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D000099311:Dentin Sialophosphoprotein
D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D011498:Protein Precursors; D012795:Sialoglycoproteins; D051785:Smad Proteins; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000099311:Dentin Sialophosphoprotein; C497791:Smad3 protein, mouse; C508961:Tgfb1 protein, mouse