Yuichiro Enoki

The Paired-box Homeodomain Transcription Factor Pax6 Binds to the Upstream Region of the TRAP Gene Promoter and Suppresses Receptor Activator of NF-κB Ligand (RANKL)-induced Osteoclast Differentiation

Background: Negative regulation of osteoclast differentiation is critical for suppression of pathological bone destruction. Results: Pax6 is induced by RANKL in osteoclasts and attenuates osteoclast differentiation via blocking TRAP gene expression. Conclusion: Pax6 functions together with its co-receptor to suppress TRAP gene expression and osteoclastogenesis. Significance: This study provides a new aspect for investigating the molecular targets linked to physiological bone resorption. Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

Expression of TLE3 by bone marrow stromal cells is regulated by canonical Wnt signaling

Transducing‐like enhancer of split 3 (TLE3), one of the Groucho/TLE family members, targets Runx2 transcription and suppresses osteoblast differentiation in bone marrow stromal cells (BMSCs). Here, we identify Wnt responsive elements of the TLE3 promoter region through comparative genomic and functional analyses and show that expression of TLE3 is increased by Wnt signaling, which is important for osteoblast differentiation. We also demonstrated that TLE3 is able to suppress canonical Wnt signaling in BMSCs. Taken together, our data suggest that induction of TLE3 by Wnt signaling is part of a negative feedback loop active during osteoblast differentiation.

Netrin-4 derived from murine vascular endothelial cells inhibits osteoclast differentiation in vitro and prevents bone loss in vivo.

Bone is a highly vascularized organ, thus angiogenesis is a vital process during bone remodeling. However, the role of vascular systems in bone remodeling is not well recognized. Here we show that netrin‐4 inhibits osteoclast differentiation in vitro and in vivo. Co‐cultures of bone marrow macrophages with vascular endothelial cells markedly inhibited osteoclast differentiation. Adding a neutralizing antibody, or RNA interference against netrin‐4, restored in vitro osteoclast differentiation. Administration of netrin‐4 prevented bone loss in an osteoporosis mouse model by decreasing the osteoclast number. We propose that vascular endothelial cells interact with bone in suppressing bone through netrin‐4.

Proteomics-based identification of novel proteins in temporal tendons of patients with masticatory muscle tendon--aponeurosis hyperplasia.

Masticatory muscle tendon-aponeurosis hyperplasia (MMTAH) is a new disease associated with limited mouth opening that is often misdiagnosed as a temporomandibular disorder; subsequently, patients are mistakenly treated with irreversible operations. Due to the poor presentation and characterization of symptoms, the underlying pathological conditions remain unclear. We have previously conducted a proteomic analysis of tendons derived from one MMTAH subject and one facial deformity subject using two-dimensional fluorescence difference gel electrophoresis and liquid chromatography coupled with tandem mass spectrometry. However, the results were obtained for only one subject. The aim of the present study was to confirm the expression of specific molecules in tendon tissues from multiple subjects with MMTAH by applying two-dimensional polyacrylamide gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Of the 19 proteins identified in tendons from both MMTAH and facial deformity patients, fibrinogen fragment D and beta-crystallin A4 were up-regulated, whereas myosin light chain 4 was down-regulated in MMTAH. We also found fibrinogen to be expressed robustly in tendon tissues of MMTAH patients. Our data provide the possibility that the distinctive expression of these novel proteins is associated with the pathology of MMTAH.

Correlation of Inflammatory Markers, Survival, and COX2 Expression in Oral Cancer and Implications for Prognosis

Objective Peripheral blood–derived inflammation-based scores, such as the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and the combination of platelet count and NLR, have recently been proposed as prognostic markers in solid tumors. The purpose of this study was to investigate the validity of inflammatory markers as predictive prognostic factors for locally advanced oral squamous cell carcinoma (OSCC). In addition, we evaluated the potential correlation between systemic inflammation and local expression of COX2. Study Design Retrospective chart review and histologic analysis. Setting Tertiary referral academic center. Subjects and Methods We conducted a retrospective analysis of 94 patients with advanced OSCC treated with surgery at our hospital between 2007 and 2015. The relationship among patient survival, systemic inflammatory markers, and local COX2 expression was evaluated. Local COX2 expression in surgical specimens was measured by immunohistochemistry. Results High NLR and high PLR were associated with significantly shorter overall survival and cancer-specific survival. Multivariate analysis revealed that cN stage, NLR, and postoperative radiation/chemoradiation were significantly associated with overall survival and cancer-specific survival. PLR and combination of platelet count and NLR were significantly correlated with tumor expression of COX2. Finally, patients with cN2 stage disease and high local COX2 expression had a significantly worse prognosis than other patient groups. Conclusion Pretreatment inflammatory markers are useful as prognostic factors in advanced OSCC. Our study suggests that local COX2 may be affected by systemic inflammation and that the prognostic impact of COX2 expression depends on host factors and tumor characteristics.

Usability of surgical treatment in cases of bisphosphonate-related osteonecrosis of the jaw stage 2 with sequestrum

Objective: This retrospective study was conducted to reveal usability of surgical treatment in the cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) stage 2 with sequestrum. Patients and Methods: Study subjects included 18 patients having BRONJ stage 2 with sequestrum and 12 non-BRONJ patients with nearly equal clinical states of BRONJ stage 2. Patient characteristics, frequency of inciting factors of osteonecrosis, and treatment results were compared between BRONJ group and non-BRONJ groups. In addition, correlation between treatment methods (conservative therapy, sequestrum curettage, and sequestrectomy) and treatment results and correlation between the administration route of bisphosphonates (BPs) (oral or intravenous) and treatment results were examined statistically. The Student′s t-test and Fisher′s exact test were performed for statistical analysis. Results: Patient characteristics, frequency of inciting factors of osteonecrosis, and treatment results showed no significant differences between the two groups. In the BRONJ group, treatment result of sequestrectomy was significantly better than conservative therapy/sequestrum curettage (P < 0.001), however, no significant difference was observed in the non-BRONJ group. No significant difference was found in correlation between the administration route of BPs and treatment results in the BRONJ group. Conclusion: Treatment outcome of sequestrectomy was better than conservative therapy/sequestrum curettage in BRONJ stage 2 cases with sequestrum.

Limited mouth opening with a square mandible configuration: a case of masticatory muscle tendon-aponeurosis hyperplasia

Most clinicians throughout the world are probably unaware of the existence of masticatory muscle tendon-aponeurosis hyperplasia (MMTAH), potentially leading to misdiagnoses such as temporomandibular joint disorder (TMD). Here, we introduce this disease from the viewpoint of education. In February 2013, a 39-year-old woman presented with limited mouth opening. Her facial configuration was characterized by a square mandible. There was no evidence of TMD. Magnetic resonance imaging (MRI) showed bilateral enlargement of the masseter muscles. Additionally, a ‘thick’ aponeurosis of the anterior aspect of the masseter muscle was noted bilaterally. On maximal mouth opening, intraoral palpation along the anterior border of the masseter muscle confirmed a hard cord-like structure, consistent with the findings on MRI. MMTAH was diagnosed. When clinicians notice limited mouth opening on oral examination, they should be knowledgeable about diseases associated with limited mouth opening and a square mandibular configuration, such as MMTAH.

Evaluation of oral wetness using an improved moisture-checking device for the diagnosis of dry mouth

Abstract Purpose In 2013, we reported the results of a third-generation oral moisture-checking device in a multicentre clinical study involving patients with dry mouth and healthy volunteers. Subsequently, several improvements have been made to the third-generation device, and a fourth-generation device is now commercially available. This study aimed to confirm the usefulness of this improved fourth-generation device in the diagnosis of dry mouth and to assess the physiological wetness of lingual mucosa by using this device. Materials and Method This multicentre study comprised subjects with dry mouth (dry mouth group) and those without dry mouth (healthy group). Results In this study, the degree of moisture was considerably different between the two groups. Receiver operating characteristic analysis revealed an area under the curve value of 0.831. Sensitivity and specificity values were close to 80% in cases where the degree of moisture ≥29.6 was defined as normal, ≤27.9 was defined as dry mouth, and 28.0–29.5 was defined as borderline dry mouth. Conclusions These results suggest that the improved fourth-generation moisture-checking device can be used for the diagnosis of oral dryness.

Donepezil prevents RANK-induced bone loss via inhibition of osteoclast differentiation by downregulating acetylcholinesterase

Objective Donepezil, an inhibitor of acetylcholinesterase (AChE) targeting the brain, is a common medication for Alzheimers disease. Interestingly, a recent clinical study found that administration of this agent is associated with lower risk of hip fracture independently of falling, suggesting its direct effect on bone tissues as well. AChE has been reported to be involved in osteoblast function, but the role of AChE on osteoclastogenesis still remains unclear. We analyzed the effect of AChE and donepezil on osteoclastogenesis in vivo and in vitro. Methods Cell-based assays were conducted using osteoclasts generated in cultures of murine bone marrow macrophages (BMMs) with receptor activator of nuclear factor-kappa B ligand (RANKL). The effect of donepezil was also determined in vivo using a mouse model of RANKL-induced bone loss. Results Recombinant AChE in BMMs cultured with RANKL further promoted RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation. RANKL also upregulated AChE expression in BMMs. RNA interference-mediated knockdown of AChE significantly inhibited RANKL-induced osteoclast differentiation and suppressed gene expression specific for osteoclasts. AChE upregulated expression of RANK, the receptor of RANKL, in BMMs. Donepezil decreased cathepsin K expression in BMMs and the resorptive function of osteoclasts on dentine slices. Donepezil decreased RANK expression in BMMs, resulting in the inhibition of osteoclast differentiation with downregulation of c-Fos and upregulation of Id2. Moreover, administration of donepezil prevented RANKL-induced bone loss in vivo, which was associated with the inhibition of bone resorption by osteoclasts. Conclusions AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimers disease.

A huge osteolipoma involving the coronoid process: a case report.

A 28-year-old man visited our hospital with the chief complaint of trismus. Computed tomography revealed a well-defined, soft tissue tumor, 66 × 45 × 21 mm, with a distinct boundary in the inner region of the zygomatic arch. The mass contained various sizes of bone-like hard tissue, some of which adhered to the right coronoid process. A contrast-enhanced magnetic resonance image showed that the mass was composed mainly of adipose tissue. Tumorectomy was performed, and the histopathological diagnosis was osteolipoma. At 2-year follow-up, mouth opening had increased from 31 mm to 50 mm. (J Oral Sci 58, 141-144, 2016).