| Transforming growth factor β-1 (TGF-β 1) is a multifunctional growth factor regulating many cellular processes, including cell proliferation, differentiation, apoptosis, matrix synthesis and the immune response. TGF-pl elicits its biological effects via heteromeric complexs of transmembrane serine/theronine kinases, type I and type II receptor. Upon ligand binding, the type II receptor recruits and activates the type I receptor. This activation causes recruitment and phosphorylation of pathway-restricted proteins, Smad2 or SmadS. The phosphorylated Smad2 or Smad3 form complexs with Smad4, a common-mediated Smad. The heteromeric Smads complexs translocate into the nucleus, where they regulate gene transcription by either association with DNA-binding protein or direct binding to promoter sequences of target genes. Smad7 belongs to the inhibitory Smads and acts as inhibitor of the TGF-β signaling pathway, probably by competitive interaction with the type I receptor or with Smad4. TGF-β 1 has been implicated in the regulation of odontoblast differentiation and dentin formation. TGF-β 1 mRNA is expressed by preodontoblasts and odontoblasts. After secreted by differentiated odontoblasts, TGF-pl is sequestered within thedentin extracellular matrix (ECM), where it can participate in tertiary dentinogenesis after pulp injury. TGF-pl has been shown to be an active component of dentin ECM associated with the regulation of cell growth, differentiation, and matrix synthesis. Studies of amelogenesis and dentinogenesis have shown an upregulation of TGF-pl mRNA at the onset of matrix formation and low levels during early odontogenesis. Other studies have shown that TGF-pl can influence the differentiation of odontoblasts within cultured dental papilla mesenchyme. TGF-pl (-/-) mice showed a progressive loss of dental hard tissues through a gradual destruction or wearing of the enamel and underlying dentin. Evidence in transgenic mice overexpressing TGF-pl suggests that TGF-pl mediates the expression of DSPP which is required for normal tooth mineralization. Despite these advances, the molecular mechanisms of TGF-pl regulating odontoblast differentiation and ECM biosynthesis remain unresolved.The objectives of this study were to investigate the role of Smad signaling in odontoblast differentiation and ECM biosynthesis regulated by TGF-pl, and to explore the molecular mechanism of TGF-pl action during odontoblast differentiation and ECM biosynthesis.The present study consists of three parts:Part One: Smads in MDPC-23: expression, regulation and roles of TGF-pl signalingIn this part, we firstly investigated the characterization of odontoblast-like cell line, MDPC-23, which is kindly provided by Dr. Jacques E. Nor. The results showed that MDPC-23 retained their specific features which included 1) epithelioid morphology of all cells with multiple cell membrane processes; 2) formation of multilayered nodules and a cell doubling time of less than 24h; 3) expression of type I collagen, ALP, OPN, OC and DSPP.Secondly, we investigated the expression, regulation and role of Smad in MDPC-23 cells. Semi-quantitive RT-RCR analysis revealed that MDPC-23 cells expressed Smad2, Smad3 and Smad7 mRNA, the level of Smad3 mRNA was down-regulated by TGF-pl after 4h of treatment, whereas Smad2 was less affected. Smad7 mRNA was rapidly induced by TGF-pl, with a maximal peak of expression after 90 min stimulation, and reduced gradually to low level thereafter. Immunohistochemical analysis demonstrated that treatment of TGF-p1 for 1h resulted in translocation of Smad2 and Smad3 from cytoplasm to the nucleus, whereas a marked increase of intensity of Smad7 in the cytoplasm, however, no significant change in the intracellular distribution of SmadV. Effects of Smads on thetranscriptional activity of TGF-pl were examined in cotransfection experiments using a TGF-p-responsive reporter gene construct, p3TP-Lux. It showed that Smad3, but not Smad2, enh...
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