Abstract
Our previous studies have demonstrated that nuclear factor I-C (NFI-C) null mice developed short molar roots that contain aberrant odontoblasts and abnormal dentin formation. Based on these findings, we performed studies to elucidate the function of NFI-C in odontoblasts. Initial studies demonstrated that aberrant odontoblasts become dissociated and trapped in an osteodentin-like mineralized tissue. Abnormal odontoblasts exhibit strong bone sialoprotein expression but a decreased level of dentin sialophosphoprotein expression when compared with wild type odontoblasts. Loss of Nfic results in an increase in p-Smad2/3 expression in aberrant odontoblasts and pulp cells in the subodontoblastic layer in vivo and primary pulp cells from Nfic-deficient mice in vitro. Cell proliferation analysis of both cervical loop and ectomesenchymal cells of the Nfic-deficient mice revealed significantly decreased proliferative activity compared with wild type mice. In addition, Nfic-deficient primary pulp cells showed increased expression of p21 and p16 but decreased expression of cyclin D1 and cyclin B1, strongly suggesting cell growth arrest caused by a lack of Nfic activity. Analysis of the pulp and abnormal dentin in Nfic-deficient mice revealed an increase in apoptotic activity. Further, Nfic-deficient primary pulp cells exhibited an increase in caspase-8 and -3 activation, whereas the cleaved form of Bid was hardly detected. These results indicate that the loss of Nfic leads to the suppression of odontogenic cell proliferation and differentiation and induces apoptosis of aberrant odontoblasts during root formation, thereby contributing to the formation of short roots.
Highlights
Rived ectomesenchyme (EM).2 The dental epithelium gives rise to the outer and inner enamel epithelium from which ameloblasts differentiate, whereas EM cells differentiate into odontoblasts
We previously reported that Nficdeficient mice develop short roots with aberrant odontoblasts that exhibit unique morphological features [29]
Transforming growth factor (TGF)-1-overexpressing transgenic mice develop distinct dentin defects similar to those seen in Nfic-deficient mice [20]
Summary
Antibodies—Antisera against NFI-C, DSP, and BSP [24] were produced by immunization of rabbit with the synthetic peptides NH2-RPTRPLQTVPLWD-COOH (amino acid residues 427ϳ439 of NFI-C), NH2-GNKSIITKESGKLSGS-COOH (amino acid residues 372ϳ387 of DSP), and NH2-RRIKAEDSEENGVFKYR-COOH (amino acid residues 24ϳ40 of BSP). The DSPP promoter (pGL3LUC791ϳϩ54) plasmid was a kind gift from Dr W.-X He (Department of Operative Dentistry, Qin Du Stomatological, Xian, China). The deparaffinized sections were immersed in 0.6% H2O2/methanol for 20 min to quench the endogenous peroxidase activity They were preincubated with 1% bovine serum albumin in PBS for 30 min and incubated overnight at 4 °C with rabbit polyclonal DSP, BSP (1:100), or p-Smad2/3 (1:200; Santa Cruz Biotechnology) antibodies. BrdUrd-labeled cells in 5-m-thick sections were identified using a BrdUrd staining kit (Zymed Laboratory) according to the manufacturer’s instructions and counterstained with hematoxylin. The endogenous peroxidase within the tissue sections was inactivated by incubation for 10 min in 3% H2O2 before enzymatic labeling. Reverse Transcription (RT)-PCR and Real Time PCR Analysis— Total RNA was extracted from the primary pulp cells with TRIzol reagent according to the manufacturer’s instructions (Invitrogen). One microliter of the reverse transcription product was subjected to
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