• 대한의생명과학회지
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• 제15권2호
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• pp.113-118
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• 2009

Actin cytoskeletal architecture is believed to be a crucially important modulator of chondrocyte phenotype. 2DG(2-Dexoy-D-glucose) induces reorganization of actin cytoskeletal architecture in chondrocytes. In this study, we have investigated the effects of 2DG on dedifferentiation and inflammation via reorganization of cytoskeletal architecture in rabbit articular chondrocytes, with a focus on p38 kinase pathway. Treatment of 2DG alone reduced type II collagen and COX-2 expression in chondrocytes. But, 2DG reduced type II collagen was recovered by CD, disruptor of actin cytoskeletal architecture, whereas did not affect on COX-2 expression and production of $PGE_2$ compared with 2DG alone treated cells. Treatment of 2DG with JAS, inducer of cytoskeletal architecture polymerization, accelerated reduction of type II collagen expression and synthesis of proteoglycan but did not affect on COX-2 expression and production of $PGE_2$. Also, 2DG stimulated activation of p38 kinase. This result showed that 2DG regulates type II collagen but not cyclooxygenase-2 expression through reorganization of cytoskeletal architecture via p38 kinase pathway in rabbit articular chondrocytes.

• Molecules and Cells
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• 제26권4호
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• pp.356-361
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• 2008

In this work, we have studied the structural and functional linkage between lamin A/C, nuclear actin, and organization of chromosome territories (CTs) in mammary carcinoma MCF-7 cells. Selective down-regulation of lamin A/C expression led to disruption of the lamin A/C perinuclear layer and disorganization of lamin-bound emerin complexes at the inner nuclear membrane. The silencing of lamin A/C expression resulted in a decrease in the volume and surface area of chromosome territories, especially in chromosomes with high heterochromatin content. Inhibition of actin polymerization led to relaxation of the structure of chromosome territories, and an increase in the volumes and surface areas of the chromosome territories of human chromosomes 1, 2 and 13. The results show an important role of polymeric actin in the organization of the nuclei and the chromosome territories.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2004년도 International Meeting of the Microbiological Society of Korea
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• pp.141-146
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• 2004

SPIN90 was identified to farm molecular complex with $\betaPIX$, WASP and Nck. This complex shows that SPIN90 interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix, but $SPIN90{\cdot}{\beta}PIX{\cdot}WASP$ complex was stable even in suspended cells. This suggests that SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the $SPIN90{\cdot}{\beta}PIX{\cdot}WASP$ complex and Nck. It thus appears that the interaction of the $SPIN90{\cdot}{\beta}PIX{\cdot}WASP$ complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERX activation. SPIN90 directly binds syndapin I, syndapin isoform II-1 and II-s via its PRD region in vitro, in vivo and also associates with endocytosis core components such as clathrin and dynamin. In neuron and fibroblast, SPIN90 colocalizes with syndapins as puntate form, consistent with a role for SPIN90 in clathrin-mediated endocytosis pathway. Overexpression of SPIN90 N-term inhibits receptor-mediated endocytosis. Interestingly, SPIN90 PRD, binding interface of syndapin, significantly blocks internalization of transferrin, demonstrating SPIN90 involvement in endocytosis in vivo by interacting syndapin. Depletion of endogenous SPIN90 by introducing $\alpha-SPIN90$ also blocks receptor-mediated endocytosis. Actin polymerization could generate farce facilitating the pinch-out event in endocytosis, detach newly formed endocytic vesicle from the plasma membrane or push out them via the cytosol on actin tails. Here we found that SPIN90 localizes to high actin turn over cortical area, actin-membrane interface and membrane ruffle in PDGF treated cells. Overexpression of SPIN90 has an effect on cortical actin rearrangement as filopodia induction and it is mediated by the Arp2/3 complex at cell periphery. Consistent with a role in actin organization, CFP-SPIN90 present in actin comet tail generated by PIP5 $kinase\gamma$ overexpression. Therefore this study suggests that SPIN90 is functional linker between endocytosis and actin cytoskeleton.

• 한국임상수의학회지
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• 제39권2호
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• pp.51-58
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• 2022

Quercetin, a flavonoid found in fruits and vegetables, exhibits a strong anti-inflammatory activity. The objective of this study was to examine the effect of quercetin on chemotactic activity of peripheral blood polymorphonuclear cells (PMNs) to culture supernatant from peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS). In addition, we determined whether this effect is related to interleukin (IL)-8 and changes in cytoskeleton. The chemotactic activity of PMNs was evaluated by a modified Boyden chamber assay. Total cellular filamentous (F)-actin levels were measured by method of fluorescence microscopy. The levels of IL-8 mRNA and protein were measured by real time polymerase reaction method and enzyme-linked immunosorbent assay, respectively. Quercetin (0-50 µM) itself has no chemoattractant effect for PMNs. The culture supernatant from PBMCs (2 × 10⁶ cells/mL) treated with LPS (1 ㎍/mL) showed remarkable increase in chemotaxis of PMNs. However, this effect was reduced dose-dependently by treatment with quercetin. In addition, PBMCs treated with LPS revealed enhanced levels in IL-8 protein and mRNA. Co-treatment of LPS with quercetin (50 µM) in PBMCs decreased IL-8 production and expression. Treatment of quercetin (0-50 µM) on PMNs to rpIL-8 (10 nM) decreased dose-dependently the chemotactic activity of PMNs. Treatment of quercetin on PMNs to IL-8 also reduced their total cellular F-actin level. These results suggested that quercetin attenuates chemotactic activity of PMNs, which is mediated by down-regulation of IL-8 production from LPS-stimulated PBMCs and inhibition of F-actin polymerization in PMNs.

• 대한의생명과학회지
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• 제12권1호
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• pp.9-16
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• 2006

We investigated the effects of 835-MHz electromagnetic field (EMF), one of the most popular communication frequency band in Korean code-division multiple-access (CDMA) mobile phone system, on cellular signal transduction. For this, we examined the change of intracellular calcium $([Ca^{2+}]_i)$, reactive oxygen species (ROS) and F-actin polymerization after exposure to 835-MHz EMF followed by the treatment of agonists in Rat-2 fibroblast cells. Culture cells were pretreated with serum-tree medium and concomitantly exposed to 835-MHz at specific absorption rate (SAR) of 4.0 W/kg for 24 hr in a specialized designed apparatus based on Transverse Electro Magnetics (TEM) wave theory. Intracellular $Ca^{2+}$ responses to lysophosphatidic acid (LPA) and epidermal growth factor (EGF) in Rat-2 fibroblast after exposure to 835-MHz EMF were shown to be similar pattern as observed in normal cultured cells. However, the LPA-induced calcium spiking was slightly delayed to 7 sec and sustained thereafter to a little higher ground level under 835-MHz EMF radiation compared to unexposed cells. ROS production level by LPA in the exposed cells was not different from that in control. Furthermore, LPA induced the production of stress fibers with no significant difference in the exposed and unexposed cells. These results suggest that mobile phone radiation (835-MHz, SAR 4.0 W/kg) may not be directly related to signal transduction in Rat-2 fibroblasts except the slight effect of calcium spiking in LPA-induced cells but remain to be further elucidated for possible indirect intervention.

• Journal of Microbiology and Biotechnology
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• 제28권10호
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• pp.1723-1729
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• 2018

The aim of this work is to investigate the protective efficacy of emodin, an active, naturally-occurring anthraquinone derivative of several traditional Chinese herbs, against Brucella abortus infection in macrophages. Brucella were incubated with different concentrations of emodin and showed that bacterial survival rates were markedly reduced in a dose-dependent manner at increasing incubation time points. Through bacterial infection assay, the highest non-cytotoxic concentration of emodin demonstrated attenuated invasion of Brucella into macrophages, however it did not inhibit the growth of these pathogens within the host cells. On the other hand, emodin effectively decreased the number of bacteria that adhered to host cells, which indicated its potential as an anti-adhesin agent. Furthermore, using immunoblotting and FACS assay for detecting MAPK signaling proteins and F-actin polymerization, respectively, the results showed that the emodin-incubated cells displayed modest reduction in the phosphorylation levels of ERK1/2 and inhibition of F-actin polymerization as compared to control cells. These findings indicate the potential use of emodin as a naturally-occurring alternative method for the prevention of animal brucellosis although this requires confirmation of safe clinical doses.

• Asian Pacific Journal of Cancer Prevention
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• 제16권8호
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• pp.3213-3222
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• 2015

Background: Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative stress have long been associated with cancer. ROS play a vital role in regulating actin dynamics that are sensitive to oxidative modification. The current work aimed at studying the effects of sub-lethal metabolic oxidative stress on actin cytoskeleton, focal adhesion and cell migration. Materials and Methods: T47D human breast cancer cells were treated with 2-deoxy-D-glucose (2DG), L-buthionine sulfoximine (BSO), or doxorubicin (DOX), individually or in combination, and changes in intracellular total glutathione and malondialdehyde (MDA) levels were measured. The expression of three major antioxidant enzymes was studied by immunoblotting, and cells were stained with fluorescent-phalloidin to evaluate changes in F-actin organization. In addition, cell adhesion and degradation ability were measured. Cell migration was studied using wound healing and transwell migration assays. Results: Our results show that treating T47D human breast cancer cells with drug combinations (2DG/BSO, 2DG/DOX, or BSO/DOX) decreased intracellular total glutathione and increased oxidized glutathione, lipid peroxidation, and cytotoxicity. In addition, the drug combinations caused a reduction in cell area and mitotic index, prophase arrest and a decreased ability to form invadopodia. The formation of F-actin aggregates was increased in treated T47D cells. Moreover, combination therapy reduced cell adhesion and the rate of cell migration. Conclusions: Our results suggest that exposure of T47D breast cancer cells to combination therapy reduces cell migration via effects on metabolic oxidative stress.

• Clinical and Experimental Reproductive Medicine
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• 제47권2호
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• pp.114-121
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• 2020

Objective: Despite extensive research on implantation failure, little is known about the molecular mechanisms underlying the crosstalk between the embryo and the maternal endometrium, which is critical for successful pregnancy. Profilin 1 (PFN1), which is expressed both in the embryo and in the endometrial epithelium, acts as a potent regulator of actin polymerization and the cytoskeletal network. In this study, we identified the specific role of endometrial PFN1 during embryo implantation. Methods: Morphological alterations depending on the status of PFN1 expression were assessed in PFN1-depleted or control cells grown on Matrigel-coated cover glass. Day-5 mouse embryos were cocultured with Ishikawa cells. Comparisons of the rates of F-actin formation and embryo attachment were performed by measuring the stability of the attached embryo onto PFN1-depleted or control cells. Results: Depletion of PFN1 in endometrial epithelial cells induced a significant reduction in cell-cell adhesion displaying less formation of colonies and a more circular cell shape. Mouse embryos co-cultured with PFN1-depleted cells failed to form actin cytoskeletal networks, whereas more F-actin formation in the direction of surrounding PFN1-intact endometrial epithelial cells was detected. Furthermore, significantly lower embryo attachment stability was observed in PFN1-depleted cells than in control cells. This may have been due to reduced endometrial receptivity caused by impaired actin cytoskeletal networks associated with PFN1 deficiency. Conclusion: These observations definitively demonstrate an important role of PFN1 in mediating cell-cell adhesion during the initial stage of embryo implantation and suggest a potential therapeutic target or novel biomarker for patients suffering from implantation failure.

• International Journal of Oral Biology
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• 제39권3호
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• pp.131-136
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• 2014

Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.

• International Journal of Oral Biology
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• 제40권1호
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• pp.27-33
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• 2015

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.