Dong-Seol Lee

Nuclear Factor I-C Is Essential for Odontogenic Cell Proliferation and Odontoblast Differentiation during Tooth Root Development

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.

The odontogenic ameloblast-associated protein (ODAM) cooperates with RUNX2 and modulates enamel mineralization via regulation of MMP-20.

We have previously reported that the odontogenic ameloblast‐associated protein (ODAM) plays important roles in enamel mineralization through the regulation of matrix metalloproteinase‐20 (MMP‐20). However, the precise function of ODAM in MMP‐20 regulation remains largely unknown. The aim of the present study was to uncover the molecular mechanisms responsible for MMP‐20 regulation. The subcellular localization of ODAM varies in a stage‐specific fashion during ameloblast differentiation. During the secretory stage of amelogenesis ODAM was localized to both the nucleus and cytoplasm of ameloblasts. However, during the maturation stage of amelogenesis, ODAM was observed in the cytoplasm and at the interface between ameloblasts and the enamel layer, but not in the nucleus. Secreted ODAM was detected in the conditioned medium of ameloblast‐lineage cell line (ALC) from days 14 to 21, which coincided with the maturation stage of amelogenesis. Interestingly, the expression of Runx2 and nuclear ODAM correlated with MMP‐20 expression in ALC. We therefore examined whether ODAM cooperates with Runx2 to regulate MMP‐20 and modulate enamel mineralization. Increased expression of ODAM and Runx2 augmented MMP‐20 expression, and Runx2 expression enhanced expression of ODAM, although overexpression of ODAM did not influence Runx2 expression. Conversely, loss of Runx2 in ALC decreased ODAM expression, resulting in down‐regulation of MMP‐20 expression. Increased MMP‐20 expression accelerated amelogenin processing during enamel mineralization. Our data suggest that Runx2 regulates the expression of ODAM and that nuclear ODAM serves an important regulatory function in the mineralization of enamel through the regulation of MMP‐20 apart from a different, currently unidentified, function of extracellular ODAM. J. Cell. Biochem. 111: 755–767, 2010.

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases.

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34(+) EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases.

Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

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.

Disruption of Nfic Causes Dissociation of Odontoblasts by Interfering With the Formation of Intercellular Junctions and Aberrant Odontoblast Differentiation

We reported previously that Nfic-deficient mice exhibit short and abnormal molar roots and severely deformed incisors. The objective of this study is to address the mechanisms responsible for these changes using morphological, IHC, and RT-PCR analysis. Nfic-deficient mice exhibited aberrant odontoblasts and abnormal dentin formation in molar roots and the labial crown analog of incisors. The most striking changes observed in these aberrant odontoblasts were the loss of intercellular junctions and the decreased expression of ZO-1 and occludin. As a result, they became dissociated, had a round shape, and lost their cellular polarity and arrangement as a sheet of cells. Furthermore, the dissociated odontoblasts became trapped in dentin-like mineralized tissue, resembling osteodentin in the overall morphology. These findings suggest that loss of the Nfic gene interferes with the formation of intercellular junctions that causes aberrant odontoblast differentiation and abnormal dentin formation. Collectively, these changes in odontoblasts contributed to development of molars with short and abnormal roots in Nfic-deficient mice. (J Histochem Cytochem 57:469–476, 2009)

NFI-C regulates osteoblast differentiation via control of osterix expression.

In bone marrow, bone marrow stromal cells (BMSCs) have the capacity to differentiate into osteoblasts and adipocytes. Age‐related osteoporosis is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow. In this study, we demonstrate that disruption of nuclear factor I‐C (NFI‐C) impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. Interestingly, NFI‐C controls postnatal bone formation but does not influence prenatal bone development. We also found decreased NFI‐C expression in osteogenic cells from human osteoporotic patients. Notably, transplantation of Nfic‐overexpressing BMSCs stimulates osteoblast differentiation and new bone formation, but inhibits adipocyte differentiation by suppressing peroxisome proliferator‐activated receptor gamma expression in Nfic−/− mice showing an age‐related osteoporosis‐like phenotype. Finally, NFI‐C directly regulates Osterix expression but acts downstream of the bone morphogenetic protein‐2‐Runx2 pathway. These results suggest that NFI‐C acts as a transcriptional switch in cell fate determination between osteoblast and adipocyte differentiation in BMSCs. Therefore, regulation of NFI‐C expression in BMSCs could be a novel therapeutic approach for treating age‐related osteoporosis. Stem Cells 2014;32:2467–2479

The effect of odontoblast conditioned media and dentin non-collagenous proteins on the differentiation and mineralization of cementoblasts in vitro

OBJECTIVE Cementum is an important mineralized tissue in root formation, however, the precise mechanism of cementum formation remains undetermined. The purpose of this study was to evaluate the effect of odontoblast conditioned media (CM) and dentin non-collagenous proteins (dNCPs) on the differentiation and mineralization of cementoblastic OCCM-30 cells. METHODS The CM of ameloblastic ALCs, odontoblastic MDPC-23 and OD-11, osteoblastic MG-63, and fibroblastic NIH3T3 cells were transferred to OCCM-30 cells. dNCPs were extracted directly from porcine and human dentin and applied to OCCM-30 cells. The results were evaluated through the analysis of the morphologic appearance, expression of cementum matrix genes, and the formation of mineralized nodules in vitro. RESULTS dNCPs hardly influenced proliferation, cell cycle modification, and chemotaxis of cementoblasts. Mineralization of cementoblasts was accelerated with dNCPs and CM from odontoblastic MDPC-23 and OD-11. RT-PCR analysis revealed the earlier and stronger expression of bone sialoprotein (BSP), alkaline phosphatase (ALP), and osteocalcin (OC) mRNAs in the MDPC23- and OD11-CM-treated OCCM-30 cells than those in the control OCCM-30 cells. The level of gene expression was also significantly higher in the dNCP-treated group than the control group. CONCLUSION These results suggest that dentin matrix proteins, or the secreted products of odontoblasts, induced cementoblast differentiation and mineralization. These findings may contribute to the development of a periodontal treatment that includes cementum regeneration.

Dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and Hertwig's epithelial root sheath/epithelial rests of Malassez cells through the Fas-Fas ligand pathway.

Hertwigs epithelial root sheath (HERS), epithelial rests of Malassez (ERM) cells, and reduced ameloblasts undergo apoptosis during tooth development. This study examined the effects of dental follicle cells and cementoblasts on the apoptosis of ameloblast-lineage and HERS/ERM cells derived from the enamel organ. We also elucidated the induction pathways and identified the apoptotic pathway involved in this process. Here, we showed terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL)-positive HERS cells and reduced ameloblasts near dental follicle cells during tooth development. Co-culturing ameloblast-lineage cell line (ALC) ameloblasts and HERS/ERM cells with either dental follicle cells or OCCM-30 cementoblasts markedly enhanced the apoptosis of ameloblasts and HERS/ERM cells compared with cells cultured alone. However, dental follicle cells and cementoblasts did not modulate the apoptotic responses of co-cultured non-odontogenic MCF10A or KB cells. When ameloblasts + HERS and cementoblasts + dental follicle cells were co-cultured, the expression of Fas ligand (FasL) increased in cementoblasts + dental follicle cells, while the expression of Fas increased in ameloblasts + HERS. Interestingly, recombinant FasL induced ameloblast apoptosis while the cementoblast-induced ameloblast apoptosis was suppressed by the Fas/FasL antagonist Kp7-6. These results suggest that during tooth development, dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and HERS/ERM cells through the Fas-FasL pathway, but do not induce the apoptosis of non-odontogenic epithelial cells.

CPNE7, a preameloblast-derived factor, regulates odontoblastic differentiation of mesenchymal stem cells

Tooth development involves sequential interactions between dental epithelial and mesenchymal cells. Our previous studies demonstrated that preameloblast-conditioned medium (PA-CM) induces the odontogenic differentiation of human dental pulp cells (hDPCs), and the novel protein Cpne7 in PA-CM was suggested as a candidate signaling molecule. In the present study, we investigated biological function and mechanisms of Cpne7 in regulation of odontoblast differentiation. Cpne7 was expressed in preameloblasts and secreted extracellularly during ameloblast differentiation. After secretion, Cpne7 protein was translocated to differentiating odontoblasts. In odontoblasts, Cpne7 promoted odontoblastic markers and the expression of Dspp in vitro. Cpne7 also induced odontoblast differentiation and promoted dentin/pulp-like tissue formation in hDPCs in vivo. Moreover, Cpne7 induced differentiation into odontoblasts of non-dental mesenchymal stem cells in vitro, and promoted formation of dentin-like tissues including the structure of dentinal tubules in vivo. Mechanistically, Cpne7 interacted with Nucleolin and modulated odontoblast differentiation via the control of Dspp expression. These results suggest Cpne7 is a diffusible signaling molecule that is secreted by preameloblasts, and regulates the differentiation of mesenchymal cells of dental or non-dental origin into odontoblasts.

Tertiary Dentin Formation after Direct Pulp Capping with Odontogenic Ameloblast-associated Protein in Rat Teeth

INTRODUCTION Odontogenic ameloblast-associated protein (ODAM) has been shown to be specifically expressed in ameloblasts and odontoblasts and has been suggested to play a role in the mineralization of the enamel, possibly through the regulation of matrix metalloproteinase 20. However, its function in dentin is not clear. The purpose of this study was to evaluate the effect of ODAM on tertiary dentin formation. METHODS MDPC-23 odontoblastic cells were cultured, and the effect of recombinant ODAM (rODAM) on mineralized nodule formation was evaluated. Pinpoint pulp exposures were made in rat teeth and then capped with rODAM mixed with a carrier (rODAM group), carrier only (Carrier group), or white mineral trioxide aggregate (WMTA group). After 1, 2, and 4 weeks, odontoblasts and tertiary dentin were investigated histologically and immunohistochemically. RESULTS Nodule formation in MDPC-23 cells was enhanced by rODAM treatment. Odontoblasts were polarized and showed a palisade arrangement in the remaining pulp from the rODAM group, but not the Carrier or WMTA groups. In the WMTA group, extensive tertiary dentin along the entire pulp-dentin border obliterated the pulp chamber. In contrast, in the rODAM group, limited reaction of odontoblasts resulted in normal pulp tissue appearance without excessive tertiary dentin formation and obliteration of the pulp cavity. In the Carrier and WMTA groups, bone sialoprotein was immunostained in most of the tertiary dentin, whereas in the rODAM group, dentin sialoprotein expression was immunostained primarily in newly formed reactionary dentin. CONCLUSIONS These results suggest that rODAM accelerates reactionary dentin formation close to the pulp exposure area, thereby preserving normal odontoblasts in the remaining pulp.