Satoshi Rokutanda

Akt regulates skeletal development through GSK3, mTOR, and FoxOs

Although Akt plays key roles in various cellular processes, the functions of Akt and Akt downstream signaling pathways in the cellular processes of skeletal development remain to be clarified. By analyzing transgenic embryos that expressed constitutively active Akt (myrAkt) or dominant-negative Akt in chondrocytes, we found that Akt positively regulated the four processes of chondrocyte maturation, chondrocyte proliferation, cartilage matrix production, and cell growth in skeletal development. As phosphorylation of GSK3beta, S6K, and FoxO3a was enhanced in the growth plates of myrAkt transgenic mice, we examined the Akt downstream signaling pathways by organ culture. The Akt-mTOR pathway was responsible for positive regulation of the four cellular processes. The Akt-FoxO pathway enhanced chondrocyte proliferation but inhibited chondrocyte maturation and cartilage matrix production, while the Akt-GSK3 pathway negatively regulated three of the cellular processes in limb skeletons but not in vertebrae due to less GSK3 expression in vertebrae. These findings indicate that Akt positively regulates the cellular processes of skeletal growth and endochondral ossification, that the Akt-mTOR, Akt-FoxO, and Akt-GSK3 pathways positively or negatively regulate the cellular processes, and that Akt exerts its function in skeletal development by tuning the three pathways in a manner dependent on the skeletal part.

Cbfb regulates bone development by stabilizing Runx family proteins.

Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA‐binding capacity. Cbfb‐deficient (Cbfb−/−) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb−/− mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfbfl/fl mice with Dermo1 Cre knock‐in mice. Cbfbfl/fl/Cre mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfbfl/fl/Cre chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfbfl/fl/Cre embryos compared with the respective protein in the respective tissue of Cbfbfl/fl embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfbfl/fl/Cre primary osteoblasts, and Runx2 protein was less stable in Cbfbfl/fl/Cre osteoblasts than Cbfbfl/fl osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons.

Overexpression of CRKII increases migration and invasive potential in oral squamous cell carcinoma

CT10 regulator of kinase (CRK) was originally identified as an oncogene product of v-CRK in a CT10 chicken retrovirus system. Overexpression of CRKII has been reported in several human cancers. CRKII regulates cell migration, morphogenesis, invasion, phagocytosis, and survival; however, the underlying mechanisms are not well understood. In the present study, we evaluated the possibility of CRKII as an appropriate molecular target for cancer gene therapy. The expression of CRKII in 71 primary oral squamous cell carcinomas and 10 normal oral mucosal specimens was determined immunohistochemically, and the correlation of CRKII overexpression with clinicopathological factors was evaluated. Overexpression of CRKII was detected in 41 of 70 oral squamous cell carcinomas, the frequency being more significant than in normal oral mucosa. In addition, CRKII overexpression was more frequent in higher-grade cancers according to the T classification, N classification, and invasive pattern. Moreover, RNAi-mediated suppression of CRKII expression reduced the migration and invasion potential of an oral squamous cell carcinoma cell line, OSC20. Downregulation of CRKII expression also reduced the expression of Dock180, p130Cas, and Rac1, and the actin-associated scaffolding protein cortactin. These results indicate that the overexpression of CRKII is tightly associated with an aggressive phenotype of oral squamous cell carcinoma. Therefore, we propose that CRKII could be a potential molecular target of gene therapy by RNAi-targeting in oral squamous cell carcinoma.

Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation

Osteoblast apoptosis plays an important role in bone development and maintenance, and is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging. Although Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis, the physiological significance of Bcl2 in osteoblast differentiation has not been fully elucidated. To investigate this, we examined Bcl2-deficient (Bcl2−/−) mice. In Bcl2−/− mice, bromodeoxyuridine (BrdU)-positive osteoblasts were reduced in number, while terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive osteoblasts were increased. Unexpectedly, osteoblast differentiation was accelerated in Bcl2−/− mice as shown by the early appearance of osteocalcin-positive osteoblasts. Osteoblast differentiation was also accelerated in vitro when primary osteoblasts were seeded at a high concentration to minimize the reduction of the cell density by apoptosis during culture. FoxO transcription factors, whose activities are negatively regulated through the phosphorylation by Akt, play important roles in multiple cell events, including proliferation, death, differentiation, longevity, and stress response. Expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated; the expression and activity of FoxOs were enhanced; and the phosphorylation of Akt and that of FoxO1 and FoxO3a by Akt were reduced in Bcl2−/− calvariae. Further, the levels of p53 mRNA and protein were increased, and the expression of p53-target genes, Pten and Igfbp3 whose proteins inhibit Akt activation, was upregulated in Bcl2−/− calvariae. However, Pten but not Igfbp3 was upregulated in Bcl2−/− primary osteoblasts, and p53 induced Pten but not Igfbp3 in vitro. Silencing of either FoxO1 or FoxO3a inhibited and constitutively-active FoxO3a enhanced osteoblast differentiation. These findings suggest that Bcl2 deficiency induces and activates FoxOs through Akt inactivation, at least in part, by upregulating Pten expression through p53 in osteoblasts, and that the enhanced expression and activities of FoxOs may be one of the causes of accelerated osteoblast differentiation in Bcl2−/− mice.

Application of polyglycolic acid sheets and fibrin glue spray to bone surfaces during oral surgery: a case series.

PURPOSE Previous studies have described the use of a combination of polyglycolic acid (PGA) sheets (a resorbable biomaterial) and fibrin glue spray to treat open soft tissue wounds during oral surgery, which have produced good results. However, there have not been any detailed investigations of the use of these materials to treat exposed hard tissue wounds. This study investigated the combination of PGA sheets and fibrin glue spray to treat exposed bone surfaces during oral surgery. MATERIALS AND METHODS PGA sheets and fibrin glue spray were applied to exposed bone surfaces after lesion resection in 8 patients (10 sites) who had been diagnosed with malignant tumors. The sheets were cut into pieces (width, 5 to 10 mm) and applied to the exposed bone surface. RESULTS PGA adhesion was confirmed for the final time on postoperative days 28 to 56 (mean, 35.8 days), and there were no cases in which the PGA sheets fell off the wound prematurely. Epithelialization of the wound surface occurred gradually and was complete by postoperative weeks 4 to 5, regardless of the size of the wound. CONCLUSION This method was considered very effective at preventing postoperative bleeding, alleviating postoperative pain, and promoting epithelialization during the reconstruction of bone surfaces after tumor resection in the oral cavity.

Histopathological and immunohistochemical study in keratocystic odontogenic tumors: Predictive factors of recurrence

The aim of the present study was to identify the most useful markers for predicting recurrence of keratocystic odontogenic tumors (KCOTs). A total of 65 tumor samples from 63 patients diagnosed with typical parakeratinized cysts and KCOTs between 1992 and 2014 were retrospectively studied. Clinical and histopathological data and treatment modality were reviewed. In addition, the expression profiles of Ki-67, cluster of differentiation (CD)34 and podoplanin were assessed using immunohistochemistry. The association between these factors and the rate of KCOT recurrence was evaluated. The presence of daughter cysts, epithelial islands and high Ki-67, CD34 and podoplanin expression levels were revealed to be associated with tumor recurrence. In particular, univariate analysis revealed that high CD34 expression levels were significantly associated with tumor recurrence (P=0.034), as was conservative surgical treatment (P=0.003). Multivariate analysis revealed that conservative treatment was the greatest independent risk factor for tumor recurrence (odds ratio=13.337; P=0.018). These results suggest that overexpression of CD34 may be a potent predictor of tumor recurrence and radical treatment of the teeth that are in contact with the tumors is recommended in order to prevent tumor recurrence.

Spindle cell variant of ameloblastic carcinoma: a case report and literature review.

Spindle cell variant of ameloblastic carcinoma is an extremely rare tumor. Severe dedifferentiated spindle cell variants are diagnostically challenging, particularly in small biopsy specimens. Here, we report a case of spindle cell variant of ameloblastic carcinoma in the mandible of a 69-year-old male patient and review the available literature. The tumor was surgically resected under general anesthesia. Histopathologic diagnosis of spindle cell carcinoma was made on incisional biopsy, and the final diagnosis was confirmed as spindle cell variant of ameloblastic carcinoma. Immunohistochemistry using cytokeratin and CK19 is helpful in determining the origin of spindle cell variant of ameloblastic carcinoma, particularly CK19 indicated that sarcomatoid spindle cells are derived from odontogenic epithelium. A review demonstrated higher mean age of patients compared with that of other types of ameloblastic carcinoma. The rates of mortality and local recurrence were concurrently 30%. No recurrence or metastasis was seen in the 23-month follow-up period in the present case.

Predictors of obstructive sleep apnoea-hypopnea severity and oral appliance therapy efficacy by using lateral cephalometric analysis.

Obstructive sleep apnoea-hypopnea (OSAH) is a common disorder characterised by repetitive complete or partial closure of the upper airway during sleep, which results in sleep fragmentation and oxygen desaturation. There is growing interest in the use of oral appliances (OAs) to treat OSAH. The purpose of this study was to clarify the cephalometric factors that are associated with OSAH severity and that predict the outcome of OA therapy. Two hundred nine patients with OSAH were recruited and analysed retrospectively. They had a polysomnographically documented apnoea-hypopnea index (AHI) of more than five respiratory events per hour. Lateral skull radiographs were used for cephalometric analysis. Only 67 of the 209 recruited patients underwent a second polysomnography (PSG) to evaluate the efficacy of OA therapy. In all recruited patients, the angle formed by the subspinal point (A) to the nasion (N) to the supramental point (B) (i.e. ANB angle) and the distance between the mandibular plane and hyoid bone (MP-H) were predictive factors of OSAH severity. In only 67 patients underwent PSG with an OA, the mean rate of decrease in the AHI was 47·8 ± 29·1%. OA therapy effectively treated OSAH in some patients with a very severe form of OSAH. However, patients who had a high position of the hyoid bone had a poor response to OA therapy. This study suggested that cephalometric analysis is useful for predicting OSAH severity and OA therapy efficacy.

Granular cell ameloblastoma: case report of a particular ameloblastoma histologically resembling oncocytoma.

Granular cell ameloblastoma is classified as a histological subtype of solid/multicystic ameloblastoma. Usual granular cell ameloblastoma is histologically characterized by granular changes of stellate‐like cells located in the inner portion of the epithelial follicles. Here we report a case of another type of granular cell ameloblastoma, showing predominant anastomosing double‐stranded trabeculae of granular cells. This type of granular cell ameloblastoma is extremely rare, and the World Health Organization classification does not contain the entity. We tentatively termed it ‘anastomosing granular cell ameloblastoma’ in this report. The present case suggests the importance of differential diagnosis because the histology of ‘anastomosing granular cell ameloblastoma’ resembles that of salivary gland oncocytoma rather than that of usual granular cell ameloblastoma. The trabeculae observed in our case continued to the peripheral cells of a small amount of epithelial sheets of plexiform ameloblastoma, and the tumor cells were positive for CK19, which is regarded as an immunohistochemical marker of odontogenic epithelium. Similar to usual granular cell ameloblastoma, the tumor cells had CD68‐positive granules. For precise diagnosis of this condition, immunohistochemistry using CK19 and CD68, as well as detailed histological observation, are recommended.

Comparison of osseous healing after sagittal split ramus osteotomy and intraoral vertical ramus osteotomy

The sagittal split ramus osteotomy (SSRO) is generally associated with greater postoperative stability than the intraoral vertical ramus osteotomy (IVRO); however, it entails a risk of inferior alveolar nerve damage. In contrast, IVRO has the disadvantages of slow postoperative osseous healing and projection of the antegonial notch, but inferior alveolar nerve damage is believed to be less likely. The purposes of this study were to compare the osseous healing processes associated with SSRO and IVRO and to investigate changes in mandibular width after IVRO in 29 patients undergoing mandibular setback. On computed tomography images, osseous healing was similar in patients undergoing SSRO and IVRO at 1year after surgery. Projection of the antegonial notch occurred after IVRO, but returned to the preoperative state within 1year. The results of the study indicate that IVRO is equivalent to SSRO with regard to both bone healing and morphological recovery of the mandible.