Transforming Growth Factor-Beta and Sonic Hedgehog Signaling in Palatal Epithelium Regulate Tenascin-C Expression in Palatal Mesenchyme During Soft Palate Development.

During palatogenesis, the palatal shelves first grow vertically on either side of the tongue before changing their direction of growth to horizontal. The extracellular matrix (ECM) plays an important role in these dynamic changes in palatal shelf morphology. Tenascin-C (TNC) is an ECM glycoprotein that shows unique expression in the posterior part of the palatal shelf, but little is known about the regulation of TNC expression. Since transforming growth factor-beta-3 (TGF-ß3) and sonic hedgehog (SHH) signaling are known to play important roles in palatogenesis, we investigated whether TGF-ß3 and SHH are involved in the regulation of TNC expression in the developing palate. TGF-ß3 increased the expression of TNC mRNA and protein in primary mouse embryonic palatal mesenchymal cells (MEPM) obtained from palatal mesenchyme dissected at embryonic day 13.5-14.0. Interestingly, immunohistochemistry experiments revealed that TNC expression was diminished in K14-cre;Tgfbr2 fl/fl mice that lack the TGF-ß type II receptor in palatal epithelial cells and exhibit cleft soft palate, whereas TNC expression was maintained in Wnt1-cre;Tgfbr2 fl/fl mice that lack the TGF-ß type II receptor in palatal mesenchymal cells and exhibit a complete cleft palate. SHH also increased the expression of TNC mRNA and protein in MEPM cells. However, although TGF-ß3 up-regulated TNC mRNA and protein expression in O9-1 cells (a cranial neural crest cell line), SHH did not. Furthermore, TGF-ß inhibited the expression of osteoblastic differentiation markers (osterix and alkaline phosphatase) and induced the expression of fibroblastic markers (fibronectin and periostin) in O9-1 cells, whereas SHH did not affect the expression of osteoblastic and fibroblastic markers in O9-1 cells. However, immunohistochemistry experiments showed that TNC expression was diminished in the posterior palatal shelves of Shh-/+ ;MFCS4 +/- mice, which have deficient SHH signaling in the posterior palatal epithelium. Taken together, our findings support the proposal that TGF-ß and SHH signaling in palatal epithelium co-ordinate the expression of TNC in the posterior palatal mesenchyme through a paracrine mechanism. This signal cascade may work in the later stage of palatogenesis when cranial neural crest cells have differentiated into fibroblast-like cells. The spatiotemporal regulation of ECM-related proteins by TGF-ß and SHH signaling may contribute not only to tissue construction but also to cell differentiation or determination along the anterior-posterior axis of the palatal shelves.

MLH1-mediated recruitment of FAN1 to chromatin for the induction of apoptosis triggered by O6 -methylguanine.

O6 -Methylguanines (O6 -meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein-dependent manner. To understand the molecular mechanism of O6 -meG-induced apoptosis, we performed functional analyses of FANCD2 and FANCI-associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N-methyl-N-nitrosourea (MNU), indicating the formation of a FAN1-MMR complex. In comparison with control cells, FAN1-knockdown cells were more resistant to MNU, and the appearances of a sub-G1 population and caspase-9 activation were suppressed. FAN1 formed nuclear foci in an MLH1-dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single-stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU-induced formation of ssDNA was dramatically suppressed in FAN1-knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O6 -meG.

CO2 laser therapy accelerates the healing of ulcers in the oral mucosa by inducing the expressions of heat shock protein-70 and tenascin C.

The treatment of ulceration or stomatitis with laser therapy is known to accelerate healing and relieve pain, but the underlying biological mechanism is not fully understood. The present study used a mouse model of ulceration to investigate the molecular mechanisms by which CO2 laser therapy accelerated the wound healing process. An ulcer was experimentally created in the palatal mucosa of the mouse and irradiated with light from a CO2 laser. Compared with controls (no irradiation), laser irradiation induced the proliferation of epithelial cells and faster re-epithelialization of the wound area. Immunohistochemistry experiments showed that heat shock protein-70 (HSP70) was expressed mainly in the epithelium of normal palatal tissue, whereas there was little tenascin C (TnC) expression in the epithelium and mesenchyme under normal conditions. Laser irradiation induced HSP70 mRNA and protein expression in the lamina propria as well as TnC expression in the mesenchyme underlying the renewing epithelium. Epithelial cells and fibroblasts were exposed to heated culture medium or laser irradiation to establish whether hyperthermia mimicked the effect of laser irradiation. Culture of fibroblasts in heated medium increased the expressions of both TnC and TGF-ß1, whereas laser irradiation induced only TnC expression. The present study indicates that CO2 laser irradiation exerts a photobiogenic effect to up-regulate TnC expression without inducing TGF-ß1 expression. We suggest that CO2 laser therapy has an advantage over thermal stimulation.

The crucial role of the TRPM7 kinase domain in the early stage of amelogenesis.

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

Hertwig's epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by TGF-ß.

In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwig's epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-ß regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-ß was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-ß1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-ß1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-ßsignaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.

Osteogenic potential for replacing cells in rat cranial defects implanted with a DNA/protamine complex paste.

Osteoinductive scaffolds are required for bone tissue engineering. The aim of the present study was to assess the osteoinductive capacity of deoxyribonucleic acid (DNA)/protamine complexes in a rat model of critical-size calvarial defects. In addition, we investigated whether cultured mesenchymal-like cells (DP-cells) outgrown from DNA/protamine complex engrafted defects could differentiate to become osteogenic cells in vitro. DNA/protamine complexes were prepared by reactions between DNA and protamine sulfate solutions with stirring. Critical-sized (8mm) calvarial defects were created in the central parietal bones of adult rats. Defects were either left empty or treated with DNA/protamine complex scaffolds. Subsequently, micro-computed tomography (micro-CT), histological, and immunohistochemical analyses were performed. Micro-CT and histological assays showed that DNA/protamine complex engrafted defects had enhanced bone regeneration. DP-cells were expanded from explants of DNA/protamine complex engrafted defects using an explant outgrowth culture system. Osteogenesis-related factors were assessed in DP-cells after treatment with an osteoblast-inducing reagent (OIR). After 3months, nearly complete healing was observed for DNA/protamine complex engrafted calvarial defects. Increased alkaline phosphatase (ALP) activity and Alizarin red staining were found for cultured DP-cells. These cells had high expression levels of osteogenic genes, including those for RUNX-2, ALP, osteopontin, and osteocalcin. These results indicated that DNA/protamine complexes could facilitate bone regeneration in calvarial defects. Moreover, in vitro osteogenic induction experiments showed that DP-cells outgrown from DNA/protamine engrafted defects had an osteogenic potential. Based on these results, we suggest that DNA/protamine complexes may recruit osteocompetent cells in these defects, where they differentiate to osteogenic cells.

Preparation of Sr-containing carbonate apatite as a bone substitute and its properties.

Sr-containing carbonate apatite (SrCAp) specimens of varied Sr contents, ranging from 0 to 13.3 mol%, were prepared through a phosphate treatment of set gypsum-and-carbonate mixture at 100°C for 7 days. Effects of Sr content in SrCAp on microstructure, osteoblast-like cell (MC3T3-E1) attachment and proliferation, and alkaline phosphatase (ALP) activity were evaluated. Sr(2+) ion substituted Ca(2+) ion in the apatite lattice. Carbonate content was about 9-13.6 wt%, increasing in content level as Sr content increased. Sr addition benefited cell attachment but had no significant influence on cell proliferation, although the latter was inhibited at the highest Sr content. ALP activity reached a peak in specimen containing 3.4 mol% of Sr. The present study revealed that SrCAp is a promising candidate for use as a bone substitute material with good resorbabilty and osteoconductivity.

Prosthetic treatment for severe open bite malocclusion and loss of several teeth.

This report describes a prosthetic approach that uses fixed partial dentures to establish stable posterior occlusion. Four fixed partial dentures were made of a silver-palladium-copper-gold alloy; the pontics were veneered with a light-curing indirect composite material. They were prepared using a thiouracil metal conditioner and seated with an adhesive luting cement. The clinical follow-up indicated that the prosthetic procedure reported here is applicable for severe anterior and posterior open bite malocclusion.