PURPOSE: In osteoclast differentiation, actin-rich membrane protrusions play a crucial role in cell adhesion. Odontogenic ameloblast-associated protein (Odam) contributes to cell adhesion by inducing actin rearrangement. Odam-mediated RhoA activity may play a significant role in multinucleation of osteoclasts. However, the precise function of Odam in osteoclast cell adhesion and differentiation remains largely unknown. Here, we identify a critical role for Odam in inducing osteoclast adhesion and differentiation. MATERIALS AND METHODS: The expression of Odam in osteoclasts was evaluated by immunohistochemistry. Primary mouse bone marrow and RAW264.7 cells were used to test the cell adhesion and actin ring formation induced by Odam. RESULT: Odam was expressed in osteoclasts around alveolar bone. Odam transfection induced actin filament rearrangement and cell adhesion compared with the control or collagen groups. Overexpression of Odam promoted actin stress fiber remodeling and cell adhesion, resulting in increased osteoclast fusion. CONCLUSION: These results suggest that Odam expression in primary mouse osteoclasts and RAW264.7 cells promotes their adhesion, resulting in the induction of osteoclast differentiation.
Actin Cytoskeleton ; Actins* ; Animals ; Bone Marrow ; Cell Adhesion ; Collagen ; Immunohistochemistry ; Membranes ; Mice ; Osteoclasts* ; Stress Fibers ; Transfection

PURPOSE: To evaluate the possible association between psychological stress and glaucoma, given that there are emerging issues and controversy regarding whether psychological stress is one of contributing factors of glaucoma development. METHODS: We used the medical records of 16,426 patients from the Korean National Health and Nutritional Examination Survey 2008–2011. Glaucoma was defined based on examinations including the intraocular pressure, optic disc, visual field, and retinal nerve fiber layer. Stress was evaluated using five questionnaires regarding sustained stress, depression, feelings of suicide, history of suicide attempt/s, and history of psychological counseling. We used univariable and multivariable logistic regression analyses after adjusting confounding factors for glaucoma. RESULTS: Univariable logistic regression analysis revealed no significant association between glaucoma and psychological stress (odds ratio [OR] = 0.84; confidence interval [CI] = 0.70–1.01), depression (OR = 1.22; CI = 0.97–1.55), suicide attempt/s (OR = 0.73, CI = 0.33–1.59), and psychological counseling (OR = 0.72, CI = 0.43–1.21). Using univariate analysis, only the feelings of suicide factor (OR = 1.28, CI = 1.02–1.60) was significantly associated with glaucoma. Using multivariable analysis after adjusting for confounding factors, no significant association was found in any psychological stress factor. CONCLUSIONS: There was no significant association between psychological stress and glaucoma. The results of this study indicated that the pathogenesis of glaucoma is more consistent with the pathogenesis of physiological causes such as age or hypertension, rather than indirect causes such as stress.
Counseling ; Depression ; Glaucoma ; Humans ; Hypertension ; Intraocular Pressure ; Korea ; Logistic Models ; Medical Records ; Nerve Fibers ; Retinaldehyde ; Stress, Psychological ; Suicide ; Visual Fields

Akt, a key protein of cell survival, can promote cell growth and survival by activations of various cellular protective factors. Ischemic preconditioning (IP) has been known to reduce ischemic injury through upregulation of phosphorylation of Akt (p-Akt). CuZn-superoxide dismutase (SOD-1), an antioxidant enzyme, scavenges reactive oxygen species and protects cell from oxidative stress by increasing the activaiton of Akt. The present study was performed to examine the effects of IP on the expression of p-Akt and SOD-1 in the ischemicreperfused rat skeletal muscles. Thirty weeks old male SD rats were divided into 4 groups, such as controls, IP, 4 hour ischemia and 4 hour ischemia with IP. For IP, commom iliac artery was occluded three times for 5 min ischemia followed by 5 min reperfusion using rodent vascular clamps. Ischemia was induced by occlusion on the same artery for 4 hours. The Tibialis anterior and Soleus were removed at 0, 1, 3, and 24 hours of reperfusion. The expressions of p-Akt (Ser 473) and SOD-1 were examined with immunohistochemistry and Western blot analysis.In the IP group, the p-Akt and SOD-1 were increased, compared to the control group. In the ischemia group, the p- Akt and SOD-1 were decreased, compared to the control group, and were more abundant when reperfusion time were increased. IP increased the p-Akt and SOD-1 after 4 hour ischemia, and the p-Akt and SOD-1 were higher in Soleus compared to Tibialis anterior. These findings suggest that IP increases p-Akt and expression of SOD-1 in the ischemic-reperfused rat skeletal muscles, and that upregulations of p-Akt and SOD-1 induced by IP were higher in the red muscle fiber, Soleus, than the white muscle fiber, Tibialis anterior.
Animals ; Arteries ; Blotting, Western ; Cell Survival ; Humans ; Iliac Artery ; Immunohistochemistry ; Ischemia ; Ischemic Preconditioning ; Male ; Muscle Fibers, Fast-Twitch ; Muscle Fibers, Slow-Twitch ; Muscle, Skeletal ; Oxidative Stress ; Phosphorylation ; Rats ; Reactive Oxygen Species ; Reperfusion ; Rodentia ; Up-Regulation

BACKGROUND/AIMS: Fascin, an actin-bundling protein found in membrane ruffles, microspikes, and stress fibers, induces membrane protrusions and increases cell motility in normal and various transformed cells. The expression of fascin in epithelial neoplasms has only been described recently, and the role of fascin in colorectal carcinoma (CRC) is still unknown. METHODS: Paraffin sections of CRC from 79 patients were immunohistochemically investigated using monoclonal anti-fascin antibody. Staining of >5% of tumor cells was recorded as positive immunoreactivity. RESULTS: Overall, fascin immunoreactivity was detected in 63 of 79 patients (79.7%). Twenty-three patients were classified as 1+ (5-25% immunoreactive tumor cells) and 24 were 2+ (>25% immunoreactive tumor cells). In these patients, 16 had 3+ (>75% immunoreactive tumor cells) fascin immunoreactivity. Fascin immunoreactivity was increased according to the TNM stage (P<0.001), positive lymph node metastasis (P<0.001), budding (P<0.001), vessel invasion (P=0.001), perineural invasion (P=0.039), overall survival (P=0.012), and disease-free survival (P=0.016); however, fascin immunoreactivity was not correlated with recurrence or depth of tumor invasion. CONCLUSIONS: This study suggests that an increased expression of fascin was associated with a poor prognosis and the immunohistochemical detection of fascin provides useful information as one of the prognostic values in CRC.
Carrier Proteins ; Cell Movement ; Colorectal Neoplasms ; Disease-Free Survival ; Glycosaminoglycans ; Humans ; Immunohistochemistry ; Lymph Nodes ; Membranes ; Microfilament Proteins ; Neoplasm Metastasis ; Neoplasms, Glandular and Epithelial ; Paraffin ; Prognosis ; Recurrence ; Stress Fibers

Proteasomes are the primary degradation machinery for oxidatively damaged proteins that compose a class of misfolded protein substrates. Cellular levels of reactive oxygen species increase with age and this cellular propensity is particularly harmful when combined with the age-associated development of various human disorders including cancer, neurodegenerative disease and muscle atrophy. Proteasome activity is reportedly downregulated in these disease conditions. Herein, we report that docosahexaenoic acid (DHA), a major dietary omega-3 polyunsaturated fatty acid, mediates intermolecular protein cross-linkages through oxidation, and the resulting protein aggregates potently reduce proteasomal activity both in vitro and in cultured cells. Cellular models overexpressing aggregation-prone proteins such as tau showed significantly elevated levels of tau aggregates and total ubiquitin conjugates in the presence of DHA, thereby reflecting suppressed proteasome activity. Strong synergetic cytotoxicity was observed when the cells overexpressing tau were simultaneously treated with DHA. Antioxidant N-acetyl cysteine significantly desensitized the cells to DHA-induced oxidative stress. DHA significantly delayed the proteasomal degradation of muscle proteins in a cellular atrophy model. Thus, the results of our study identified DHA as a potent inducer of cellular protein aggregates that inhibit proteasome activity and potentially delay systemic muscle protein degradation in certain pathologic conditions.
Atrophy ; Cells, Cultured ; Cysteine ; Humans ; In Vitro Techniques* ; Muscle Fibers, Skeletal* ; Muscle Proteins ; Muscular Atrophy* ; Neurodegenerative Diseases ; Oxidative Stress ; Proteasome Endopeptidase Complex* ; Protein Aggregates* ; Reactive Oxygen Species ; Ubiquitin

Most of the structures in the lumbar region including the visceral organs could be the sources of low back pain. The management of low back pain starts from a thorough understanding of the anatomical structures and the underlying pathophysiologic processes related to the generation of the pain. Mechanical stresses applying to the lumbar spine and the inflammatory changes contribute to the generation of low back pain. Many nerves branching from the spinal and autonomic nerves supply all of the musculoskeletal structures in the lumbar area. There are extensive nociceptive nerve fibers in the facet joints and some small fibers in the outer layer of discs and ligaments of the lumbar vertebrae. They respond to the mechanical, chemical and other stimuli. Acute pain caused by tissue trauma or inflammation is well controlled by the removal or elimination of its causes. In idiopathic, uncontrolled and chronic pain, however, the long-lasting nociceptive stimuli and many chemical mediators released from the tissue injury and inflammation sensitize the local nervous system. They change the normal process of pain transmission to neuropathic pain. For the proper treatment of low back pain, not only the knowledge of anatomical structures but also the understanding of the pathophysiology of chronic neuropathic pain is necessary.
Acute Pain ; Autonomic Pathways ; Chronic Pain ; Inflammation ; Ligaments ; Low Back Pain* ; Lumbar Vertebrae ; Lumbosacral Region ; Nerve Fibers ; Nervous System ; Neuralgia ; Spine ; Stress, Mechanical ; Zygapophyseal Joint

BACKGROUND: The normal functions of the cell cycle inhibitor p16INK4a are frequently inactivated in many human cancers. Over 80% of hepatocellular carcinoma (HCC) cases lack a functional p16/Rb pathway. p16/Rb pathway, as well as p53 pathway, is considered as one of key components of tumor suppression. METHODS: To study the roles of p16INK4a in HCC, a stable cell line expressing exogenous p16 was generated from SNU-449 hepatocellular carcinoma cells lacking endogenous p16, and suppression subtractive hybridization (SSH) was performed in parallel with the control cells. RESULTS: 1) SSH identifies fibronectin (FN1), crystallin alphaB (CRYAB), Rac1, WASP, RhoGEF, and CCT3 as differentially-expressed genes. 2) Among the selected genes, the up- regulation of FN1 and CRYAB was confirmed by Northern blot, RT-PCR and by proteomic methods. CONCLUSION: These genes are likely to be associated with the induction of stress fiber and stabilization of cytoskeleton. Further studies are required to clarify the possible role of p16 in the signal transduction pathway.
Blotting, Northern ; Carcinoma, Hepatocellular* ; Cell Cycle ; Cell Line ; Crystallins ; Cytoskeleton ; Fibronectins ; Gene Expression* ; Humans ; Rho Guanine Nucleotide Exchange Factors ; Signal Transduction ; Stress Fibers ; Wasps

Rho small GTPases control multiple aspects of neuronal morphogenesis by regulating the assembly and organization of the actin cytoskeleton. Although they are negatively regulated by GTPase activating proteins (GAPs), the roles of RhoGAPs in the nervous system have not been fully investigated. Here we describe a characterization of Drosophila RhoGAP68F that is mainly expressed in the embryonic central nervous system. RNA in situ hybridization analysis showed that expression of RhoGAP68F is highly restricted to the embryonic brain and ventral nerve cord. Database search revealed that RhoGAP68F contains an N-terminal Sec14 domain and a C-terminal RhoGAP domain. Rho-GTP pull-down assay demonstrated that the RhoGAP domain of RhoGAP68F inactivates RhoA but not Rac1 or Cdc42 in HEK293 cells. In addition, expression of RhoGAP68F in NIH3T3 cells suppressed LPA-induced stress fiber formation, which is mediated by RhoA. Finally, neuronal overexpression of RhoGAP68F causes synaptic overgrowth at the larval neuromuscular junction (NMJ). Taken together, these results suggest that RhoGAP68F may play a role in synaptic growth regulation by inactivating RhoA.
Actin Cytoskeleton ; Actins ; Brain ; Central Nervous System ; Drosophila ; GTPase-Activating Proteins ; HEK293 Cells ; In Situ Hybridization ; Monomeric GTP-Binding Proteins ; Morphogenesis ; Nervous System ; Neuromuscular Junction ; Neurons ; RNA ; Stress Fibers

BACKGROUNDSIRT3 is an important regulator in cell metabolism, and recent studies have shown that it may be involved in the pharmacological effects of metformin. However, the molecular mechanisms underlying this process are unclear.

METHODSThe effects of SIRT3 on the regulation of oxidative stress and insulin resistance in skeletal muscle were evaluated in vitro. Differentiated L6 skeletal muscle cells were treated with 750 µmol/L palmitic acid to induce insulin resistance. SIRT3 was knocked down and overexpressed in L6 cells. SIRT3, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, c-Jun N-terminal kinase 1 (JNK1), and superoxide dismutase 2 (SOD2) were evaluated by Western blotting.

RESULTSOver expression of SIRT3 increased glucose uptake and decreased ROS production in L6-IR cells as well as in L6 cells. Knock-down of SIRT3 induced increased production of ROS while decreased glucose uptake in both L6 and L6-IR cells, and these effects were reversed by N-acetyl-L-cysteine (NAC). Metformin increased the expression of SIRT3 (1.5-fold) and SOD2 (2-fold) while down regulating NF-κB p65 (1.5-fold) and JNK1 (1.5-fold). Knockdown of SIRT3 (P < 0.05) reversed the metformin-induced decreases in NF-κB p65 and JNK1 and the metformin-induced increase in SOD2 (P < 0.05).

CONCLUSIONSUpregulated SIRT3 is involved in the pharmacological mechanism by which metformin promotes glucose uptake. Additionally, SIRT3 may function as an important regulator of oxidative stress and a new alternative approach for targeting insulin resistance-related diseases.

Animals ; Cell Line ; Insulin Resistance ; physiology ; Metformin ; pharmacology ; Muscle Fibers, Skeletal ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Rats ; Sirtuin 3 ; metabolism ; Transcription Factor RelA ; metabolism

Recent investigations consider adipose-derived stemcells (ASCs) as a promising source of stemcells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated.Due to proper availability ofASCs, results can be employed in cardiovascular engineeringwhen production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.
Actin Cytoskeleton ; Cell Body ; Cytoskeleton ; Microscopy, Atomic Force ; Muscle Cells ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Polymerization ; Polymers ; Stem Cells* ; Stress Fibers ; Tissue Engineering