OBJECTIVETo investigate the relationship between c-fos gene and filamentous actin (F-actin) in MG-63 osteoblasts under cyclic tensile stress.

METHODSMG-63 osteoblasts were subjected to cyclic tensile stress (0.5 Hz, 2 000 microstrain) for 3, 6, and 12 h. The changes of c-fos gene were investigated by fluorescent quantitation polymerase chain reaction. Then the best loading time group was screened as the experimental group compared with 0 h group. The changes of F-actin and c-fos were investigated with or without cytochalasin D treatment.

RESULTSCyclic tensile stress induced high expression of c-fos mRNA, and peaked at 3 h. After loading, F-actin had a structure reorganization, but had no change in expression. After cytochalasin D treatment, the formation of stress fibers and the fluorescence intensity of F-actin cytoskeleton significantly reduced, meanwhile the c-fos mRNA expression was inhibited.

CONCLUSIONAfter loading, there is only structure reorganization for F-actin, and the expression has not any change. That means the remodeling F-actin is the existing one. F-actin reorganization is an important part in c-fos gene expression induced by stress.

Actin Cytoskeleton ; Actins ; Cytochalasin D ; Cytoskeleton ; Genes, fos ; Humans ; Microtubules ; Osteoblasts ; RNA, Messenger ; Stress, Mechanical

In the present study, we investigated the effect of staurosporine on the formation of cellular processes in human gingival fibroblasts and rat astrocytes. Staurosporine caused a rapid induction of process formation in human gingival fibroblasts and rat astrocytes in a concentration dependent manner. The process formation of human gingival fibroblasts and rat astrocytes was prevented by the pretreatment with N-acetylcysteine, suggesting that staurosporine-induced ROS production was responsible for the process formation. Colchicine, a microtubule depolymerizing agent, inhibited the staurosporine-induced process formation, whereas cytochalasin D, an actin filament breakdown agent, failed to suppress the formation of cellular processes. This result indicated that polymerization of microtubule, and not actin filament, was responsible for the formation of cellular processes induced by staurosporine. In support of this hypothesis, Western blot analysis was conducted using anti-tubulin antibody, and the results showed that the amount of polymerized microtubule was increased by the treatment with staurosporine while that of depolymerized beta-tubulin in soluble fraction was decreased. These results indicate that staurosporine induces ROS-mediated, microtubule-dependent formation of cellular processes in human gingival fibroblasts and rat astrocytes.
Acetylcysteine ; Actin Cytoskeleton ; Animals ; Astrocytes* ; Blotting, Western ; Colchicine ; Cytochalasin D ; Fibroblasts* ; Humans ; Microtubules ; Polymerization ; Polymers ; Rats* ; Staurosporine* ; Tubulin

BACKGROUND: Mesenchymal stem cells (MSCs) have strong self-renewal ability and multiple differentiation potential. Some studies confirmed that spreading shape and area of single MSCs influence cell differentiation, but few studies focused on the effect of the circularity of cell shape on the osteogenic differentiation of MSCs with a confined area during osteogenic process.METHODS: In the present study, MSCs were seeded on a micropatterned island with a spreading area lower than that of a freely spreading area. The patterns had circularities of 1.0 or 0.4, respectively, and areas of 314, 628, or 1256 µm² . After the cells were grown on a micropatterned surface for 1 or 3 days, cell apoptosis and F-actin were stained and analyzed. In addition, the expression of β-catenin and three osteogenic differentiation markers were immunofluorescently stained and analyzed, respectively.RESULTS: Of these MSCs, the ones with star-like shapes and large areas promoted the expression of osteogenic differentiation markers and the survival of cells. The expression of F-actin and its cytosolic distribution or orientation also correlated with the spreading shape and area. When actin polymerization was inhibited by cytochalasin D, the shape-regulated differentiation and apoptosis of MSCs with the confined spreading area were abolished.CONCLUSION: This study demonstrated that a spreading shape of low circularity and a larger spreading area are beneficial to the survival and osteogenic differentiation of individual MSCs, which may be regulated through the cytosolic expression and distribution of F-actin.
Actins ; Antigens, Differentiation ; Apoptosis ; Cell Differentiation ; Cell Shape ; Cytochalasin D ; Cytosol ; Mesenchymal Stromal Cells ; Osteogenesis ; Polymerization ; Polymers

The microfilaments of hepatocyte are distributed throughout the vicinity of cell membranes, especially numerous around the region of bile canaliculus, and provide the maintenance of cell shape, cellular wall tension, canalicular motility, the secretion for bile, etc. To evaluate the relationship between the microfilament and alteration of cell shape, we examined the morphological changes of cultured rat hepatocytes, following treatments with phalloidin or cytochalasin D with fluorescent and electron microscopes. 1. In the fluorescent micrographs, actin microfilament was distributed near the plasma membrane and bile canaliculus. 2. Both drugs, phalloidin or cytochalasin D, produce the cytoplasmic protrusions from the surface. Their shapes were pedunculated with narrow neck or bulged with broad base, respectively. 3. In the phalloidin treated group, cytoplasmic protrusion was seperated from the internal cytoplasm by microfila-ments networks at the narrow base. In contrast, in the cytochalasin D treated group, cytoplasm was bulged with broad base and kept in direct continuity with the canalicular ectoplasm. 4. Pericanalicular ectoplasm of phalloidin treated group was widened and accumulated with microfilaments. But, bile canaliculus of cytochalasin D treated group was markedly dilated and devoid of microvilli, and the ectoplasm was almost disappeared. Considering above results, dysfunction of microfilaments leads to the structural changes and inhibition of bile secretion of hepatocytes.
Actin Cytoskeleton ; Animals ; Bile ; Bile Canaliculi ; Cell Membrane ; Cell Shape ; Cytochalasin D* ; Cytoplasm ; Hepatocytes* ; Microvilli ; Neck ; Phalloidine* ; Rats*

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-1beta and TNF-alpha was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-kappaB activation and cytokine expression. Treatment with M. leprae significantly increased NF-kappaB activation and expression of TNF-alpha and IL-1beta in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.
Cytochalasin D ; Cytokines ; Humans ; Leprosy ; Macrophage Activation ; Macrophages ; Mycobacterium ; Mycobacterium leprae ; NF-kappa B ; Phagocytosis ; Proteins ; Receptors, Cytoplasmic and Nuclear ; Tumor Necrosis Factor-alpha

BACKGROUND: Cytoskeletons, the complex set of protein fibers found in the cytoplasm, have important roles in the movement of cells and subcellular structures and the generation of shapes. Melanocytes have numerous dendritic processes which are in direct contact with many keratinocytes and transfer the melanosomes into the neighboring keratinocytes. Little information is available on the structure and function of cytoskeletons, and the effects of ultraviolet light on the cytoskeletons of the melanocytes. OBJECTIVE: The purpose of this study was to investigate the general cytoskeletal system of cultured melanocytes and to find out the effects of the cytoskeletal antagonists and UVB on the cytoskeletal system of the cultured melanocytes. METHODS: Melanocytes were cultured from adult foreskin and then exposed to various cytoskeletal antagonists and UVB radiation. The changes of the cultured melanocytes were evaluated by using phase contrast microscopy, immunofluorescence staining methods and electron microscopic examinations. RESULTS: Colchicine produced shortening of dendrites, stellate cellular contour and granular fluorescence of the tubulin. Cytochalasin D produced round cellular contour and granular fluorescence of the actin. Acrylamide produced disorganization of cytoplasmic constituents, but no specific fluorescent change was observed. Colchicine also had inhibitory effects on the vimentin. Cellular responses induced by these agents were reversible. UVB caused morphological changes of the melanocytes, but their effects on the organization of the cytoskeletal system could not be detected in this method. CONCLUSION: Microtubules are related to the dendritic movement of the melanocytes. Vimentin may be involved in the transfer of cellular organelles, probably including the melanosomes. Cytoskeletal antagonists produce their characteristic morphological changes to cultured melanocytes.
Acrylamide ; Actins ; Adult ; Colchicine ; Cytochalasin D ; Cytoplasm ; Cytoskeleton* ; Dendrites ; Fluorescence ; Fluorescent Antibody Technique ; Foreskin ; Humans* ; Keratinocytes ; Melanocytes* ; Melanosomes ; Methods ; Microscopy, Phase-Contrast ; Microtubules ; Organelles ; Tubulin ; Ultraviolet Rays ; Vimentin

To examine the role of actin microfilaments which are located at beneath the plasma membrane, we observed the ultrastructural changes of rat hepatocyte induced by alteration of the microfilamentous integrity. The isolated hepatocytes from Sprague-Dawley were cultured in the L-15 medium containing phalloidin (agent that cause polymerization of actin) or cytochalasin D (agent that cause depolymerization of actin) for 30 min, 1 hour, 2 hours, 4 hours, 10 hours and 20 hours, respectively. The results observed with scanning and transmission electron microscope were as follows. 1. Following the alteration of actin microfilaments, bile canaliculi were dilated and devoid of microvilli. In phalloidin treated group, the thickening of microfilamentous ectoplasm was more marked than that of cytochalasin D treated group. Whereas, the dilation of bile canaliculi was more marked in cytochalasin D group. 2. Both drugs, phalloidin or cytochalasin D, produced the alteration of cell shape to form cytoplasmic protrusions at the cell surface. In the phalloidin treated group, protrusions were pedunculated, and the microfilament networks were accumulated at the narrow neck region. 3. In cytochalasin D treated group, no microfilament barrier was seen at the broad base of protrusion which exhibit direct continuity with the internal cytoplasm. 4. Single hepatocyte tend to recover their structural integrity as those in vivo. The new bile canaliculus was sealed off at the intercellular space by tight junctions, and intercellular contacts were established by the junctional complexes. The results demonstrated that excessive accumulation or depletion of microfilaments induced by phalloidin or cytochalasin D altered the cell shape different, respectively. The microfilaments of ectoplasm play an important role in the maintenance of the structural integrity of cultured hepatocytes.
Actin Cytoskeleton* ; Animals ; Bile Canaliculi ; Cell Membrane ; Cell Shape ; Cytochalasin D ; Cytoplasm ; Extracellular Space ; Hepatocytes* ; Microvilli ; Neck ; Phalloidine ; Polymerization ; Polymers ; Rats ; Rats, Sprague-Dawley ; Tight Junctions

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates the func-tions of beta1 integrin, suggesting that it may play a role in tumor cell invasion. In this study, the effects of CD98 signaling on the adhesion and invasion of tumor cells were investigated. The expression of CD98 on MCF-7 human breast carcinoma cells was confirmed by immunohistochemistry. The effects of CD98 activation on the adhesion to extracellular matrix (ECM) and invasion of MCF-7 cells were determined by adhesion assay and cell invasion assay. Dominant negative forms of focal adhesion kinase (FAK) were transiently transfected into MCF-7 cells using liposome reagents. CD98 stimulation increased the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV. Activation of CD98 augmented the invasion rate of MCF-7 cells through ECM. EDTA or a function-blocking anti-beta1 integrin mAb suppressed the effect of CD98 on invasiveness. Inhibition of phosphorylation of FAK by PP2, an inhibitor of Src family kinase, reduced CD98-induced invasion of MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, cytochalasin D or phalloidin inhibited CD98-mediated induction of tumor cell invasion. Inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated invasion of MCF-7 cells were diminished by pretreatment of cells with Mn++, which is shown to induce conformational change of beta1 intgerin. These results provide the first evidence that CD98 activation increases tumor cell invasion by activating beta1 integrin affinity, and that FAK phosphorylation and subsequent cytoskeletal reorganization may be essential for CD98-mediated regulation of cell motility.
Actins ; Antigens, CD29 ; Breast Neoplasms* ; Breast* ; Cell Movement ; Collagen ; Cytochalasin D ; Cytoskeleton ; Edetic Acid ; Extracellular Matrix ; Fibronectins ; Focal Adhesion Protein-Tyrosine Kinases ; Glycoproteins ; Humans* ; Immunohistochemistry ; Indicators and Reagents ; Laminin ; Liposomes ; MCF-7 Cells* ; Phalloidine ; Phosphorylation ; Phosphotransferases

Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-alpha. The present study investigated the mechanisms involved in TNF-alpha production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-alpha. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF-alpha by E. faecalis. In addition, antioxidant treatment reduced TNF-alpha production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF-alpha expression by RAW 264.7 cells. Furthermore, activation of NF-kappaB and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF-alpha in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-kappaB, and AP-1.
Bacteria ; Cell Line ; Cytochalasin D ; Cytokines ; Endocytosis ; Enterococcus ; Enterococcus faecalis ; Gene Expression ; Macrophages ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Periapical Periodontitis ; Phosphotransferases ; Reactive Oxygen Species ; RNA, Messenger ; Transcription Factor AP-1 ; Tumor Necrosis Factor-alpha ; Up-Regulation

Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-alpha. The present study investigated the mechanisms involved in TNF-alpha production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-alpha. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF-alpha by E. faecalis. In addition, antioxidant treatment reduced TNF-alpha production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF-alpha expression by RAW 264.7 cells. Furthermore, activation of NF-kappaB and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF-alpha in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-kappaB, and AP-1.
Bacteria ; Cell Line ; Cytochalasin D ; Cytokines ; Endocytosis ; Enterococcus ; Enterococcus faecalis ; Gene Expression ; Macrophages ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Periapical Periodontitis ; Phosphotransferases ; Reactive Oxygen Species ; RNA, Messenger ; Transcription Factor AP-1 ; Tumor Necrosis Factor-alpha ; Up-Regulation