Yuka Hotokezaka

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-α, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate‐resistant acid phosphatase (TRAP)‐positive mononuclear cells into multinuclear cells. TRAP‐positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS‐induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)‐α and peptidoglycan (PGN) induced cell fusion, but M‐CSF did not. The cell fusion induced by RANKL, TNF‐α, and LPS was specifically blocked by osteoprotegerin (OPG), anti‐TNF‐α antibody, and polymyxin B, respectively. LPS‐ and PGN‐induced cell fusion was partly inhibited by anti‐TNF‐α antibody but not by OPG. When TRAP‐positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal‐regulated kinase (ERK), p38MAPK (p38), and c‐Jun NH2‐terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK‐ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T‐cells c1 (NFATc1) and dendritic cell‐specific transmembrane protein (DC‐STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP‐positive mononuclear cells treated with or without M‐CSF, RANKL, TNF‐α, LPS, or PGN. Collectively, RANKL, TNF‐α, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC‐STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion‐inducing factors other than DC‐STAMP might be necessary for the cell fusion. J. Cell. Biochem. 101: 122–134, 2007.

αNAC depletion as an initiator of ER stress-induced apoptosis in hypoxia

Accumulation of unfolded proteins triggers endoplasmic reticulum (ER) stress and is considered a part of the cellular responses to hypoxia. The nascent polypeptide-associated complex (NAC) participates in the proper maturation of newly synthesized proteins. However, thus far, there have been no comprehensive studies on NAC involvement in hypoxic stress. Here, we show that hypoxia activates glycogen synthase kinase-3β (GSK-3β) and that the activated GSK-3β destabilizes αNAC with the subsequent apoptosis of the cell. Hypoxia of various cell types and the mouse ischemic brain was associated with rapid downregulation of αNAC and ER stress responses involving PERK, ATF4, γ-taxilin, elF2α, Bip, and CHOP. Depletion of αNAC by RNA interference specifically activated ER stress responses and caused mitochondrial dysfunction, which resulted in apoptosis through caspase activation. Interestingly, we found that the hypoxic conditions activated GSK-3β, and that GSK-3β inhibition prevented αNAC protein downregulation in hypoxic cells and rescued the cells from apoptosis. In addition, αNAC overexpression increased the viability of hypoxic cells. Taken together, these results suggest that αNAC degradation triggers ER stress responses and initiates apoptotic processes in hypoxic cells, and that GSK-3β may participate upstream in this mechanism.

An In Vivo 3D Micro-CT Evaluation of Tooth Movement After the Application of Different Force Magnitudes in Rat Molar

OBJECTIVE To investigate the precise longitudinal change in the periodontal ligament (PDL) space width and three-dimensional tooth movement with continuous-force magnitudes in living rats. MATERIALS AND METHODS Using nickel-titanium closed-coil springs for 28 days, 10-, 25-, 50-, and 100-g mesial force was applied to the maxillary left first molars. Micro-CT was taken in the same rat at 0, 1, 2, 3, 10, 14, and 28 days. The width of the PDL was measured in the pressure and tension sides from 0 to 3 days. Angular and linear measurements were used to evaluate molar position at day 0, 10, 14, and 28. The finite element model (FEM) was constructed to evaluate the initial stress distribution, molar displacement, and center of rotation of the molar. RESULTS The initial evaluation of PDL width showed no statistical differences among different force magnitudes. Tooth movement was registered 1 hour after force application and gradually increased with time. From day 10, greater tooth movement was observed when 10 g of force was applied. The FEM showed that the center of rotation in the molar is located in the center of five roots at the apical third of the molar roots. CONCLUSION The rats molar movement mainly consists of mesial tipping, extrusion of distal roots, intrusion of mesial root, palatal inclination, and mesial rotation. Although the initial tooth movement after the application of different force magnitudes until day 3 was not remarkably different, 10 g of force produced more tooth movement compared with heavier forces at day 28.

Ionizing Radiation Induces Macrophage Foam Cell Formation and Aggregation Through JNK-Dependent Activation of CD36 Scavenger Receptors

PURPOSE Irradiated arteries of cancer patients can be associated with atherosclerosis-like lesions containing cholesterol-laden macrophages (foam cells). Endothelial cell damage by irradiation does not completely explain the foam cell formation. We investigated the possible underlying mechanisms for ionizing radiation (IR)-induced foam cell formation. METHODS AND MATERIALS Human peripheral blood monocytes were activated by macrophage colony-stimulating factor and then treated with varying doses of IR in vitro in the absence of endothelial cells. Scavenger receptor expression and foam cell formation of IR-treated macrophages were investigated in the presence or absence of oxidized low-density lipoprotein. We also assessed the importance of mitogen-activated protein kinase activity in the macrophage colony-stimulating factor-activated human monocytes (macrophages) for the foam cell formation. RESULTS We found that IR treatment of macrophage colony-stimulating factor-activated human peripheral blood monocytes resulted in the enhanced expression of CD36 scavenger receptors and that cholesterol accumulated in the irradiated macrophages with resultant foam cell formation in the presence of oxidized low-density lipoprotein. Furthermore, when cultured on collagen gels, human macrophages formed large foam cell aggregates in response to IR. Antibodies against CD36 inhibited the IR-induced foam cell formation and aggregation, indicating that the IR-induced foam cell formation and the subsequent aggregation are dependent on functional CD36. In addition, we found that IR of human macrophages resulted in c-Jun N-terminal kinase activation and that c-Jun N-terminal kinase inhibition suppressed IR-induced CD36 expression and the subsequent foam cell formation and aggregation. CONCLUSION Taken together, these results suggest that IR-induced foam cell formation is mediated by c-Jun N-terminal kinase-dependent CD36 activation.

Simple and Reliable Determination of Intravoxel Incoherent Motion Parameters for the Differential Diagnosis of Head and Neck Tumors

Intravoxel incoherent motion (IVIM) imaging can characterize diffusion and perfusion of normal and diseased tissues, and IVIM parameters are authentically determined by using cumbersome least-squares method. We evaluated a simple technique for the determination of IVIM parameters using geometric analysis of the multiexponential signal decay curve as an alternative to the least-squares method for the diagnosis of head and neck tumors. Pure diffusion coefficients (D), microvascular volume fraction (f), perfusion-related incoherent microcirculation (D*), and perfusion parameter that is heavily weighted towards extravascular space (P) were determined geometrically (Geo D, Geo f, and Geo P) or by least-squares method (Fit D, Fit f, and Fit D*) in normal structures and 105 head and neck tumors. The IVIM parameters were compared for their levels and diagnostic abilities between the 2 techniques. The IVIM parameters were not able to determine in 14 tumors with the least-squares method alone and in 4 tumors with the geometric and least-squares methods. The geometric IVIM values were significantly different (p<0.001) from Fit values (+2±4% and −7±24% for D and f values, respectively). Geo D and Fit D differentiated between lymphomas and SCCs with similar efficacy (78% and 80% accuracy, respectively). Stepwise approaches using combinations of Geo D and Geo P, Geo D and Geo f, or Fit D and Fit D* differentiated between pleomorphic adenomas, Warthin tumors, and malignant salivary gland tumors with the same efficacy (91% accuracy = 21/23). However, a stepwise differentiation using Fit D and Fit f was less effective (83% accuracy = 19/23). Considering cumbersome procedures with the least squares method compared with the geometric method, we concluded that the geometric determination of IVIM parameters can be an alternative to least-squares method in the diagnosis of head and neck tumors.

Length of intact plasma membrane determines the diffusion properties of cellular water.

Molecular diffusion in a boundary-free medium depends only on the molecular size, the temperature, and medium viscosity. However, the critical determinant of the molecular diffusion property in inhomogeneous biological tissues has not been identified. Here, using an in vitro system and a high-resolution MR imaging technique, we show that the length of the intact plasma membrane is a major determinant of water diffusion in a controlled cellular environment and that the cell perimeter length (CPL) is sufficient to estimate the apparent diffusion coefficient (ADC) of water in any cellular environment in our experimental system (ADC = −0.21 × CPL + 1.10). We used this finding to further explain the different diffusion kinetics of cells that are dying via apoptotic or non-apoptotic cell death pathways exhibiting characteristic changes in size, nuclear and cytoplasmic architectures, and membrane integrity. These results suggest that the ADC value can be used as a potential biomarker for cell death.

GSK-3β-dependent downregulation of γ-taxilin and αNAC merge to regulate ER stress responses

The signaling pathway leading to the endoplasmic reticulum (ER) stress responses has not been fully elucidated. Here we showed that glycogen synthase kinase-3β (GSK-3β)-dependent downregulation of γ-taxilin and nascent polypeptide-associated complex α-subunit (αNAC) mediates hypoxia-induced unfolded protein responses (UPRs) and the subsequent apoptotic and autophagic pathways. The degradation of γ-taxilin or αNAC was sufficient to initiate UPRs in normoxic cells. However, the ER stress signaling pathways initiated by γ-taxilin or αNAC were distinct, triggering different ER stress sensors and activating different downstream pathways. Hypoxia caused GSK-3β-dependent tau hyperphosphorylation and cleavage in neuronal cells, but γ-taxilin ablation induced tau hyperphosphorylation alone and αNAC ablation induced neither changes. Notably, downregulation of γ-taxilin and αNAC occurs in the brain of patients with Alzheimer’s disease. These results suggest that GSK-3β-dependent downregulation of γ-taxilin and αNAC, which differently activate the UPRs, merge to regulate hypoxia-induced ER stress responses and provide a new insight into the pathogenesis of neurodegenerative diseases.

Oral and maxillofacial radiology teaching file on the World Wide Web

An oral and maxillofacial radiology teaching file on the World Wide Web was created effective June 15, 1995. The file contains 106 cases (279 images) of 94 diseases in the oral and maxillofacial region.

Comparison between ultrasonography and MR imaging for discriminating squamous cell carcinoma nodes with extranodal spread in the neck

OBJECTIVE To compare the diagnostic ability of ultrasonography (US) and MR imaging for discriminating squamous cell carcinoma (SCC) nodes with extranodal spread (ENS) in the neck. METHODS US and MR imaging was retrospectively evaluated for differentiating ENS-positive (n=28) from ENS-negative (n=26) SCC nodes (>10mm short-axis diameter) in 50 patients with head and neck SCCs. We assessed nodal size on US and MR images; irregular nodal margin on US; and vanishing nodal border, flare, and shaggy nodal margin signs on T1-, fat-suppressed T2-, and contrast-enhanced T1-weighted MR images, respectively. US and MR images were analyzed by 3 radiologists in consensus and the results were compared between ENS-positive and ENS-negative SCC nodes. RESULTS The nodal sizes of ENS-positive nodes (21±9 mm) were significantly larger than those of ENS-negative SCC nodes (14±4 mm) (p<0.001). Irregular nodal margins were more frequently observed in ENS-positive SCC nodes (75%) than in ENS-negative SCC nodes (12%). The vanishing nodal margin, flare, and shaggy nodal margin signs were more frequently observed in ENS-positive SCC nodes (93%, 89%, and 82%, respectively) than in ENS-negative nodes (46%, 19%, and 19%, respectively). A combination of size (≥22 mm) and imaging criteria (irregular margin or flare sign) best discriminated ENS-positive SCC nodes with 82% sensitivity, 89% specificity, and 85% accuracy for US and 89% sensitivity, 81% specificity, and 85% accuracy for MR imaging. CONCLUSION US discriminated ENS-positive from ENS-negative SCC nodes with comparable accuracy and higher specificity than MR imaging.

Magnetic resonance imaging-based differentiation between juvenile recurrent parotitis and juvenile Sjögren’s syndrome

Differentiation between juvenile recurrent parotitis and juvenile Sjögren’s syndrome may be difficult on the basis of clinical symptoms and imaging examinations, such as sialography and ultrasonography. Histological findings of the labial glands may also not be definitive for this purpose. Here, we report three cases of patients with juvenile recurrent parotitis who underwent magnetic resonance imaging examinations. The imaging results suggest that magnetic resonance imaging may be an effective tool for differentiating between these two distinct salivary gland diseases.