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1.
J Oral Biosci ; 66(1): 90-97, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38246420

ABSTRACT

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.


Subject(s)
Acrocephalosyndactylia , Receptor, Fibroblast Growth Factor, Type 2 , Animals , Mice , Acrocephalosyndactylia/genetics , Morphogenesis/genetics , Mutation , Receptor, Fibroblast Growth Factor, Type 2/genetics , Submandibular Gland
2.
Int J Mol Sci ; 24(19)2023 Oct 02.
Article in English | MEDLINE | ID: mdl-37834290

ABSTRACT

The differentiation and function of osteocytes are controlled by surrounding cells and mechanical stress; however, the detailed mechanisms are unknown. Recent findings suggest that IL-33 is highly expressed in periodontal tissues in orthodontic tooth movement. The present study aimed to elucidate the effect of IL-33 on the expression of regulatory factors for bone remodeling and their molecular mechanisms in the osteocyte-like cell line MLO-Y4. MLO-Y4 cells were treated with IL-33, and the activation of intracellular signaling molecules and transcriptional factors was determined using Western blot analysis and chromatin immunoprecipitation assay. IL-33 treatment enhanced the expression of IL-6 in MLO-Y4 cells, which was suppressed by the knockdown of the IL-33 receptor ST2L. Additionally, IL-33 treatment induced activation of NF-κB, JNK/AP-1, and p38 MAPK signaling pathways in MLO-Y4 cells. Moreover, pretreatment with specific inhibitors of NF-κB, p38 MAPK, and JNK/AP-1 attenuated the IL-33-induced expression of IL-6. Furthermore, chromatin immunoprecipitation indicated that IL-33 increased c-Jun recruitment to the IL-6 promoter. Overall, these results suggest that IL-33 induces IL-6 expression and regulates osteocyte function via activation of the NF-κB, JNK/AP-1, and p38 MAPK pathways through interaction with ST2L receptors on the plasma membrane.


Subject(s)
Interleukin-6 , NF-kappa B , NF-kappa B/metabolism , Interleukin-6/metabolism , Interleukin-33/pharmacology , Interleukin-33/metabolism , Transcription Factor AP-1/metabolism , Carrier Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Osteocytes/metabolism
3.
Biochem Biophys Res Commun ; 642: 75-82, 2023 01 29.
Article in English | MEDLINE | ID: mdl-36566565

ABSTRACT

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.


Subject(s)
MSX1 Transcription Factor , Tooth , Mice , Animals , MSX1 Transcription Factor/genetics , MSX1 Transcription Factor/metabolism , Mandible , Tooth/metabolism , Morphogenesis/genetics , Signal Transduction
4.
J Dent Sci ; 17(4): 1714-1721, 2022 Oct.
Article in English | MEDLINE | ID: mdl-36299324

ABSTRACT

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.

5.
Am J Orthod Dentofacial Orthop ; 161(6): e507-e523, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35337704

ABSTRACT

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.


Subject(s)
Mouthwashes , Orthodontic Appliances , Humans , Nickel , Orthodontic Appliances/adverse effects , Orthodontic Appliances, Fixed , Water
6.
J Oral Biosci ; 63(2): 184-191, 2021 06.
Article in English | MEDLINE | ID: mdl-33684522

ABSTRACT

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.


Subject(s)
Acrocephalosyndactylia , Receptor, Fibroblast Growth Factor, Type 2 , Animals , Humans , Hyperplasia/genetics , Mice , Mice, Transgenic , Receptor, Fibroblast Growth Factor, Type 2/genetics , Salivary Glands
7.
Cell Tissue Res ; 383(2): 667-675, 2021 Feb.
Article in English | MEDLINE | ID: mdl-32960355

ABSTRACT

Mammalian taste bud cells have a limited lifespan and differentiate into type I, II, and III cells from basal cells (type IV cells) (postmitotic precursor cells). However, little is known regarding the cell lineage within taste buds. In this study, we investigated the cell fate of Mash1-positive precursor cells utilizing the Cre-loxP system to explore the differentiation of taste bud cells. We found that Mash1-expressing cells in Ascl1CreERT2::CAG-floxed tdTomato mice differentiated into taste bud cells that expressed aromatic L-amino acid decarboxylase (AADC) and carbonic anhydrase IV (CA4) (type III cell markers), but did not differentiate into most of gustducin (type II cell marker)-positive cells. Additionally, we found that Mash1-expressing cells could differentiate into phospholipase C ß2 (PLCß2)-positive cells, which have a shorter lifespan compared with AADC- and CA4-positive cells. These results suggest that Mash1-positive precursor cells could differentiate into type III cells, but not into most of type II cells, in the taste buds.


Subject(s)
Aging/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation , Phospholipase C beta/metabolism , Taste Buds/cytology , Taste Buds/metabolism , Animals , Biomarkers/metabolism , Mice
8.
FEBS Open Bio ; 10(8): 1612-1623, 2020 08.
Article in English | MEDLINE | ID: mdl-32592329

ABSTRACT

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.


Subject(s)
Nucleotide Transport Proteins/metabolism , Osteoblasts/metabolism , 3T3 Cells , Animals , Cell Differentiation , Cells, Cultured , Mice , Nucleotide Transport Proteins/genetics , Osteoblasts/cytology
9.
Arch Oral Biol ; 110: 104607, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31810015

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate , Nociception , Nucleotide Transport Proteins , Adenosine Triphosphate/metabolism , Animals , Fibroblasts , Humans , Nucleotide Transport Proteins/physiology , Nucleotides , Periodontal Ligament/physiology , Rats , Tooth Movement Techniques
10.
Free Radic Biol Med ; 147: 175-186, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31866360

ABSTRACT

Orthodontic patients complain of pain for the first few days after insertion of appliances. Mechanical force has been reported to produce oxidants in periodontal ligament (PDL) cells. It has not been studied whether orthodontic force-induced oxidative stress elicits nociception. Herein, we focused on the role of the oxidant-sensitive channel TRPA1 on nociception in orthodontic pain. In a rat model of loaded orthodontic force between the maxillary first molar and incisor, the behavioral signs of orofacial nociception, facial rubbing and wiping, increased to a peak on day 1 and gradually diminished to the control level on day 5. Administration of free radical scavengers (Tempol and PBN) and TRPA1 antagonist (HC-030031) inhibited nociceptive behaviors on day 1. In the PDL, the oxidative stress marker 8-OHdG was highly detected on day 1 and recovered on day 5 to the sham-operated level. The dental pulp showed similar results as the PDL. TRPA1 mRNA was abundantly expressed in the trigeminal ganglion relative to PDL tissue, and there were TRPA1-immunopositive neuronal fibers in the PDL and pulp. In dissociated trigeminal ganglion neurons, H2O2 at 5 mM induced a Ca2+ response that was inhibited by HC-030031. Although H2O2 at 100 µM did not yield any response, it enhanced the mechanically activated TRPA1-dependent Ca2+ response. These results suggest that oxidative stress in the PDL and dental pulp following orthodontic force activates and/or mechanically sensitizes TRPA1 on nociceptive fibers, resulting in orthodontic nociception. Later, the disappearance of nociception seems to be related to a decrease in oxidative stress, probably due to tissue remodeling.


Subject(s)
Dental Pulp , Nociception , Animals , Humans , Hydrogen Peroxide , Oxidative Stress , Periodontal Ligament , Rats , TRPA1 Cation Channel/genetics
11.
Acta Histochem Cytochem ; 49(1): 21-8, 2016 Feb 27.
Article in English | MEDLINE | ID: mdl-27006518

ABSTRACT

Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X3, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp.

12.
Arch Oral Biol ; 62: 86-92, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26655952

ABSTRACT

OBJECTIVE: During orthodontic tooth movement, bone resorption and inhibition of bone formation occur on the compressed side, thereby preventing ankylosis. Periodontal ligament (PDL) cells control bone metabolism and inhibition of bone formation on the compressed side by secreting bone-formation inhibitory factors such as asporin (ASPN) or sclerostin (encoded by SOST). The aim of this study was to identify the inhibitory factors of bone formation in PDL cells. DESIGN: In vitro, the changes in expression of ASPN and SOST and subsequent protein release in human PDL (hPDL) cells were assessed by semi-quantitative polymerase chain reaction (PCR), real-time PCR, and immunofluorescence in hPDL cells subjected to centrifugal force using a centrifuge (45, 90, 135, and 160 × g). In vivo, we applied a compressive force using the Waldo method in rats, and examined the distribution of ASPN or sclerostin by immunohistochemistry. RESULTS: In vitro, hPDL cells subjected to 90 × g for 24h demonstrated upregulated ASPN and downregulated SOST expressions, which were confirmed by immunofluorescent staining. In addition, the formation of mineralized tissue by human osteoblasts was significantly inhibited by the addition of medium from hPDL cells cultured during compressive force as well as the addition of equivalent amounts of ASPN peptide. In vivo, asporin-positive immunoreactive PDL cells and osteoclasts were found on the compressed side, whereas few sclerostin-positive PDL cells were observed. CONCLUSIONS: PDL cells subjected to an optimal compressive force induce the expression and release of ASPN, which inhibits bone formation during orthodontic tooth movement on the compressed side.


Subject(s)
Extracellular Matrix Proteins/metabolism , Osteogenesis/physiology , Periodontal Ligament/physiology , Adaptor Proteins, Signal Transducing , Animals , Bone Morphogenetic Proteins/biosynthesis , Bone Morphogenetic Proteins/metabolism , Cell Line , Cells, Cultured , Extracellular Matrix Proteins/biosynthesis , Gene Expression , Genetic Markers , Humans , Male , Osteoblasts/cytology , Osteoblasts/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Periodontal Ligament/cytology , Periodontal Ligament/metabolism , Pressure , Rats , Rats, Sprague-Dawley , Stress, Mechanical , Tooth Movement Techniques
13.
J Dent Sci ; 11(3): 272-278, 2016 Sep.
Article in English | MEDLINE | ID: mdl-30894984

ABSTRACT

BACKGROUND/PURPOSE: Bone resorption and inhibition of bone formation occur on the compressed side during orthodontic tooth movement. Bone formation inhibitory factors such as sclerostin (encoded by SOST) are secreted on the compressed side by periodontal ligament (PDL) cells. PDL cells control bone metabolism, and compressed PDL cells inhibit bone formation during orthodontic tooth movement. The aim of this study was to identify the inhibitory factors of bone formation in PDL cells. MATERIALS AND METHODS: Changes in SOST expression and subsequent protein release from human PDL (hPDL) cells were assessed using the real-time polymerase chain reaction (PCR), semiquantitative PCR, and immunofluorescence in hPDL cells subjected to centrifugal force (40g and 90g). To confirm the effects on bone formation, human alveolar bone-derived osteoblasts (hOBs) were grown with the addition of sclerostin peptide. In vivo, a compressive force was applied using the Waldo method in rats, and the distribution of sclerostin in PDL tissues was examined by immunohistochemistry. RESULTS: SOST expression was downregulated in vitro by centrifugation at 90g for 24 hours but upregulated by centrifugation at 40g based on real-time PCR, as was confirmed by immunofluorescence staining. The addition of sclerostin peptide significantly decreased the mineralized area in hOBs. However, slightly weakly sclerostin-positive PDL cells were observed on the compressed side in vivo. CONCLUSION: These results indicate that PDL cells subjected to light compressive force exhibit increased expression of SOST/sclerostin, which inhibits bone formation on the compressed side during orthodontic tooth movement.

14.
Neuropeptides ; 47(4): 251-9, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23660339

ABSTRACT

Hemokinin-1 (HK-1) is a novel member of the tachykinin family that is encoded by preprotachykinin 4 (TAC4) and shares the neurokinin-1 receptor (NK1-R) with substance P (SP). Although HK-1 is thought to be an endogenous peripheral SP-like endocrine or paracrine molecule in locations where SP is not expressed, neither the distribution of HK-1 in the maxillofacial area nor the role HK-1 in bone tissue have been examined. In this study, we investigated the distribution of HK-1 in trigeminal ganglion (TG) and maxillary bone, and assessed the expression of HK-1 during osteoclast differentiation. In vivo, rat molars were loaded for 5 days using the Waldo method. In vitro, rat osteoclast-like cells were induced from bone marrow cells. HK-1 distribution and expression were examined by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). In vivo, HK-1 was localized in rat TG neurons; however, the number of HK-1-positive neurons was less than that of SP-positive neurons. In the maxillary bone, nerve fibers, blood vessels, and osteocytes were immunopositive for HK-1. Furthermore, HK-1-positive immunoreactivity was found in osteoclasts on the pressure side. In vitro, PCR showed that TAC4 and NK1-R mRNA was expressed in osteoclasts as well as in bone marrow cells. Although SP (10⁻7 M) treatment led to an increased number of osteoclasts, HK-1 (10⁻7 M) treatment did not. The numbers of biotin-labeled HK-1 peptides bound osteoclasts significantly decreased upon incubation with unlabeled SP and biotin-labeled HK-1 compared with biotin-labeled HK-1 alone. These results suggest that HK-1 may not stimulate the differentiation and function of osteoclasts. SP-stimulated osteoclast formation is competitively regulated by peripheral HK-1 through NK1-Rs.


Subject(s)
Maxilla/metabolism , Osteoclasts/metabolism , Substance P/pharmacology , Tachykinins/metabolism , Trigeminal Ganglion/metabolism , Animals , Male , Molar/drug effects , Molar/metabolism , Osteoclasts/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Neurokinin-1/metabolism , Tachykinins/pharmacology
15.
Acta Histochem Cytochem ; 46(2): 65-73, 2013 Apr 30.
Article in English | MEDLINE | ID: mdl-23720605

ABSTRACT

Nerve growth factor (NGF) plays a critical role in the trigeminal ganglion (TG) following peripheral nerve damage in the oral region. Although neurons in the TG are surrounded by satellite glial cells (SGCs) that passively support neural function, little is known regarding NGF expression and its interactions with TG neurons and SGCs. This study was performed to examine the expression of NGF in TG neurons and SGCs with nerve damage by experimental tooth movement. An elastic band was inserted between the first and second upper molars of rats. The TG was removed at 0-7 days after tooth movement. Using in situ hybridization, NGF mRNA was expressed in both neurons and SGCs. Immunostaining for NGF demonstrated that during tooth movement the number of NGF-immunoreactive SGCs increased significantly as compared with baseline and reached maximum levels at day 3. Furthermore, the administration of the gap junction inhibitor carbenoxolone at the TG during tooth movement significantly decreased the number of NGF-immunoreactive SGCs. These results suggested that peripheral nerve damage may induce signal transduction from neurons to SGCs via gap junctions, inducing NGF expression in SGCs around neurons, and released NGF may be involved in the restoration of damaged neurons.

16.
Mol Endocrinol ; 26(3): 414-22, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22282470

ABSTRACT

We previously reported that alymphoplasia (aly/aly) mice, which have a natural loss-of-function mutation in the Nik gene, which encodes a kinase essential for the processing of p100 to p52 in the alternative nuclear factor-κB (NF-κB) pathway, show mild osteopetrosis with an increase in several parameters of bone formation: bone formation rate, mineral apposition rate, and osteoblast number. We therefore investigated the molecular mechanisms triggered by the alternative NF-κB pathway in the regulation of osteoblast differentiation using primary osteoblasts (POB) prepared from aly/aly mice. Alkaline phosphatase (ALP) activity and mineralization induced by the presence of ß-glycerophosphate and ascorbic acid were enhanced in POB from aly/aly compared with wild-type (WT) mice. Furthermore, osteoblastic differentiation induced by bone morphogenetic protein 2 (BMP2), as shown by ALP activity, mRNA expression of osteocalcin, Id1, Osterix and Runx2, and Sma- and Mad-related protein (Smad)1/5/8 phosphorylation, was also enhanced in POB from aly/aly mice. The ectopic bone formation in vivo that was induced by BMP2 was enhanced in aly/aly mice compared with controls. Transfection of a mutant form of p100, p100ΔGRR, which cannot be processed to p52, stimulated ALP activity and Smad phosphorylation. In contrast to p100ΔGRR, overexpression of p52 inhibited these events. Both BMP2-induced ALP activity and Smad phosphorylation were reduced in POB from p100-deficient mice, which carry a homozygous deletion of the COOH-terminal ankyrin repeats of p100 but still express functional p52 protein. p52 and p100ΔGRR interacted with a BMP receptor, ALK2, in overexpressed COS7 cells and changed the ALK2 protein levels in opposite directions: p52 reduced ALK2 and p100 increased it. Thus, the alternative the NF-κB pathway via the processing of p52 from p100 negatively regulates osteoblastic differentiation and bone formation by modifying BMP activity.


Subject(s)
Cell Differentiation , NF-kappa B p52 Subunit/physiology , Osteoblasts/physiology , Osteogenesis , Alkaline Phosphatase/metabolism , Animals , Bone Morphogenetic Protein 2/physiology , Bone Morphogenetic Protein Receptors/genetics , Bone Morphogenetic Protein Receptors/metabolism , Cells, Cultured , Female , Humans , MAP Kinase Signaling System , Male , Mice , Mice, Knockout , NF-kappa B p52 Subunit/metabolism , Osteoblasts/enzymology , Osteoblasts/metabolism , Phosphorylation , Primary Cell Culture , Prostheses and Implants , Protein Binding , Skull/cytology , Smad Proteins/metabolism
17.
Neuropeptides ; 45(3): 239-44, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21514667

ABSTRACT

Neurokinin B (NKB) is a neuropeptide in the tachykinin family that acts as a neurotransmitter and neuromodulator, primarily in the central nervous system. The distribution and role of NKB and its receptor, the neurokinin-3 receptor (NK-3R), in peripheral tissues are poorly understood. In this study, we investigated the distribution of NKB and NK-3R in peripheral tissues as well as the role of NKB in bone metabolism, especially in osteoclast formation and bone resorption activity through NK-3R. The distributions of NKB in intact rat neurons of the trigeminal ganglion (TG) and in axons of periodontal tissue were investigated by immunohistochemistry. Osteoclasts from cultured rat bone marrow cells were used to examine the distribution of NK-3R by immunocytochemistry and RT-PCR and to investigate the effects of NKB on the resorption activity of osteoclasts on ivory slices. We found that NKB immunopositive neurons were localized in the rat TG and that NKB immunopositive axons were distributed in periodontal tissues. Immunoreactivity for NK-3R was found in cultured osteoclasts, and NK-3R mRNA expression in the osteoclasts was confirmed by RT-PCR. The addition of NKB significantly increased the number of osteoclasts and the resorption area compared with the control. These findings suggest that NKB was localized in peripheral neurons and may involve the activation of osteoclast formation and bone resorption through NK-3R.


Subject(s)
Bone Resorption , Neurokinin B/metabolism , Neurokinin B/pharmacology , Osteoclasts/drug effects , Osteoclasts/physiology , Animals , Cells, Cultured , Male , Neurokinin B/genetics , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Osteoclasts/cytology , Periodontium/cytology , Periodontium/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Neurokinin-3/metabolism , Trigeminal Ganglion/cytology
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