Eui-Sic Cho

β-catenin is Required in Odontoblasts for Tooth Root Formation:

The tooth root is an important part of the tooth that works together with the surrounding periodontium to maintain the tooth in the alveolar socket. The root develops after crown morphogenesis. While the molecular and cellular mechanisms of early tooth development and crown morphogenesis have been extensively studied, little is known about the molecular mechanisms controlling tooth root formation. Here, we show that β-catenin is strongly expressed in odontoblast-lineage cells and is required for root formation. Tissue-specific inactivation of β-catenin in developing odontoblasts produced molars lacking roots and aberrantly thin incisors. At the beginning of root formation in the mutant molars, the cervical loop epithelium extended apically to form Hertwig’s epithelial root sheath (HERS), but root odontoblast differentiation was disrupted and followed by the loss of some HERS inner layer cells. However, the outer layer of the HERS extended without the root, and the mutant molars finally erupted. The periodontal tissues extensively invaded the dental pulp. These results indicate that there is a cell-autonomous requirement for Wnt/β-catenin signaling in the dental mesenchyme for root formation.

Excessive Wnt/β-catenin signaling disturbs tooth-root formation

BACKGROUND AND OBJECTIVE Wingless-type MMTV integration site family (Wnt)/β-catenin signaling plays an essential role in cellular differentiation and matrix formation during skeletal development. However, little is known about its role in tooth-root formation. In a previous study, we found excessive formation of dentin and cementum in mice with constitutive β-catenin stabilization in the dental mesenchyme. In the present study we analyzed the molar roots of these mice to investigate the role of Wnt/β-catenin signaling in root formation in more detail. MATERIAL AND METHODS We generated OC-Cre:Catnb(+/lox(ex3)) mice by intercrossing Catnb(+/lox(ex3)) and OC-Cre mice, and we analyzed their mandibular molars using radiography, histomorphometry and immunohistochemistry. RESULTS OC-Cre:Catnb(+/lox(ex3)) mice showed impaired root formation. At the beginning of root formation in mutant molars, dental papilla cells did not show normal differentiation into odontoblasts; rather, they were prematurely differentiated and had a disorganized arrangement. Interestingly, SMAD family member 4 was upregulated in premature odontoblasts. In 4-wk-old mutant mice, molar roots were about half the length of those in their wild-type littermates. In contrast to excessively formed dentin in crown, root dentin was thin and hypomineralized in mutant mice. Biglycan and dentin sialophosphoprotein were downregulated in root dentin of mutant mice, whereas dentin matrix protein 1 and Dickkopf-related protein 1 were upregulated. Additionally, ectonucleotide pyrophosphatase/phosphodiesterase 1 was significantly downregulated in the cementoblasts of mutant molars. Finally, in the cementum of mutant mice, bone sialoprotein was downregulated but Dickkopf-related protein 2 was upregulated. CONCLUSION These results suggest that temporospatial regulation of Wnt/β-catenin signaling plays an important role in cell differentiation and matrix formation during root and cementum formation.

The role of PIN1 on odontogenic and adipogenic differentiation in human dental pulp stem cells.

Recently, the involvement of PIN1, a peptidyl-prolyl cis/trans isomerase, has been reported in age-related bone homeostasis and adipogenesis. However, the role of PIN1 during odontogenic and adipogenic differentiation remains to be fully understood, particularly regarding human dental pulp stem cells (HDPSCs). Thus, in the present study, we have investigated the role of PIN1 in odontogenic and adipogenic differentiation of HDPSCs and signaling pathways possibly involved. PIN1 mRNA and protein level were upregulated in a time-dependent manner during adipogenic differentiation, increasing until 1 day of odontogenic induction and then steadily declined during odontogenic differentiation. Treatment of a known PIN1 inhibitor, juglone, significantly increased odontogenic differentiation as confirmed by alkaline phosphatase (ALP) activity, calcium deposition, and mRNAs induction of odontogenic markers [ALP, osteopontin (OPN), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP-1)]. On the contrary, adipogenic differentiation was dramatically reduced upon juglone treatment, with concomitant downregulation of lipid droplet accumulation and adipogenic marker genes [peroxisome proliferation-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (AP2)]. In contrast to PIN1 inhibition, the overexpression of PIN1 via adenoviral infection (Ad-PIN1) in HDPSCs inhibited odontogenic differentiation but increased adipogenic differentiation, in which stem cell property markers such as stage-specific embryonic antigen-4 (SSEA-4) and STRO-1 were upregulated during odontogenic differentiation but downregulated in adiopogenic differentiation. Consistently, juglone-mediated inhibition of PIN1 augmented the osteogenic medium (OM)-induced activation of bone morphogenetic protein (BMP), Wnt/β-catenin, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB) pathway, which response was reversed by Ad-PIN1. Moreover, juglone blocked the adipogenic induction medium-induced activation of PPARγ, C/EBPα, C/EBPβ, ERK, and NF-κB pathways, which was rescued by Ad-PIN1 infection. In summary, the present study shows for the first time that PIN1 acts as an important modulator of odontogenic and adipogenic differentiation of HDPSCs and may have clinical implications for regenerative dentistry.

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 effects of rosiglitazone on osteoblastic differentiation, osteoclast formation and bone resorption

Rosiglitazone has the potential to activate peroxisome proliferator-activated receptor-γ (PPARγ), which in turn can affect bone formation and resorption. However, the mechanisms by which rosiglitazone regulates osteoclastic or osteoblastic differentiation are not fully understood. This study examines how rosiglitazone affects osteoclast formation, bone resorption and osteoblast differentiation from mouse bone marrow. Rosiglitazone treatment not only inhibited the formation of tartrate-resistant acid phosphatase-positive cells, but also prevented pit formation by bone marrow cells in a dose- and time-dependent manner. Rosiglitazone also suppressed the receptor activator of nuclear factor (NF)-κB ligand (RANKL) receptor (RANK) expression but increased PPARγ2 expression in the cells. In addition, rosiglitazone diminished RANKL-induced activation of NF-κB-DNA binding by blocking IκBα phosphorylation. Furthermore, it reduced collagen and osteocalcin levels to nearly zero and prevented mRNA expression of osteoblast-specific proteins including runtrelated transcription factor-2, osteocalcin, and type I collagen. However, mRNA levels of adipocyte-specific marker, aP2, were markedly increased in the cells co-incubated with rosiglitazone. These results suggest that PPARγ activation by rosiglitazone inhibits osteoblast differentiation with increased adipogenesis in bone marrow cells and also may prevent osteoclast formation and bone resorption in the cells.

Wntless Regulates Dentin Apposition and Root Elongation in the Mandibular Molar

Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of OC-Cre;WlsCO/CO mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, β-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the Wls gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation.

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

Osterix Regulates Tooth Root Formation in a Site-specific Manner

Bone and dentin share similar biochemical compositions and physiological properties. Dentin, a major tooth component, is formed by odontoblasts; in contrast, bone is produced by osteoblasts. Osterix (Osx), a zinc finger-containing transcription factor, has been identified as an essential regulator of osteoblast differentiation and bone formation. However, it has been difficult to establish whether Osx functions in odontoblast differentiation and dentin formation. To understand the role of Osx in dentin formation, we analyzed mice in which Osx was subjected to tissue-specific ablation under the control of either the Col1a1 or the OC promoter. Two independent Osx conditional knockout mice exhibited similar molar abnormalities. Although no phenotype was found in the crowns of these teeth, both mutant lines exhibited short molar roots due to impaired root elongation. Furthermore, the interradicular dentin in these mice showed severe hypoplastic features, which were likely caused by disruptions in odontoblast differentiation and dentin formation. These phenotypes were closely related to the temporospatial expression pattern of Osx during tooth development. These findings indicate that Osx is required for root formation by regulating odontoblast differentiation, maturation, and root elongation. Cumulatively, our data strongly indicate that Osx is a site-specific regulator in tooth root formation.

Molecular regulation of kidney development.

Genetically engineered mice have provided much information about gene function in the field of developmental biology. Recently, conditional gene targeting using the Cre/loxP system has been developed to control the cell type and timing of the target gene expression. The increase in number of kidney-specific Cre mice allows for the analysis of phenotypes that cannot be addressed by conventional gene targeting. The mammalian kidney is a vital organ that plays a critical homeostatic role in the regulation of body fluid composition and excretion of waste products. The interactions between epithelial and mesenchymal cells are very critical events in the field of developmental biology, especially renal development. Kidney development is a complex process, requiring inductive interactions between epithelial and mesenchymal cells that eventually lead to the growth and differentiation of multiple highly specialized stromal, vascular, and epithelial cell types. Through the use of genetically engineered mouse models, the molecular bases for many of the events in the developing kidney have been identified. Defective morphogenesis may result in clinical phenotypes that range from complete renal agenesis to diseases such as hypertension that exist in the setting of grossly normal kidneys. In this review, we focus on the growth and transcription factors that define kidney progenitor cell populations, initiate ureteric bud branching, induce nephron formation within the metanephric mesenchyme, and differentiate stromal and vascular progenitors in the metanephric mesenchyme.

Alteration of Conserved Alternative Splicing in AMELX Causes Enamel Defects

Tooth enamel is the most highly mineralized tissue in vertebrates. Enamel crystal formation and elongation should be well controlled to achieve an exceptional hardness and a compact microstructure. Enamel matrix calcification occurs with several matrix proteins, such as amelogenin, enamelin, and ameloblastin. Among them, amelogenin is the most abundant enamel matrix protein, and multiple isoforms resulting from extensive but well-conserved alternative splicing and postsecretional processing have been identified. In this report, we recruited a family with a unique enamel defect and identified a silent mutation in exon 4 of the AMELX gene. We show that the mutation caused the inclusion of exon 4, which is almost always skipped, in the mRNA transcript. We further show, by generating and characterizing a transgenic animal model, that the alteration of the ratio and quantity of the developmentally conserved alternative splicing repertoire of AMELX caused defects in enamel matrix mineralization.