Response of TGF-ß isoforms in epithelial-mesenchymal transition of enamel epithelial cells.

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.

Pediatric dentists' perspectives of children with special health care needs in Japan: developmental disabilities, phobia, maltreatment, and multidisciplinary collaboration.

BACKGROUND: The number of children diagnosed with developmental disabilities (DDs) or other chronic difficulties has risen. However, each professional's awareness of children with developmental, emotional and behavioural difficulties may differ, allowing their special needs to be overlooked at child health checkups until secondary difficulties appear. Therefore, it is necessary to explore the multi-professional views of children with such chronic difficulties. This study investigates pediatric dentists' perception of children with potential chronic difficulties. METHODS: Interviews were conducted with 21 pediatric dentists, and the transcripts were analyzed using grounded theory to develop categories for the theoretical assessment. RESULTS: Four themes emerged regarding the children with potential chronic difficulties: children exhibiting possible DDs with awkward social communication and interaction; severe rampant caries possibly derived from maltreatment; dental phobia possibly derived from mental health problems; a complicated home environment where their mothers exhibit poor oral health literacy. CONCLUSIONS: This study's findings imply that participants' concept of children of concern included the risks of poor oral health and mental health problems that other healthcare professionals might overlook. It is recommended that multidisciplinary professionals engaging in child health checkups be aware of children's oral and mental health status as well as potential DDs and child maltreatment.

Effects of Zoledronate on Local and Systemic Production of IL-1ß, IL-18, and TNF-α in Mice and Augmentation by Lipopolysaccharide.

Bisphosphonates (BPs) containing nitrogen (N-BPs) exhibit far stronger anti-bone-resorptive effects than non-N-BPs. However, repeated administration of N-BPs causes osteonecrosis selectively in jawbones. As BPs accumulate in large amounts within inflamed bones, any N-BP released from the pool accumulated within jawbones might directly act on cells in the surrounding soft-tissues and induce inflammation or necrosis. Here, we examined the local and systemic effects of zoledronate (the most potent N-BP with the highest incidence of jawbone-necrosis) on inflammatory cytokines in mice. Locally within ear-pinnas: (i) zoledronate induced long-lasting accumulation of interleuikin-1ß (IL-1ß) and IL-18, but not tumor necrosis factor-α (TNF-α), (ii) zoledronate and lipopolysaccharide (LPS, a cell-wall component of Gram-negative bacteria) mutually augmented the productions of IL-1ß, IL-18, and TNF-α, and (iii) oxidronate (a toxic non-N-BP) by itself produced not only IL-1ß and IL-18, but also TNF-α. In systemic experiments using intraperitoneal injection of zoledronate and/or LPS, (i) zoledronate by itself increased none of the above cytokines in serum, and (ii) in mice pretreated (3 d before) with zoledronate, the LPS-induced increases in serum IL-1ß and IL-18 were greatly augmented with a delayed slight TNF-α augmentation. These results, together with previous ones, suggest that (a) pro-IL-1ß and pro-IL-18 accumulate within cells in soft-tissues exposed to N-BPs, and infection may augment not only their production, but also the release of their mature forms, (b) IL-1ß and IL-18 (possibly together with TNF-α) may play important roles in N-BP-induced inflammation and/or necrosis, and (c) mechanisms underlying the cytotoxic effects of BPs may differ between N-BPs and non-N-BPs.

Unicystic Ameloblastoma in a Child Treated with a Combination of Conservative Surgery and Orthodontic Treatment: A Case Report.

Unicystic ameloblastoma (UAM) is a variant of intraosseous ameloblastoma that occurs as a single cystic cavity. This report describes a case of UAM of the mandible in a seven-year-old girl. The lesion radiographically mimicked a dentigerous cyst. Under the primary diagnosis of a dentigerous cyst, marsupialization was performed to erupt the first molar involved in the cystic lesion and to obtain a definitive diagnosis. The biopsy specimen revealed ameloblastoma. During careful observation, orthodontic treatment, which was performed to upright and promote the eruption of the first molar involved in the tumor, maintained the space needed for enucleation of the tumor. Finally, the second primary molar was extracted, and the lesion was enucleated at 3 years and 4 months after marsupialization. The results of the histological examination revealed UAM. Conclusively, the treatment course not only avoids a resection of the mandible but also induces eruption of the teeth involved in the tumor. Thus, the combination of conservative surgery and orthodontic treatment was effective in the management of UAM that mimics a dentigerous cyst.

Requirement of Zinc Transporter SLC39A7/ZIP7 for Dermal Development to Fine-Tune Endoplasmic Reticulum Function by Regulating Protein Disulfide Isomerase.

Skin is the first area that manifests zinc deficiency. However, the molecular mechanisms by which zinc homeostasis affects skin development remain largely unknown. Here, we show that zinc-regulation transporter-/iron-regulation transporter-like protein 7 (ZIP7) localized to the endoplasmic reticulum plays critical roles in connective tissue development. Mice lacking the Slc39a7/Zip7 gene in collagen 1-expressing tissue exhibited dermal dysplasia. Ablation of ZIP7 in mesenchymal stem cells inhibited cell proliferation thereby preventing proper dermis formation, indicating that ZIP7 is required for dermal development. We also found that mesenchymal stem cells lacking ZIP7 accumulated zinc in the endoplasmic reticulum, which triggered zinc-dependent aggregation and inhibition of protein disulfide isomerase, leading to endoplasmic reticulum dysfunction. These results suggest that ZIP7 is necessary for endoplasmic reticulum function in mesenchymal stem cells and, as such, is essential for dermal development.

Critical involvement of ZEB2 in collagen fibrillogenesis: the molecular similarity between Mowat-Wilson syndrome and Ehlers-Danlos syndrome.

Mowat-Wilson syndrome (MOWS) is a congenital disease caused by de novo heterozygous loss of function mutations or deletions of the ZEB2 gene. MOWS patients show multiple anomalies including intellectual disability, a distinctive facial appearance, microcephaly, congenital heart defects and Hirschsprung disease. However, the skin manifestation(s) of patients with MOWS has not been documented in detail. Here, we recognized that MOWS patients exhibit many Ehlers-Danlos syndrome (EDS)-like symptoms, such as skin hyperextensibility, atrophic scars and joint hypermobility. MOWS patients showed a thinner dermal thickness and electron microscopy revealed miniaturized collagen fibrils. Notably, mice with a mesoderm-specific deletion of the Zeb2 gene (Zeb2-cKO) demonstrated redundant skin, dermal hypoplasia and miniaturized collagen fibrils similar to those of MOWS patients. Dermal fibroblasts derived from Zeb2-cKO mice showed a decreased expression of extracellular matrix (ECM) molecules, such as collagens, whereas molecules involved in degradation of the ECM, such as matrix metalloproteinases (MMPs), were up-regulated. Furthermore, bleomycin-induced skin fibrosis was attenuated in Zeb2-cKO mice. We conclude that MOWS patients exhibit an EDS-like skin phenotype through alterations of collagen fibrillogenesis due to ZEB2 mutations or deletions.

TGF-ß and Physiological Root Resorption of Deciduous Teeth.

The present study was performed to examine how transforming growth factor ß (TGF-ß) in root-surrounding tissues on deciduous teeth regulates the differentiation induction into odontoclasts during physiological root resorption. We prepared root-surrounding tissues with (R) or without (N) physiological root resorption scraped off at three regions (R1-R3 or N1-N3) from the cervical area to the apical area of the tooth and measured both TGF-ß and the tartrate-resistant acid phosphatase (TRAP) activities. The TGF-ß activity level was increased in N1-N3, whereas the TRAP activity was increased in R2 and R3. In vitro experiments for the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that proteins from N1-N3 and R1-R3 enhanced the TRAP activity in RAW264 cells. A genetic study indicated that the mRNA levels of TGF-ß1 in N1 and N2 were significantly increased, and corresponded with levels of osteoprotegerin (OPG). In contrast, the expression level of RANKL was increased in R2 and R3. Our findings suggest that TGF-ß is closely related to the regulation of OPG induction and RANKL-mediated odontoclast differentiation depending on the timing of RANKL and OPG mRNA expression in the root-surrounding tissues of deciduous teeth during physiological root resorption.

TGF-ß1 autocrine signalling and enamel matrix components.

Transforming growth factor-ß1 (TGF-ß1) is present in porcine enamel extracts and is critical for proper mineralization of tooth enamel. Here, we show that the mRNA of latent TGF-ß1 is expressed throughout amelogenesis. Latent TGF-ß1 is activated by matrix metalloproteinase 20 (MMP20), coinciding with amelogenin processing by the same proteinase. Activated TGF-ß1 binds to the major amelogenin cleavage products, particularly the neutral-soluble P103 amelogenin, to maintain its activity. The P103 amelogenin-TGF-ß1 complex binds to TGFBR1 to induce TGF-ß1 signalling. The P103 amelogenin-TGF-ß1 complex is slowly cleaved by kallikrein 4 (KLK4), which is secreted into the transition- and maturation-stage enamel matrix, thereby reducing TGF-ß1 activity. To exert the multiple biological functions of TGF-ß1 for amelogenesis, we propose that TGF-ß1 is activated or inactivated by MMP20 or KLK4 and that the amelogenin cleavage product is necessary for the in-solution mobility of TGF-ß1, which is necessary for binding to its receptor on ameloblasts and retention of its activity.

[Zinc signaling-mediated regulation of dentin and periodontal tissues].

An essential trace element zinc is required for variety of cellular functions and physiological responses, therefore, downregulation of zinc homeostasis cause serious problems in health, such as growth retardation and abnormal bone formation. Recent technical advances contributed to reveal that zinc ion regulated by zinc transporters acts as a signaling mediator, called zinc signaling that involves in mammalian physiology and pathogenesis. This review will address the current understanding of the roles of zinc signaling in regulation of dentin formation and periodontal tissues homeostasis.

Functions of miR-1 and miR-133a during the postnatal development of masseter and gastrocnemius muscles.

The present study investigated the function of miR-1 and miR-133a during the postnatal development of mouse skeletal muscles. The amounts of miR-1 and miR-133a were measured in mouse masseter and gastrocnemius muscles between 1 and 12 weeks after birth with real-time polymerase chain reaction and those of HDACs, MEF2, MyoD family, MCK, SRF, and Cyclin D1 were measured at 2 and 12 weeks with Western blotting. In both the masseter and gastrocnemius muscles, the amount of miR-1 increased between 1 and 12 weeks, whereas the amount of HADC4 decreased between 2 and 12 weeks. In the masseter muscle, those of MEF2, MyoD, Myogenin, and MCK increased between 2 and 12 weeks, whereas, in the gastrocnemius muscle, only those of MRF4 and MCK increased. The extent of these changes in the masseter muscle was greater than that in the gastrocnemius muscle. The amounts of miR-133a, SRF, and Cyclin D1 did not change significantly in the masseter muscle between 1 and 12 weeks after birth. By contrast, in the gastrocnemius muscle, the amounts of miR-133a and Cyclin D1 increased, whereas that of SRF decreased. Our findings suggest that the regulatory pathway of miR-1 via HDAC4 and MEF2 plays a more prominent role during postnatal development in the masseter muscle than in the gastrocnemius muscle, whereas that of miR-133a via SRF plays a more prominent role in the gastrocnemius muscle than in the masseter muscle.

Association of DLX3 gene polymorphism and dental caries susceptibility in Japanese children.

OBJECTIVE: In this study, we investigated whether single nucleotide polymorphisms (SNPs) in DLX3 are associated with dental caries susceptibility in Japanese children. DESIGN: Genomic DNA of 201 Japanese children was extracted from buccal epithelial cells. The subjects were divided into two groups: 'low level' group with <10,000 colony forming units (CFU) of Streptococcus mutans/mL saliva (level 0) and 'high level' group with ≥ 10,000 CFU/mL (more than level 1). Each group was further divided according to decayed, missing, filled teeth (dmft) into low caries experience (dmft ≤2) and high caries experience (dmft ≥ 3). Seven SNPs in DLX3 were genotyped using TaqMan1® SNP Genotyping Assay. RESULTS: Statistical significant association was observed between DLX3 (rs2278163) and caries experience in 'high level Mutans streptococci' group. CONCLUSION: These findings suggest that rs2278163 SNP of DLX3 might be associated with dental caries susceptibility in Japanese children. T and C alleles of rs2278163 SNP may potentially be involved in caries susceptibility and caries protection respectively.

Release and systemic accumulation of heavy metals from preformed crowns used in restoration of primary teeth.

Preformed crowns for restoration of primary teeth are used in various treatments and are essential for restoring the crowns of primary molars. However, there are concerns that mechanical, chemical, and thermal stimulation may cause release of components of such crowns. We examined systemic accumulation of heavy metals associated with preformed crowns (3M Stainless Steel Primary Molar Crowns) used in primary tooth restoration. The participants were 37 children who had visited the Pediatric Dental Clinic of Tsurumi University Dental Hospital. They were divided into two groups: 22 participants without a history of being fitted with a preformed crown for primary tooth restoration (controls), and 15 participants with preformed crowns for primary tooth restoration. Analysis of hair samples showed a significant difference in the level of the trace element Cr - an important component of the preformed crowns - between children with and without preformed crowns, but no significant differences in Fe or Ni levels. Levels of the trace elements Ni, Cr, and Fe were within allowable ranges, indicating that these minerals were not likely to be harmful.

Association between the lack of teeth and the expression of myosins in masticatory muscles of microphthalmic mouse.

The purposes of the present study were to elucidate the influences of the deficiency of teeth on masticatory muscles, such as the masseter, temporalis and digastric muscles and compare the influence among masticatory muscles. We analysed the expressions of myosin heavy chain (MyHC) isoform messenger RNA (mRNA) and protein in these muscles in the microphthalmic (mi/mi) mouse, whose teeth cannot erupt because of a mutation in the mitf gene locus. The expression levels of MyHC mRNA and protein in the masseter, temporalis, digastric, tibialis anterior and gastrocnemius muscles of +/+ and mi/mi mice were analysed with real-time polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The mi/mi masseter muscle at 8 weeks of age expressed 4.1-fold (p < 0.05) and 3.3-fold (p < 0.01) more MyHC neonatal mRNA and protein than that in the +/+, respectively; the expression level of MyHC neonatal protein was 19% of the total MyHC protein in the masseter muscle of mi/mi mice. In the digastric muscle, the expression levels of MyHC I mRNA and protein in the mi/mi mice were 4.7-fold (p < 0.05) and 5-fold (p < 0.01) higher than those in the +/+ mice. In the temporalis, tibialis anterior and gastrocnemius muscles, there was no significant difference in the expression levels of any MyHC isoform mRNA and protein between +/+ and mi/mi mice. These results indicate associations between the lack of teeth and the expression of MyHC in the masseter and digastric muscles but not such associations in the temporalis muscle, suggesting that the influence of tooth deficiency varies among the masticatory muscles.

Genetic study of gutter-shaped root (GSR) in AKXL RI mouse strains using QTL analysis.

In this study, quantitative trait locus (QTL) analysis was used to identify candidate chromosomes and for detecting the regions that include the gene or genes causing gutter-shaped root (GSR) in AKXL recombinant inbred mouse strains. One potential QTL was detected on chromosome 5 within a region of 13.0 cM, where the likelihood ratio statistic (LRS) score was higher than a suggestive level. This indicates that one of the candidate genes causing mouse GSR may be located in this region.

Enhanced induction of a histamine-forming enzyme, histidine decarboxylase, in mice primed with NOD1 or NOD2 ligand in response to various Toll-like receptor agonists.

We investigated the immunopharmacological aspects of innate immune responses via Toll-like receptors (TLRs), NOD1 and NOD2, in terms of induction of the histamine-forming enzyme, histidine decarboxylase (HDC), activity in mice. Intravenous injection of TLR4-agonistic synthetic lipid A definitely induced HDC activity in the liver, spleen, and lungs, especially the lungs, in mice, where maximum activity was induced about 3 h after the injection of lipid A. The TLR2/6 agonistic synthetic diacyl-type lipopeptide FSL-1 and TLR3-agonistic poly I:C were also effective in inducing HDC, while the NOD2-agonistic synthetic muramyldipeptide (MDP) and NOD1-agonistic synthetic FK156 and FK565 exhibited only weak activities in this respect. Mice primed with intravenous injection of NOD1 or NOD2 agonists produced higher HDC activity following the 4-6 h later intravenous challenge with the above TLR agonists. Among the priming agents, FK565 exhibited the strongest activity, and it was effective via various administration routes - intraperitoneal, subcutaneous, intramuscular, as well as intravenous injection; furthermore, oral (gastric) administration was effective, although it needed a dose 10 times higher than that required for other administration routes. These findings suggest that HDC is induced in association with TLRs and NOD1/2, and that the newly formed histamine by the induced HDC might play important roles in the regulation of inflammatory and immune responses in various organs.

Pharmacological characterization of anaphylaxis-like shock responses induced in mice by mannan and lipopolysaccharide.

Intravenous injection of lipopolysaccharide (LPS, a component of the Gram-negative bacterial cell-surface) or mannan (Man, a component of the fungal cell-surface) into mice reportedly induces anaphylaxis-like shock (ALS) via complement-associated platelet degradation and platelet-activating factor (PAF), respectively. However, it is unclear whether PAF is involved in LPS-ALS or whether complements and/or platelets are involved in Man-ALS. Here, using preparations of Man from Saccharomyces cerevisiae and LPS from Klebsiella O3, we characterized and compared LPS-ALS and Man-ALS, with the following results. (1) ALS depended on mouse strain (ddY and BALB/c being highly responsive to Man and LPS, respectively), but not on Toll-like receptors 2 and 4. (2) In ddY mice, Man had little effect on platelets, K76 (C5a-inhibitor) did not prevent Man-ALS, and Man-ALS was augmented by prior platelet depletion. (3) CV-3988 (PAF antagonist) prevented Man-ALS, but not LPS-ALS. (4) LPS-ALS and Man-ALS were each augmented by prior injection of a muramyl dipeptide (MDP, a constituent abundant in the Gram-positive bacterial cell-surface), but prevented by prior macrophage depletion. (5) Co-administration of Man and LPS induced an augmented ALS in both ddY and BALB/c mice. These results indicate that (i) Man and LPS each induces ALS in mice in strain-dependent and macrophage-dependent (but not TLR-dependent) ways by stimulating a platelet-non-associated PAF pathway and a platelet-associated complement pathway, respectively, and (ii) these pathways are primed by MDP and exhibit mutually augmenting actions. Man-ALS and LPS-ALS may therefore serve as models for diseases involving augmentation by multiple or mixed infections.

The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.

BACKGROUND: Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-beta signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. CONCLUSIONS/SIGNIFICANCE: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-beta signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-beta signaling and connective tissue dysfunction.

Characterization of excess hard tissue occurring in the mesio-buccal surface of the mandibular first molar in microphthalmic mouse.

OBJECTIVE: The aim of the present study was to characterize the excess hard tissue on the mandible of the microphthalmic mouse having a mutation at the mitf locus. DESIGN: Homozygous mutant (mi/mi) and wild-type (+/+) mice were obtained by mating a breeding pair (strain name, B6C3Fe a/a-Mitf(mi)/J). We used mi/mi and +/+ mice at ages 6, 7, 8, 9, 28, and 49 days for micro-computed tomographic and histologic analyses. RESULTS: Excess hard tissue was found on the mesio-buccal surface of the mandibular first molar in all 11mi/mi mice, but none was found in the 8mi/+ or 14 +/+ mice. The excess hard tissue was located in the mental foramen connected to the mandibular canal. The mandibular canal passed near the basal part of the incisor and the root of the mandibular first molar due to aberrant development of the teeth and mandible. The excess hard tissue contained predentine immunostained for dentine sialoprotein, a marker for early stages of dentinogenesis, which was first observed at about 7 days of age. Dentine, predentine, pulp, and root-like structures were observed in the excess hard tissue, but neither enamel nor enamel organ was observed. CONCLUSION: Odontogenic cells in the basal part of the incisor and/or the mandibular first molar with the ability to develop into odontoblasts and pulp cells appeared to migrate through the mandibular canal to the mental foramen, where they developed into odontoblasts and pulp-like cells, and then formed dentine and predentine-like structures.

Structural relationship of child behavior and its evaluation during dental treatment.

The present study uses structural equation modeling to explore the structural relationship of child behavior type and its evaluation during dental treatment. The study population consisted of 33 children at their first visit to a pediatric dentist at the Dental Hospital of Tsurumi University. Child behavior was evaluated by the Frankl Behavior Rating Scale and the behavior evaluation scale developed by Kurosu. Factor analysis extracted 3 behavior types: escape, self-defense, and facial expression. The path diagram of structural relationships between child behavior and the Frankl Behavior Rating Scale indicated that facial expression had the strongest correlation to the Frankl Behavior Rating Scale.