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.