Tooth root morphogenesis involves two biological processes, root elongation and dentinogenesis, which are guaranteed by downgrowth of Hertwig's epithelial root sheath (HERS) and normal odontoblast differentiation. Ubiquitin-dependent protein degradation has been reported to precisely regulate various physiological processes, while its role in tooth development is still elusive. Here we show ubiquitin-specific protease 34 (USP34) plays a pivotal role in root formation. Deletion of Usp34 in dental mesenchymal cells leads to short root anomaly, characterized by truncated roots and thin root dentin. The USP34-deficient dental pulp cells (DPCs) exhibit decreased odontogenic differentiation with downregulation of nuclear factor I/C (NFIC). Overexpression of NFIC partially restores the impaired odontogenic potential of DPCs. These findings indicate that USP34-dependent deubiquitination is critical for root morphogenesis by stabilizing NFIC.
Cell Differentiation ; Female ; Morphogenesis ; NFI Transcription Factors ; Odontogenesis ; Tooth Root

Nuclear factor I-C (NFI-C) plays a pivotal role in various cellular processes such as odontoblast and osteoblast differentiation. Nfic-deficient mice showed abnormal tooth and bone formation. The transplantation of Nfic-expressing mouse bone marrow stromal cells rescued the impaired bone formation in Nfic(-/-) mice. Studies suggest that NFI-C regulate osteogenesis and dentinogenesis in concert with several factors including transforming growth factor-β1, Krüppel-like factor 4, and β-catenin. This review will focus on the function of NFI-C during tooth and bone formation and on the relevant pathways that involve NFI-C.
Animals ; Bone Development* ; Dentinogenesis ; Mesenchymal Stromal Cells ; Mice ; NFI Transcription Factors* ; Odontoblasts ; Osteoblasts ; Osteogenesis ; Osteoporosis ; Tooth*

PURPOSE: Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. Deletion of the Wntless (Wls) gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation. However, it remains unclear if autonomous Wnt ligands are necessary for differentiation of dental pulp cells into odontoblast-like cells to induce reparative dentinogenesis, one of well-known feature of pulp repair to form tertiary dentin. MATERIALS AND METHODS: To analyze the autonomous role of Wls for differentiation of dental pulp cells into odontoblast-like cells, we used primary dental pulp cells from unerupted molars of Wls-floxed allele mouse after infection with adenovirus for Cre recombinase expression to knockout the floxed Wls gene or control GFP expression. The differentiation of dental pulp cells into odontoblast-like cells was analyzed by quantitative real-time polymerase chain reaction. RESULT: Proliferation rate was significantly decreased in dental pulp cells with Cre expression for Wls knockout. The expression levels of Osterix (Osx), runt-related transcription factor 2 (Runx2), and nuclear factor I-C (Nfic) were all significantly decreased by 0.3-fold, 0.2-fold, and 0.3-fold respectively in dental pulp cells with Wls knockout. In addition, the expression levels of Bsp, Col1a1, Opn, and Alpl were significantly decreased by 0.7-fold, 0.3-fold, 0.8-fold, and 0.6-fold respectively in dental pulp cells with Wls knockout. CONCLUSION: Wnt ligands produced autonomously are necessary for proper proliferation and odontoblastic differentiation of mouse dental pulp cells toward further tertiary dentinogenesis.
Adenoviridae ; Alleles ; Animals ; Dental Pulp* ; Dentin ; Dentinogenesis ; Epithelium ; Ligands ; Mesoderm ; Mice* ; Molar ; Morphogenesis ; NFI Transcription Factors ; Odontoblasts* ; Real-Time Polymerase Chain Reaction ; Recombinases ; Tooth ; Transcription Factors

Molecular interactions between epithelium and mesenchyme are important for root formation. Nuclear factor I-C (Nfic) has been identified as a key regulator of root formation. However, the mechanisms of root formation and their interactions between Hertwig's epithelial root sheath (HERS) and mesenchyme remain unclear. In this study, we investigated the role of Nfic in root patterning and growth during molar root development. The molars of Nfic knockout mice exhibited an enlarged pulp chamber and apical displacement of the pulpal floor, characteristic features of taurodontism, due to delayed furcation formation. In developing molar roots of mutant mice at P14, BrdU positive cells decreased in the apical mesenchyme of the elongation region whereas those cells increased in the dental papilla of the furcation region. Whereas cytokeratin 14 and laminin were localized in HERS cells of mutant molars, Smoothened (Smo) and Gli1 were downregulated in preodontoblasts. In contrast, cytokeratin 14 and Smo were localized in the cells of the furcation region of mutant molars. These results indicate that Nfic regulates cell proliferation in the dental mesenchyme and affects the fate of HERS cells in a site-specific manner. From the results, it is suggested that Nfic is required for root patterning and growth during root morphogenesis.
Animals ; Bromodeoxyuridine ; Cell Proliferation ; Dental Papilla ; Dental Pulp Cavity ; Epithelium ; Keratin-14 ; Laminin ; Mesoderm ; Mice ; Mice, Knockout ; Molar ; Morphogenesis ; NFI Transcription Factors ; Tooth Root* ; Tooth*

OBJECTIVETo investigate the effect of nuclear factor I-C (NFI-C) on platelet-derived growth factor (PDGF)-induced up-regulation of TGF-β receptor II (TβRII) in dermal fibroblasts.

METHODSA lentiviral vector containing NFI-C sequence (Lenti-GFP-NFI-C) was transfected into a human foreskin fibroblast cell line (HFF-1). Cultured HFF-1 cells, cells transfected with Lenti-GFP-NFI-C, and cells transfected with a negative virus were stimulated with PDGF-BB, and Western blotting and RT-qPCR were used to detect the expression levels of TβRII in the treated cells.

RESULTSPDGF treatment significantly increased the expression level of TβRII in HFF-1 cells (P<0.05). The cells transfected with Lenti-GFP-NFI-C expressed a significantly lower level of TβRII than non-transfected cells in response to PDGF stimulation (P<0.05), but the negative virus showed no such inhibitory effect (P>0.05). No significant difference was found in the expression level of TβRII protein between cells transfected with Lenti-GFP-NFI-C-transfection before PDGF stimulation and the blank control cells.

CONCLUSIONNFI-C can inhibit PDGF-induced up-regulation of TβRII and thus reduce the sensitivity of the dermal fibroblasts to TGF-β.

Cell Line ; Fibroblasts ; drug effects ; Genetic Vectors ; Humans ; Lentivirus ; NFI Transcription Factors ; genetics ; Platelet-Derived Growth Factor ; pharmacology ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-sis ; Receptors, Transforming Growth Factor beta ; metabolism ; Transfection ; Transforming Growth Factor beta ; pharmacology ; Up-Regulation

OBJECTIVETo study the genetic pathogenesis of myasthenia gravis (MG) caused by cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene polymorphisms and regulation function of transcription factor.

METHODSELISA assay was used to determine the expression level of serum sCTLA-4 in MG. Four single nucleotide polymorphisms (SNPs) of CTLA-4 at exon 1 +49, promoter -318, -1661, -1772 were analyzed by restriction fragment length polymorphism (RFLP). Transcription factor nuclear factor 1(NF-1) and c/EBPbeta binding site were confirmed by chromatin immunoprecipitation(CHIP) assay.

RESULTSIt was found that the frequencies of the GG+49 genotype and G+49 allele are higher in MG patients with thymoma than those in patients of thymic hyperplasia and normal thymus subgroups. T/C-318 is not correlated with MG. The frequency of CT-1772 genotype is significantly higher in MG patients, especially in MG patients with thymoma, when compared with that in healthy controls. Meanwhile, the frequency of the G-1661 allele and GG-1661 genotype is lower in MG patients. Linkage disequilibrium (LD) between each SNPs in promoter -1772, -1661, -318 and coding sequence 1 (CDS 1) +49 is apparent. sCTLA-4 levels in patients' sera are correlated with the haplotype and genotype. T/C-1772 and A/G-1661 SNPs change the sequence of transcription factor NF-1 and c/EBPbeta binding sites. DNA variants lose site-specific binding activity of transcription factor regulated by lectin ConA and PHA.

CONCLUSIONThere are strong positive linkages among four SNPs. C/T-1772 and A/G-1661 polymorphisms can result in inefficient transcription of CTLA-4 gene. T>C-1772 mutation also affects gene splicing. These SNPs may constitute a factor of susceptibility to disease.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; Antigens, Differentiation ; blood ; genetics ; CCAAT-Enhancer-Binding Protein-beta ; genetics ; CCAAT-Enhancer-Binding Proteins ; genetics ; CTLA-4 Antigen ; Exons ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Myasthenia Gravis ; genetics ; immunology ; NFI Transcription Factors ; Point Mutation ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Thymoma ; genetics ; Thymus Hyperplasia ; genetics ; Thymus Neoplasms ; genetics ; Transcription Factors ; genetics

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.
Actins ; analysis ; Activating Transcription Factor 2 ; genetics ; Age Factors ; Ameloblasts ; pathology ; Amelogenesis ; genetics ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; Cell Adhesion Molecules ; analysis ; Cell Differentiation ; genetics ; Cementogenesis ; genetics ; Dental Cementum ; pathology ; Dental Pulp ; blood supply ; Fluorescent Dyes ; Green Fluorescent Proteins ; Male ; Mice ; Mice, Knockout ; Microvessels ; pathology ; Molar ; growth & development ; Molar, Third ; growth & development ; NFI Transcription Factors ; analysis ; Odontoblasts ; pathology ; Odontogenesis ; genetics ; Periodontal Ligament ; growth & development ; Sp7 Transcription Factor ; Stem Cells ; physiology ; Tooth Root ; growth & development ; Transcription Factors ; genetics ; Vascular Endothelial Growth Factor A ; analysis ; Zinc Fingers ; genetics