Age-dependent role of SIRT6 in jawbone via regulating senescence and autophagy of bone marrow stromal cells.

Age-related jawbone loss directly impact the function of oral cavity resulted from tooth loss, implant failure, and jaw fracture. Numerous evidences show that age-related senescence of bone marrow stromal cells (BMSCs) play a critical role in bone loss, but little attention has been paid to jawbone. Here, we delineated the critical role of sirtuin family protein 6 (SIRT6) in senescence, autophagy, and osteogenesis of BMSCs from jawbones. Radiography analysis showed less jawbone quality in elderly than young people. We also showed that SIRT6 expression decreased in bone tissue and BMSCs from the elderly by immunochemical staining. BMSCs from the elderly exhibited decreased osteogenic differentiation and inclined senescence which these phenotypes could be simulated by SIRT6 knockdown. Furthermore, accompanied with the inhibition of SIRT6, the autophagy level and ostogenesis of BMSCs was also decreased. However, using rapamycin, an autophagy activator, could rescue these adverse effects of BMSCs caused by SIRT6 inhibition. Mechanistically, SIRT6 regulated the autophagy and osteogenesis of BMSCs by activating AKT-mTOR pathway, at least in part. Finally, a decreased jawbone quality was shown in SIRT6 haploinsufficiency mice by Wnt1 specific tissue knockdown (Wnt1-Cre;SIRT6fl/+) model. Taken together, our data revealed that SIRT6 adjusted senescence and osteogenesis of BMSCs via altering autophagy level, and associated with age-related bone loss. SIRT6 could be as a promising therapeutic target for age-related osteoporosis of jawbone.

Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone.

Ameloblastoma is the most common benign odontogenic tumor in Japan. It is believed that it expands in the jaw bone through peritumoral activation of osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) released from the ameloblastoma, as in bone metastases of cancer cells. However, the clinical features of ameloblastoma, including its growth rate and patterns of invasion, are quite different from those of bone metastasis of cancer cells, suggesting that different underlying mechanisms are involved. Therefore, in the present study, we examined the possible mechanisms underlying the invasive expansion of ameloblastoma in the jaw bone. Expression levels of RANKL assessed by western blotting were markedly lower in ameloblastoma (AM-1) cells than in highly metastatic oral squamous cell carcinoma (HSC-3) cells. Experiments coculturing mouse macrophages (RAW264.7) with AM-1 demonstrated low osteoclastogenic activity, as assessed by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation, probably because of low release of RANKL, whereas cocultures of RAW264.7 with HSC-3 cells exhibited very high osteoclastogenic activity. Thus, RANKL release from AM-1 appeared to be too low to generate osteoclasts. However, AM-1 cultured directly on calcium phosphate-coated plates formed resorption pits, and this was inhibited by application of bafilomycin A1. Furthermore, vacuolar-type H+-ATPase (V-ATPase) and H+/Cl- exchange transporter 7 (CLC-7) were detected on the surface of AM-1 cells by plasma membrane biotinylation and immunofluorescence analysis. Immunohistochemical analysis of clinical samples of ameloblastoma also showed plasma membrane-localized V-ATPase and CLC-7 in the epithelium of plexiform, follicular and basal cell types. The demineralization activity of AM-1 was only 1.7% of osteoclasts demineralization activity, and the growth rate was 20% of human normal skin keratinocytes and HSC-3 cells. These results suggest that the slow expansion of several typical types of ameloblastomas in jaw bone is attributable to its slow growth and low demineralization ability.

GAD65/GAD67 double knockout mice exhibit intermediate severity in both cleft palate and omphalocele compared with GAD67 knockout and VGAT knockout mice.

Inhibitory neurotransmitters, γ-aminobutyric acid (GABA) and glycine, are transported into synaptic vesicles by the vesicular GABA transporter (VGAT). Glutamate decarboxylase (GAD) is a GABA-synthesizing enzyme and two isoforms of GAD, GAD65 and GAD67 are encoded by two independent genes. There was virtually no GABA content in GAD65/GAD67 double knockout (GADs DKO) mouse brains. Neither GABAergic nor glycinergic inhibitory postsynaptic currents were almost detected in VGAT knockout (KO) mouse cultured neurons and spinal cords. GAD67 KO and VGAT KO mice displayed developmental abnormalities, cleft palate and omphalocele, suggesting that GABAergic transmission is involved in palate and abdominal wall formations. However, the incidence and severity of both failures in GAD67 KO mice were lower and less than those in VGAT KO mice. These results raise the possibility that GABAergic transmission mediated by GAD65-produced GABA and/or glycinergic transmission contributed to both palate and abdominal wall formations. However, it still remains unclear whether GABAergic transmission mediated by GAD65 and glycinergic transmission contribute to those formations. Here, to answer these questions, we generated GADs DKO mice and compared the phenotypes of GADs DKO mice with those of GAD67 KO and VGAT KO mice. Our anatomical analyses demonstrated that the incidence of cleft palate and omphalocele in GAD67 KO mice was 65.8% and 58.9%, respectively, but the incidence of both phenotypes in GADs DKO and VGAT KO mice was 100%. The severity of cleft palate and omphalocele was evaluated by elevation of palate shelves and size and liver inclusion of omphalocele, respectively. We observed that the phenotypes of cleft palate and omphalocele in GADs DKO mice were more and less severe than those in GAD67 KO and VGAT KO mice, respectively. These results indicate the significant contribution of not only GAD65-mediated GABAergic but also glycinergic transmissions to both palate and abdominal wall formations.

An aromatase polymorphism (g.132810C>T) predicts risk of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: Bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) is an unpredictable, debilitating adverse effect. Recently, genetic polymorphisms have arisen as promising tools to identify patients with a higher risk of drug-related adverse events. AIM: We aimed to examine the association between the aromatase polymorphism g.132810C>T, and the estrogen receptor polymorphisms g.156705T>C and g.156751A>G, and the risk of BP-related ONJ. METHODS: Eighty-three subjects were included in the study. A clinical and radiological examination was conducted on oncologic subjects treated with zoledronic acid. Subjects with histologically confirmed ONJ were included in the test group (n = 30) whereas subjects with good oral health were included in control group (n = 53). Aromatase and estrogen receptor polymorphisms from blood samples were analyzed. RESULTS: The aromatase g.132810C>T polymorphism displayed an over-representation of the TT genotype in the test group (36.67 vs 16.98%; p < 0.05). There was no significant difference in either estrogen receptor polymorphism genotype frequency between the test and control groups. CONCLUSION: Our data suggest a role for the g.132810C>T polymorphism in predicting ONJ risk. These results can pave the way to the personalization of BP therapy, based on individual genotype.

Expression of the RSK2 gene during early human development.

The 90 kDa ribosomal S6 serine/threonine kinase 2 gene (RSK2, U08316) has been recently identified as a disease-causing gene in an X-linked disorder, the Coffin-Lowry Syndrome (MIM 303600) characterized by severe mental retardation, facial dysmorphisms and progressive skeletal malformations. To investigate its possible role in cerebral cortex development, we performed RNA in situ hybridization at three stages of human development: day 32 (Carnegie 15), 9 weeks (Carnegie 23) and 13 weeks. RSK2 expression is detected in the embryonic anterior and posterior telencephalon (hippocampus anlagen), mesencephalon, rhombencephalon and cerebellum. RSK2 gene expression is also observed in dorsal root ganglia, cranial nerve ganglia, and sensory epithelium of the inner ear, liver, lung and jaw anlagen. This pattern of expression may be involved in cognitive impairment and facial dysmorphisms found in Coffin-Lowry Syndrome.

The distribution of retinaldehyde dehydrogenase-2 in rat and human orodental tissues.

Retinoic acid is an important signalling molecule in embryological development and continues to be important in the adult animal because it modulates growth and differentiation in many epithelial tissues. The distribution of the enzyme retinaldehyde dehydrogenase-2 (RALDH 2), which is involved in the synthesis of retinoic acid, was studied using immunocytochemical techniques in: (1) the developing orodental region of rats aged between 15 days in utero and 6 months; and (2) in archival human autopsy material consisting of abdominal skin and mucosa from various regions of the mouth. In developing tooth germs, RALDH 2 was absent in the enamel organ and dental papilla, its presence only being noted at the periphery of the dental follicle adjacent to parts of the developing alveolar crypt. In adult teeth, the presence of RALDH 2 was limited to blood vessels in the periodontal ligament. In embryos, the connective tissue beneath the nasal epithelium and the meninges stained strongly positively for RALDH 2, as did the connective tissue beneath nasal epithelium in an adult rat. Both keratinized and non-keratinized human oral epithelia and abdominal skin stained positively for RALDH 2. Staining was present throughout the stratified epithelium, except in the keratinized layer and in the basal layer associated with the dorsal surface of the tongue. In addition, the adnexia as well as the ductal lining of mucous glands stained positively for RALDH 2.

Tonic fiber in Mauremys (Clemmys) japonica jaw muscle.

The jaw muscles of a turtle [Mauremys (Clemmys) japonica (MCJ)], have been analyzed for the histochemical characteristics of their fiber types. Six jaw muscles of the MCJ have been analyzed for histochemical characteristics of their fiber types. In this study, fiber types in two groups of muscles are classified; 1) fast twitch glycolytic (FG), fast twitch oxidative glycolytic (FOG), slow twitch oxidative (SO), and tonic fibers, according to the system of Putnam et al. (1980); 2) reacted differently in similar histochemical tests and classified, respectively, as various types. Especially tonic fibers constituted low percentage in the MAMP (ca., 0.3%), MI (ca., 3.9%) and MPtp (ca., 1.0%) were found. These results suggested that the tonic fiber plays a small part of the function in jaw movement.

Mast cell-derived tryptase in odontogenic cysts.

Inflammatory and developmental cysts of the jaws are relatively common bone destructive lesions in the human maxillofacial skeleton but their pathogenesis is still poorly understood. In this study the role of mast cells (MC), and mast cell tryptase in particular, was evaluated in the pathophysiology of bone resorption and jaw cyst formation in different types of cysts. The distribution of MC and the amount of tryptase in histological tissue sections were determined by immunohistochemistry using monoclonal antihuman tryptase antibodies and the results were quantitated by using an image analyzing system. The amount of tryptase was further studied by Western-blotting and measurement of trypsin-like activity from the neutral salt extracts obtained from different types of jaw cysts. In contrast to control tissue, high trypsin-like activities and abundant immunoreactive tryptase were observed in the extracts of all types of cysts studied (radicular, dentigerous and keratocyst). In tissue sections the highest amount of tryptase-positive staining was observed in radicular cysts (mean 6.2% of reference area) and the lowest amount in keratocysts (mean 2.1% of reference area, P < 0.01). MC were found to be located in inflammatory cell-rich tissue areas and just beneath the cyst epithelium. Importantly, MC located at the border of bone were observed to be degranulated, indicating high activity of MC and release of tryptase at the regions of early bone destruction. Based on previous findings addressing the role of mast cell tryptase in proteolytic cascades, and the known association of MC with osteoporosis, we suggest that mast cells and mast cell tryptase may contribute significantly to jaw cyst tissue remodelling during growth of a cyst, and to the destruction of the surrounding bone, resulting in jaw cyst expansion.

Prenatal diagnosis by isoenzymic differentiation of Treacher Collins' syndrome induced by retinoids in rats.

A series of branchial arch malformations was induced in 618 embryos from 72 pregnant rats by a single intraperitoneal injection of 10 mg/kg etretinate at 8.5 days of gestation. The litters developed several malformations, including microtia, low set and dorsally placed outer ears, defective middle ear ossicles, short cochleas, defectively differentiated Meckel's cartilages, micrognathia, rudimentary malar bones, lateral facial clefts, fistulas and skin tags, all of which were similar to Treacher Collins' syndrome in man. The defects were accompanied by a pathological differentiation pattern of various isoenzymes in maxillary and mandibular processes. These isoenzymes could be detected in amniotic fluid from the 9th to the 20th days of pregnancy and showed a pathological differentiation pattern here as well. We conclude that a teratogenically induced syndrome affecting the first and second branchial arches is accompanied by a pathological differentiation pattern that can be traced by determinations of isoenzymes in the branchial arches as well as in amniotic fluid.

Cyclic AMP in dental and periodontal tissues during tooth eruption in kittens.

Cyclic adenosine 3',5'-monophosphate (cAMP) concentrations and cellular distribution were studied in dental and periodontal tissues during tooth eruption in kittens. Although the mean levels of cAMP around developing teeth were similar in all the tissue samples, there were marked differences in cAMP stainability of tissues apical and occlusal to the erupting teeth.

Sarcoplasmic masses. Enzyme histochemistry and autoradiography.

A comparative enzyme-histochemical study was made of the sarcoplasmic masses in a muscle biopsy specimen from a patient with myotonic dystrophy and those in the jaw-muscle of the Rana temporaria tadpole. The enzyme-histochemical patterns of the two methods proved to be identical. An autoradiographic study was made of the sarcoplasmic masses in the R temporaria tadpole with DL-thyroxine-2-14C and levomethionine 35S. The radioactivity labeled compounds were found to be stored or incorporated mainly in the myofibrillae, and virtually not at all in the sarcoplasmic masses.