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Glia and Orofacial Pain: Progress and Future Directions

Ye, Yi; Salvo, Elizabeth; Romero-Reyes, Marcela; Akerman, Simon; Shimizu, Emi; Kobayashi, Yoshifumi; Michot, Benoit; Gibbs, Jennifer
Orofacial pain is a universal predicament, afflicting millions of individuals worldwide. Research on the molecular mechanisms of orofacial pain has predominately focused on the role of neurons underlying nociception. However, aside from neural mechanisms, non-neuronal cells, such as Schwann cells and satellite ganglion cells in the peripheral nervous system, and microglia and astrocytes in the central nervous system, are important players in both peripheral and central processing of pain in the orofacial region. This review highlights recent molecular and cellular findings of the glia involvement and glia-neuron interactions in four common orofacial pain conditions such as headache, dental pulp injury, temporomandibular joint dysfunction/inflammation, and head and neck cancer. We will discuss the remaining questions and future directions on glial involvement in these four orofacial pain conditions.
PMID: 34069553
ISSN: 1422-0067
CID: 4891382

Skeletal Response to Insulin in the Naturally Occurring Type 1 Diabetes Mellitus Mouse Model

Dixit, Manisha; Liu, Zhongbo; Poudel, Sher Bahadur; Yildirim, Gozde; Zhang, Yanjiao Zhang; Mehta, Shilpa; Murik, Omer; Altarescu, Geona; Kobayashi, Yoshifumi; Shimizu, Emi; Schaffler, Mitchell B; Yakar, Shoshana
Patients with type 1 diabetes mellitus (T1DM) exhibit reduced BMD and significant increases in fracture risk. Changes in BMD are attributed to blunted osteoblast activity and inhibited bone remodeling, but these cannot fully explain the impaired bone integrity in T1DM. The goal of this study was to determine the cellular mechanisms that contribute to impaired bone morphology and composition in T1DM. Nonobese diabetic (NOD) mice were used, along with μCT, histomorphometry, histology, Raman spectroscopy, and RNAseq analyses of several skeletal sites in response to naturally occurring hyperglycemia and insulin treatment. The bone volume in the axial skeleton was found to be severely reduced in diabetic NOD mice and was not completely resolved with insulin treatment. Decreased bone volume in diabetic mice was associated with increased sclerostin expression in osteocytes and attenuation of bone formation indices without changes in bone resorption. In the face of blunted bone remodeling, decreases in the mineral:matrix ratio were found in cortical bones of diabetic mice by Raman microspectroscopy, suggesting that T1DM did not affect the bone mineralization process per se, but rather resulted in microenvironmental alterations that favored mineral loss. Bone transcriptome analysis indicated metabolic shifts in response to T1DM. Dysregulation of genes involved in fatty acid oxidation, transport, and synthesis was found in diabetic NOD mice. Specifically, pyruvate dehydrogenase kinase isoenzyme 4 and glucose transporter 1 levels were increased, whereas phosphorylated-AKT levels were significantly reduced in diabetic NOD mice. In conclusion, in addition to the blunted bone formation, osteoblasts and osteocytes undergo metabolic shifts in response to T1DM that may alter the microenvironment and contribute to mineral loss from the bone matrix. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
PMID: 33977201
ISSN: 2473-4039
CID: 4875852

IGF-1 Mediates EphrinB1 Activation in Regulating Tertiary Dentin Formation

Matsumura, S; Quispe-Salcedo, A; Schiller, C M; Shin, J S; Locke, B M; Yakar, S; Shimizu, E
Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.
PMID: 28489485
ISSN: 1544-0591
CID: 2549352

Effects of ephrin B1 gene ablation in osteoblasts or osteocytes on cortical and trabecular bone morphology [Meeting Abstract]

Raisingani, M; Liu, Z; Han, T; Shimizu, E; Yakar, S
Eph receptors belong to a subfamily of receptor tyrosine kinases activated by membrane bound ligands called ephrins. Recent studies have shown that osteoblasts express the EphB4 receptor and it's ligand ephrinB2 and B1, while osteoclasts express a few members of the ephrinBs, suggesting that these proteins are involved in bone modeling during growth. Past studies have shown that parathyroid hormone (PTH) stimulates bone modeling via enhanced expression of the insulin-like growth factor-1 (IGF-I) and the ephrinB2 and EphB4 in osteoblasts. To define the interactions between PTH/IGF-1/Eph signaling pathways in bone, male mice at 4 weeks of age were injected 80 mcg/kg/day for 5 days. To our surprise we found 3 fold increase in the expression of Ephrin B1 in the femoral cortical shells from male mice, while the expression of ephrinB2 or EphB4 did not differ significantly between PTH treated and untreated groups. Thus, we used the osteoblast- and osteocyte-specific ephrinB1 knockout (KO) mice (using the osteocalcin-cre and the dentin matrix protein (DMP)-1-cre, respectively) to investigate their bone response to intermittent PTH treatment. We used micro Computer Tomography (CT) to characterize the basal morphology of femurs dissected from male mice. We found decreases in tissue mineral density in cortical bone of the mid-shaft femur in both Osteocalcin-ephrinB1 (1.45 +/- 0.05 g/cm3 , p=0.01) and DMP-1-ephrinB1 KO mice (1.465 +/- 0.05 mg/cm3 , p=0.02) as compared to controls (1.64 +/- 0.02 mg/cm3 ) . Similarly, we found decreased bone mineral density in the trabecular bone assessed at the distal femur (Osteocalcin-eprinB1 0.147+/-0.005 p=0.004, DMP-1-ephrinB1 0.175 +/- 0.02 p=0.06, and controls 0.248 +/- 0.02 mg/cm3 ) . However, cortical and trabecular bone morphology of males did not differ between the groups. Female DMP-ephrinB1 KO mice showed decreased total cross-sectional area (1.14 +/- 0.09 vs 1.27+/-0.06 mm2 , p = 0.02), polar moment of inertia (0.12 +/- 0.03 vs 0.14+/- 0.01 mm4 , p= 0.03), and marrow area (0.76 +/- 0.03 vs 0.86 +/- 0.06 mm2 , p = 0.03) as compared to control mice with no significant difference in tissue mineral density. Female DMPephrinB1 KO mice did not show any trabecular bone phenotype. Bone anabolic response to intermittent PTH treatment in Osteocalcin-eprinB1 and DMP-1-ephrinB1 KO mice is under investigation
ISSN: 0163-769x
CID: 2631942

The Histone-Deacetylase-Inhibitor Suberoylanilide Hydroxamic Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix Metalloproteinase-13 Activity

Duncan, Henry F; Smith, Anthony J; Fleming, Garry J P; Partridge, Nicola C; Shimizu, Emi; Moran, Gary P; Cooper, Paul R
Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular-reparative events. We have previously demonstrated that HDACis (Valproic acid, Trichostatin A) increase mineralization in dental papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 muM); while low concentrations (1 muM) SAHA did not increase apoptosis. HDACi-exposure for 24 h induced mineralization-per-cell dose-dependently after 2 weeks; however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24 h and 14d) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24 h, which reduced to 36 genes at 14d. 59% of genes were down-regulated at 24 h and 36% at 14d, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein expression (7d, 14 d) and enzyme activity (48 h, 14d). Selective MMP-13-inhibition (MMP-13i) dose-dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i-supplementation promoted expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit
PMID: 26264761
ISSN: 1097-4652
CID: 1721482

Sirtuin 1 is a Negative Regulator of Parathyroid Hormone Stimulation of Matrix Metalloproteinase 13 Expression in Osteoblastic Cells

Fei, Yurong; Shimizu, Emi; McBurney, Michael W; Partridge, Nicola C
PTH is the only current anabolic treatment for osteoporosis in the USA. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases. Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knockout mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH induced-Mmp13 expression. Two hours of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.
PMID: 25631045
ISSN: 0021-9258
CID: 1447882

Parathyroid Hormone Regulates HDAC4 through PKA-mediated Phosphorylation and Dephosphorylation in Osteoblastic Cells

Shimizu, Emi; Nakatani, Teruyo; He, Zhiming; Partridge, Nicola C
Histone deacetylases (HDACs) are crucial regulators of gene expression in transcriptional co-repressor complexes. Previously, we reported that HDAC4 was a basal repressor of matrix metalloproteinase-13 (MMP-13) transcription and parathyroid hormone (PTH) regulates HDAC4 to control MMP-13 promoter activity through dissociation from Runx2. Here, we show that PTH induces the protein kinase A (PKA)-dependent phosphorylation of HDAC4 in the nucleus of the rat osteoblastic cell line, UMR 106-01. We demonstrate that PKA-dependent phosphorylated HDAC4 is released from Runx2 bound to the MMP-13 promoter in these cells. Point mutation of Ser-740 in rHDAC4 prevents the release of HDAC4 from Runx2 on the MMP-13 promoter and also prevents the PTH stimulation of MMP-13 transcription. Thus, PTH-induced phosphorylation of rHDAC4 at Ser-740 is crucial for regulating MMP-13 transcription in osteoblasts. PTH causes degradation of HDAC4 and this product appears in the cytoplasm. The cytoplasmic degradation of HDAC4 is blocked by PKA and lysosomal inhibitors, but is not affected by proteasome, caspase-3, serine and aspartic protease inhibitors. In addition, the phosphatase inhibitor, okadaic acid, prevents degradation indicating that dephosphorylation is associated with degradation. These mechanisms regulating HDAC4 and their roles in such processes are crucial for bone and chondrocyte development. Our data support a link between PTH regulating HDAC4 phosphorylation by PKA, trafficking, partial degradation and the control of MMP-13 transcription through association with Runx2.
PMID: 24904057
ISSN: 0021-9258
CID: 1032602

Histologic and histobacteriologic observations of failed revascularization/revitalization therapy: a case report

Lin, Louis M; Shimizu, Emi; Gibbs, Jennifer L; Loghin, Simona; Ricucci, Domenico
INTRODUCTION: Mechanical debridement plays an important role in eliminating intracanal bacteria, such as biofilm on the canal walls and bacteria in the dentinal tubules. Mechanical debridement is not recommended for root canal disinfection in revascularization/revitalization therapy. Here we report a failed revascularization/revitalization case, which could be due to inadequate root canal disinfection without mechanical removal of biofilm and bacteria in dentinal tubules. METHODS: A 6-year-old boy had a traumatic injury to tooth #9, which was avulsed and replanted within 40 minutes. The tooth subsequently developed a local swelling in the periapical area. The patient was referred to the Postgraduate Endodontic Clinic for revascularization/revitalization therapy on tooth #9. The treated tooth remained asymptomatic for 16 months and then developed pain and local periapical swelling. The oral surgeon extracted the revascularized/revitalized tooth. On request, the extracted tooth was processed for histologic and histobacteriologic examination. RESULTS: The tissue in the canal was completely destroyed. Most bacteria were observed in the apical portion and not in the coronal portion of the canal and formed biofilm on the canal walls and penetrated into the dentinal tubules. CONCLUSIONS: On the basis of histobacteriologic observations, the failure of revascularized/revitalized tooth could be due to inadequate root canal disinfection without mechanical debridement. It may be important to perform mechanical debridement as part of the revascularization/revitalization therapy to disrupt the biofilm on the canal walls and remove bacteria in the dentinal tubules because revascularization/revitalization therapy is able to increase thickening of the canal walls.
PMID: 24461421
ISSN: 0099-2399
CID: 777862

Triclosan Blocks Mmp 13 Expression in Hormone-Stimulated Osteoblasts

Barnes, Virginia Monsul; Xu, Tao; Shimizu, Emi; Nakatani, Teruyo; Jefcoat, Steven; Vasilov, Anatoliy; Qin, Ling; Partridge, Nicola C
Background: Matrix metalloproteinase-13 (Mmp-13) is an important enzyme for the modulation of bone turnover and gingival recession. Elevated levels of Mmp-13 are associated with alveolar bone resorption, periodontal ligament destruction, and gingival attachment loss, which are the clinical symptoms of periodontal disease. Continued evidence suggests periodontal disease contributes to oral tissue destruction and is linked to numerous systemic conditions. Triclosan is a long standing, proven antibacterial and anti-inflammatory agent found in the only FDA-approved dentifrice for the treatment of plaque and gingivitis. Methods: This study examined the inhibitory effects of triclosan on lipopolysaccharide (LPS), parathyroid hormone (PTH) and prostaglandin E(2) (PGE(2)) induced expression of Mmp-13 in UMR 106-01 cells, an osteoblastic osteosarcoma cell line. The cells were stimulated with PTH or PGE(2) to induce Mmp-13 mRNA expression and Real Time RT-PCR was performed to determine gene expression levels. Western blot analysis assessed the presence or absence of protein degradation or inhibition of protein synthesis. Mmp-13 Promoter Reporter Assay was utilized to explore possible direct effects of triclosan on the Mmp-13 promoter. Results: Triclosan significantly reduced PTH or PGE(2) elevated expression of Mmp-13 in osteoblastic cells without affecting basal levels of the mRNA. Surprisingly, triclosan enhanced the expression of c-fos and amphiregulin mRNA. A promoter assay indicated triclosan directly inhibits the activation of the PTH-responsive minimal promoter of Mmp-13. Conclusion: Our data appear to have identified a nuclear mechanism of action of triclosan which accounts for triclosan's ability to inhibit PTH or PGE(2) induced Mmp-13 expression in osteoblastic cells.
PMID: 23368947
ISSN: 0022-3492
CID: 223642

EphB-EphrinB Interaction Controls Odontogenic/Osteogenic Differentiation with Calcium Hydroxide

Wang, Xiaozhe; Jong, George; Lin, Louis M; Shimizu, Emi
INTRODUCTION: Calcium hydroxide is used in direct pulp capping of uncontaminated exposed vital pulps caused by mechanical or traumatic injury. Calcium hydroxide creates a high alkaline pH environment and initiates a mineralized tissue formation in the pulp. The exact mechanism by which calcium hydroxide induces the reparative dentin formation is unknown. Because Eph receptors and ephrin ligands play a role in pulp stem cell migration and proliferation, our hypothesis is that calcium hydroxide-related odontogenic/osteogenic differentiation may be associated with Eph-ephrin interaction. The aim of this study was to investigate whether Eph-ephrin interaction regulates odontogenic/osteogenic differentiation with calcium hydroxide. METHODS: Primary pulp cells were harvested from the molars of C57BL/6 mice. The cells were treated with calcium hydroxide. Immunofluorescence was used to detect protein expression. A knockout of the ephrinB1 or EphB2 gene was performed with short hairpin RNAs. Cell migration, proliferation, and gene expression were then analyzed. RESULTS: Calcium hydroxide stimulated EphB2 gene expression but suppressed ephrinB1 gene expression at the proliferation stage. However, calcium hydroxide stimulated both ephrinB1 and EphB2 gene expression at the differentiation stage. In addition, EphB2 localized at ephrinB1-positive cells at the area of Dentin sialoprotein (DSP) staining, which increased with calcium hydroxide treatment. Knockdown of ephrinB1-EphB2 significantly suppressed cell proliferation. Additionally, knockdown of the ephrinB1 gene caused cell migration, whereas a lack of the EphB2 gene suppressed calcium hydroxide-induced mineralization from primary pulp cells. CONCLUSIONS: EphrinB1-EphB2 interaction contributes to calcium hydroxide-induced odontogenic/osteogenic differentiation. This observation is the first finding of the mechanism of calcium hydroxide-induced odontogenic/osteogenic differentiation.
PMID: 24041387
ISSN: 0099-2399
CID: 541662