-deficient mice, both the expression of TGF-β1 and TGFβ-RI, and the phosphorylation of Smad2/3 increase during odontoblast differentiation, leading to formation of abnormal dentin similar to osteodentin 
. Mice in which NFI-C is disrupted are morphologically similar in terms of tooth dentin to TGF-β1-overexpressing transgenic mice 
. The results of this study imply that a functional relationship exists between NFI-C and TGF-β1 signaling during odontoblast differentiation. Here, we investigated the mechanism of odontoblast differentiation by examining the mutual interaction between NFI-C and TGF-β1 signaling, and the degradation and ubiquitination of NFI-C by TGF-β1.
TGF-β1 induces cell growth arrest by activating p21, the cyclin-dependent kinase inhibitor, through the Smad signaling pathway 
. Activation of p21 also induces keratinocyte differentiation 
. Interestingly, NFI is an important factor in the inhibition of p21 expression in proliferating Hela cells 
. In the present study, TGF-β1 induced the degradation of the NFI-C protein in both the cytoplasm and nucleus through the proteasome pathway, and NFI-C was degraded by the overexpression of Smad2 and Smad3. However, when Smad3 was inactivated by knockdown, TGF-β1 could not affect the degradation of NFI-C. Based on these findings, we suggest that TGF-β1 induces odontoblast differentiation through the Smad signaling pathway by increasing p21 and TGF-β-responsive gene expression levels via the degradation of NFI-C, which suppresses p21 expression.
In the present study, we showed that NFI-C interacts with Smad3 and is degraded in the cytoplasm and nucleus, which increases in response to stimulation with TGF-β1. Interestingly, the amount of Smad3 binding to NFI-C after TGF-β1 stimulation is increased in cytoplasm but decreased in the nucleus. Therefore, we suggest that in the cytoplasm, phosphorylated Smad3 induced by TGF-β1 stimulation increases binding to NFI-C, and results in the degradation of NFI-C.
NFI-C does not contain a polyproline-tyrosine motif, whereas a database search identified many SP sites and Ser/Thr protein kinase phosphorylation sites (SSXS), in the C-terminal region of NFI-C 
. Recently, Smurf1 binding was shown to require the phosphorylation of SP sites located upstream of the PPAY motif in Smad1. MAPK-induced phosphorylation of the Smad1 linker region through bone morphogenetic protein signaling is recognized by Smurf1 and leads to the ubiquitination and degradation of Smad1 
. TGF-β1 also enhances the phosphorylation of the Smad2/3 linker regions by MAPK, which is involved with E3 Ub ligases, such as Nedd4L 
. In the present study, we show that the interaction between NFI-C and Smurf1/Smurf2 were also required for the activation of MAPK-ERK by TGF-β1 signaling. Interestingly, TGF-β1-induced NFI-C binding to Smurf1 and Smurf2 was inhibited not by a combination of a p38 inhibitor but by a JNK inhibitor (data not shown). It suggests that JNK pathway is also involved in the interaction between NFI-C and Smurf1/Smurf2 induced by TGF-β1. Therefore, JNK pathway needs further investigation. Possible interaction motifs regulating the binding of NFI-C and either Smurf1 or Smurf2 are also currently under investigation.
When Smad signaling is continuously inhibited by the absence of TGF-β signaling, endogenous levels of Smad2 and Smad3 are negatively regulated by a number of proteins, including Smurfs. Smad2 and Smad3 show enhanced ubiquitination and degradation by Smurf2 and WW domain-containing protein 2 
. In addition, the Axin and Ub ligase complex ROCK1 induces the degradation and ubiquitination of Smad3 
. Therefore, we investigated whether the expression of Smad2/3 and/or Smad3 protein was affected by NFI-C in odontoblasts. NFI-C overexpression remarkably decreased the level of p-Smad2/3 and p-Smad3 protein via the dephosphorylation of p-Smad2/3 and p-Smad3 in MDPC-23 cells. These results suggest that NFI-C is a novel negative regulator of TGF-β1 signaling during odontoblast differentiation in tooth development. Recently, it was reported that NFI-C acts primarily to repress gene expression in response to TGF-β1 during wound healing 
. This report has also provided evidence in support of our data.
To investigate whether the NFI-C interaction with TGF-β1 occurs in cell types other than odontoblasts, we measured the effects of TGF-β1 treatment on endogenous NFI-C protein levels in the human breast epithelial cell line, MCF10A. Expression of NFI-C was clearly observed in MCF10A cells. The level of NFI-C protein decreased in response to TGF-β1 treatment (Figure S7
). These results demonstrate that degradation of NFI-C induced by TGF-β1 occurs generally in cell types other than odontoblasts in normal human breast epithelial cells.
In conclusion, as shown in , these results demonstrate that interactions between NFI-C and TGF-β1 signaling play key roles in the regulation of odontoblast differentiation and homeostasis.
A proposed model of the function of NFI-C and TGF-β1 during odontoblast differentiation and homeostasis.