Abstract
Transforming growth factor-β1 (TGF-β1) signaling plays a key role in vertebrate development, homeostasis, and disease. Nuclear factor I-C (NFI-C) has been implicated in TGF-β1 signaling, extracellular matrix gene transcription, and tooth root development. However, the functional relationship between NFI-C and TGF-β1 signaling remains uncharacterized. The purpose of this study was to identify the molecular interactions between NFI-C and TGF-β1 signaling in mouse odontoblasts. Real-time polymerase chain reaction and western analysis demonstrated that NFI-C expression levels were inversely proportional to levels of TGF-β1 signaling molecules during in vitro odontoblast differentiation. Western blot and immunofluorescence results showed that NFI-C was significantly degraded after TGF-β1 addition in odontoblasts, and the formation of the Smad3 complex was essential for NFI-C degradation. Additionally, ubiquitination assay results showed that Smurf1 and Smurf2 induced NFI-C degradation and polyubiquitination in a TGF-β1-dependent manner. Both kinase and in vitro binding assays revealed that the interaction between NFI-C and Smurf1/Smurf2 requires the activation of the mitogen-activated protein kinase pathway by TGF-β1. Moreover, degradation of NFI-C induced by TGF-β1 occurred generally in cell types other than odontoblasts in normal human breast epithelial cells. In contrast, NFI-C induced dephosphorylation of p-Smad2/3. These results show that crosstalk between NFI-C and TGF-β1 signaling regulates cell differentiation and homeostatic processes in odontoblasts, which might constitute a common cellular mechanism.
Highlights
Tooth formation is regulated by sequential and reciprocal epithelial-mesenchymal interactions
To determine the stage of odontoblast differentiation in vitro, MDPC-23 cells were cultured in differentiation medium for up to 3 weeks and the expression levels of odontoblast differentiation markers were analyzed by western blot and realtime polymerase chain reaction (PCR)
The results of this study imply that a functional relationship exists between Nuclear factor I-C (NFI-C) and Transforming growth factor-b1 (TGF-b1) signaling during odontoblast differentiation
Summary
Tooth formation is regulated by sequential and reciprocal epithelial-mesenchymal interactions. An essential role of odontoblasts is the production of a thick dentin layer that forms the bulk of the tooth. The nuclear factor I (NFI) family of site-specific transcription factors, encoded by four genes in vertebrates (i.e., Nfia, Nfib, Nfic, and Nfix), plays essential developmental roles in the transcriptional modulation of various cell types [4]. Disruption of the Nfi genes in mice leads to developmental defects in brain (Nfia) [5], lung and brain (Nfib) [6,7], and brain and skeleton (Nfix) [8]. Knockout of the NFI-C gene in mice results in tooth defects, including abnormal odontoblast differentiation during molar root formation and short root formation [9]
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