Ser252Trp mutation in fibroblast growth factor receptor 2 promotes branching morphogenesis in mouse salivary glands.

OBJECTIVES: The purpose of this study was to perform morphological and immunohistochemical (IHC) analysis of the submandibular glands (SMGs) in early development in Apert syndrome model mice (Ap mice). METHODS: ACTB-Cre homozygous mice were mated with fibroblast growth factor receptor 2 (Fgfr2+/Neo-S252W) mice; ACTB-Cre heterozygous mice (ACTB-Cre mice) at embryonic day (E) 13.5 served as the control group, and Fgfr2+/S252W mice (Ap mice) served as the experimental group. Hematoxylin and eosin (H&E) staining was performed on SMGs; Total SMG area and epithelial area were determined, and the epithelial occupancy ratio was calculated. Immunostaining was performed to assess the localization of FGF signaling-related proteins. Next, bromodeoxyuridine (BrdU)-positive cells were evaluated to assess cell proliferation. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess apoptosis in SMGs. RESULTS: The epithelial occupancy ratio was significantly higher in SMGs of Ap mice compared with that in SMGs of controls. FGF7 and bone morphogenetic protein 4 (BMP4) exhibited different localizations in SMGs of Ap mice compared with SMGs of controls. Cell proliferation was higher in SMGs of Ap mice compared with that of controls; however, apoptosis did not different significantly between the two groups. CONCLUSION: Our results suggest that enhanced FGF signaling conferred by missense mutations in FGFR2 promotes branching morphogenesis in SMGs of Ap mice.

Msx1 is essential for proper rostral tip formation of the mouse mandible.

The right and left mandibular processes derived from the first branchial arch grow toward the midline and fuse to create the rostral tip region of the mandible during mandibular development. Severe and mild cases of failure in this process results in rare median cleft of the lower lip and cleft chin, respectively. The detailed molecular mechanisms of mandibular tip formation are unknown. We hypothesize that the Msx1 gene is involved in mandibular tip development, because Msx1 has a central role in other craniofacial morphogenesis processes, such as teeth and the secondary palate development. Normal Msx1 expression was observed in the rostral end of the developing mandible; however, a reduced expression of Msx1 was observed in the soft tissue of the mandibular tip than in the lower incisor bud region. The rostral tip of the right and left mandibular processes was unfused in both control and Msx1-null (Msx1-/-) mice at embryonic day (E) 12.5; however, a complete fusion of these processes was observed at E13.5 in the control. The fused processes exhibited a conical shape in the control, whereas the same region remained bifurcated in Msx1-/-. This phenotype occurred with 100% penetrance and was not restored at subsequent stages of development. Furthermore, Meckel's cartilage in addition to the outline surface soft tissues was also unfused and bifurcated in Msx1-/- from E14.5 onward. The expression of phosho-Smad1/5, which is a mediator of bone morphogenetic protein (Bmp) signaling, was downregulated in the mandibular tip of Msx1-/- at E12.5 and E13.5, probably due to the downregulated Bmp4 expression in the neighboring lower incisor bud. Cell proliferation was significantly reduced in the midline region of the mandibular tip in Msx1-/- at the same developmental stages in which downregulation of pSmad was observed. Our results indicate that Msx1 is indispensable for proper mandibular tip development.

Fibrillin-1 regulates periostin expression during maintenance of periodontal homeostasis.

Background/purpose: Human periodontal ligament consists of elastic system fibers, mainly fibrillin-1 (FBN1). Periostin (POSTN) maintains periodontal homeostasis. A previous study showed that the expression of Postn in periodontal ligament cells was decreased in mice underexpressing Fbn1. However, the relationship between FBN1 and POSTN is not fully understood in the context of mechanical stress. FBN1 contributes to transforming growth factor ß1 (TGF-ß1) activation; TGF-ß1 upregulates the expression of POSTN in human periodontal ligament cells. This study examined whether FBN1 contributed to the maintenance of periodontal homeostasis in cultured human periodontal ligament cells. Materials and methods: Human periodontal ligament fibroblasts (HPDLFs) were exposed to mechanical force via centrifugation. The expression of POSTN was examined by quantitative reverse transcription polymerase chain reaction. The phosphorylation of Smad2 in the TGF-ß/Smad signaling pathway was monitored by western blotting. Results: The expression levels of FBN1 and POSTN were not significantly decreased by centrifugation. However, the expression of POSTN after centrifugation significantly decreased upon knockdown of FBN1. The phosphorylation of Smad2 after centrifugation was decreased, regardless of FBN1 knockdown. Supplementation with 0.1 ng/ml recombinant human TGF-ß1 rescued POSTN expression after centrifugation in HPDLFs upon knockdown of FBN1. Conclusion: FBN1 regulates the expression of POSTN to maintain periodontal homeostasis via TGF-ß/Smad signaling during centrifugation.

Applicability of neutral electrolyzed water for cleaning contaminated fixed orthodontic appliances.

INTRODUCTION: We investigated whether water jet washing with neutral electrolyzed water (NW) can be an easy and safe self-performed cleaning method for oral environments of fixed orthodontic appliance-wearing patients. In line with this, we examined the bactericidal effects and dissolution behaviors of metal elements released from appliances. METHODS: A metal or resin bracket ligated with a metal wire and metal bracket adhered to an apatite-pellet were used as specimens. The bacteria and plaque removal effects of the 30 seconds of NW (30, 100 ppm) jet washing for contaminated specimens were examined via an agar-plate method and the observation of the residual plaque, comparing with other treatments (brushing and flow washing), those treatments with tap water (TW), and flow washings with commercial mouthwashes, Listerine Total Care + (LS) and ConCool F (CC). The amounts of metal released from metal specimens during the 1-week immersion in NW were analyzed and compared with those in TW, LS, and CC. RESULTS: NW jet washing produced larger decreases of surviving bacteria than the treatments with TW and CC (P <0.05) and equal or larger decreases than the treatment with LS (P <0.05). NW jet washing yielded the highest plaque removal level. The amounts of nickel and chromium released from metal specimens after the 1-week immersion in NW (30 ppm) were less than or equal to those with LS. CONCLUSIONS: NW jet washing could be applicable for cleaning fixed orthodontic appliances because of its higher bactericidal effects than the treatments with commercial mouthwashes, inducing no or a slight metal release in actual usage time.

A Ser252Trp substitution in mouse FGFR2 results in hyperplasia of embryonic salivary gland parenchyma.

OBJECTIVES: Mutations in the fibroblast growth factor receptor 2 (FGFR2) gene are responsible for several severe forms of craniosynostotic disorders, such as Apert and Crouzon syndromes. Patients with craniosynostotic disorders caused by a mutation in Fgfr2 present with several clinical symptoms, including hypersalivation. Here we used a transgenic mouse model of Apert syndrome (Fgfr2+/S252W mice) to evaluate the morphology of the submandibular glands at embryonic day 15.5 (E15.5), the time point reported to mark the start of lumen formation. METHODS: Fgfr2+/S252W mice were generated by crossing ACTB-Cre+/+ and Fgfr2+/Neo-S252W mice. After measuring body weight, the submandibular glands were collected at E15.5. H&E staining, immunostaining, and RT-qPCR were performed to investigate the development of the submandibular gland. RESULTS: The number of ducts and acini in Fgfr2+/S252W mice was significantly higher than in control littermates; however, lumen formation was not affected. The mRNA expression of Fgf1, Fgfr1, Mmp2, Bmp4, Bmp7, Dusp6, and Etv5 in Fgfr2+/S252W mice was significantly higher compared to control littermates. Immunoreactivity for FGF3, FGF1, BMP4, and F4/80 was detected in the parenchyma of Fgfr2+/S252W mice. The area of apoptotic cells stained with TUNEL in Fgfr2+/S252W mice was significantly larger than that of the control littermates. CONCLUSIONS: These results suggested that increased FGFR1 signaling and apoptosis in the submandibular glands of Fgfr2+/S252W mice occurred at E15.5, leading to parenchymal hyperplasia. This study demonstrated that a Ser252Trp substitution in mouse FGFR2 resulted in hyperplasia of the submandibular gland parenchyma during development.

VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation.

Osteoblasts release adenosine triphosphate (ATP) out of the cell following mechanical stress. Although it is well established that extracellular ATP affects bone metabolism via P2 receptors [such as purinergic receptor P2X7 (P2X7R) and purinergic receptor P2Y2 (P2Y2R)], the mechanism of ATP release from osteoblasts remains unknown. Recently, a vesicular nucleotide transporter [VNUT, solute carrier family 17 member 9 (SLC17A9)] that preserves ATP in vesicles has been identified. The purpose of this study was to elucidate the role of VNUT in osteoblast bone metabolism. mRNA and protein expression of VNUT were confirmed in mouse bone and in osteoblasts by quantitative real-time PCR (qPCR) and immunohistochemistry. Next, when compressive force was applied to MC3T3-E1 cells by centrifugation, the expression of Slc17a9, P2x7r, and P2y2r was increased concomitant with an increase in extracellular ATP levels. Furthermore, compressive force decreased the osteoblast differentiation capacity of MC3T3-E1 cells. shRNA knockdown of Slc17a9 in MC3T3-E1 cells reduced levels of extracellular ATP and also led to increased osteoblast differentiation after the application of compressive force as assessed by qPCR analysis of osteoblast markers such as Runx2, Osterix, and alkaline phosphatase (ALP) as well as ALP activity. Consistent with these observations, knockdown of P2x7r or P2y2r by siRNA partially rescued the downregulation of osteoblast differentiation markers, caused by mechanical loading. In conclusion, our results demonstrate that VNUT is expressed in osteoblasts and that VNUT inhibits osteoblast differentiation in response to compressive force by mechanisms related to ATP release and P2X7R and/or P2Y2R activity.

Vesicular nucleotide transporter mediates adenosine triphosphate release in compressed human periodontal ligament fibroblast cells and participates in tooth movement-induced nociception in rats.

OBJECTIVE: Pain control is imperative in orthodontic treatment. Adenosine triphosphate (ATP) is a key mediator released from periodontal ligament cells that excites nociceptive nerve endings. Vesicular nucleotide transporter (VNUT), encoded by the Solute carrier family 17 member 9 (SLC17A9) gene, participates in ATP uptake into secretory vesicles; thus, it may mediate tooth movement-induced pain. In the present study, we examined whether VNUT in periodontal ligament cells participates in tooth movement-induced nociception. DESIGN: Expression levels of SLC17A9, connexin 43, and pannexin 1 in human periodontal ligament fibroblasts (HPDLFs) were examined by quantitative reverse transcription-polymerase chain reaction. Mechanical force via centrifugation-induced ATP release was measured using an ATP bioluminescence assay. Inhibitors were used to evaluate the role of ATP transporters. Face-grooming behaviors were assessed as indicators of nociceptive responses after experimental tooth movement in rats, as well as the effects of drugs for the pain-like behavior. RESULTS: After HPDLFs underwent mechanical stimulation by centrifugation, SLC17A9 mRNA expression in the cells was significantly upregulated. Increased ATP release from HPDLFs after mechanical stimulation was suppressed by treatment with clodronic acid, a VNUT inhibitor, at concentrations of 0.1 and 1.0 µM. In rats, face-grooming behaviors (indicators of nociception) were significantly increased on day 1 after experimental tooth movement. Increased face-grooming behaviors were suppressed by systemic administration of clodronic acid (0.1 mg/kg). CONCLUSIONS: These results indicate that release of ATP from periodontal ligament cells via VNUT is important for nociceptive transduction during orthodontic treatment. Thus, VNUT may provide a novel drug target for tooth movement-induced pain.