TGF-β activated Smad signalling leads to a Smad3-mediated down-regulation of DSPP in an odontoblast cell line

Objective: Transforming growth factor-β (TGF-β) regulates odontoblast differentiation and stimulates dentine extracellular matrix synthesis. However, until recently, the molecular mechanisms of action of TGF-β have been unknown. Smad proteins have recently been identified as intracellular signalling mediators of TGF-β. In this study, we characterise the role of Smad proteins as mediators of TGF-β 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-β1 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-β1. The activity of the TGF-β-responsive p3TP-Lux reporter construct was stimulated by 12.7-fold with TGF-β1 treatment. Over-expression of wild-type Smad3 promoted TGF-β1-induced luciferase activity, whereas dominant negative Smad3 inhibited it. TGF-β1 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-β1 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-β1 regulation of the promoter activity of DSPP. Conclusions: Smad2, Smad3 and Smad4 are present and activated by TGF-β1 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-β1. These findings raise the possibility that Smad signalling plays a role in dentinogenesis.