Ezrin Promotes the Proliferation, Migration, and Invasion of Ovarian Cancer Cells / 生物医学与环境科学（英文）

Objective@#The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms.@*Methods@#We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells.@*Results@#The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial-mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation.@*Conclusion@#Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.

Cyclic Stretch Effects on Adipose-Derived Stem Cell Stiffness, Morphology and Smooth Muscle Cell Gene Expression

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.

Effect of Palmitoyl-Pentapeptide (Pal-KTTKS) on Wound Contractile Process in Relation with Connective Tissue Growth Factor and α-Smooth Muscle Actin Expression

To evaluate whether Palmitoyl-pentapeptide (Pal-KTTKS), a lipidated subfragment of type 1 pro-collagen (residues 212–216), plays a role in fibroblast contractility, the effect of Pal-KTTKS on the expression of pro-fibrotic mediators in hypertropic scarring were investigated in relation with trans-differentiation of fibroblast to myofibroblast, an icon of scar formation. α-SMA was visualized by immunofluorescence confocal microscopy with a Cy-3-conjugated monoclonal antibody. The extent of α-SMA-positive fibroblasts was determined in collagen lattices and in cell culture study. Pal-KTTKS (0–0.5 µM) induced CTGF and α-SMA protein levels were determined by western blot analysis and fibroblast contractility was assessed in three-dimensional collagen lattice contraction assay. In confocal analysis, fibroblasts were observed as elongated and spindle shapes while myofibroblast observed as squamous, enlarged cells with pronounced stress fibers. Without Pal-KTTKS treatment, three quarters of the fibroblasts differentiates into the myofibroblast; α-SMA-positive stress fibers per field decreased twofold with 0.1 µM Pal-KTTKS treatment (75 ± 7.1 vs 38.6 ± 16.1%, n = 3, p<0.05). The inhibitory effect was not significant in 0.5 µM Pal-KTTKS treatment. Stress fiber level and collagen contractility correlates with α-SMA expression level. In conclusion, Pal-KTTKS (0.1 µM) reduces α-SMA expression and trans-differentiation of fibroblasts to myofibroblast. The degree of reduction is dose-dependent. An abundance of myofibroblast and fibrotic scarring is correlated with excessive levels of α-SMA and collagen contractility. Delicate balance between the wound healing properties and pro-fibrotic abilities of pentapeptide KTTKS should be considered for selecting therapeutic dose for scar prevention.

Odontogenic Ameloblast-Associated Protein (Odam) Plays Crucial Roles in Osteoclast Differentiation via Control of Actin Ring Formation

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.

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.

Characterization of the Rho GTPase-Activating Protein RhoGAP68F

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.

Synovial fluid of patients with rheumatoid arthritis induces alpha-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells through a TGF-beta1-dependent mechanism

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. Transforming growth factor-beta1 (TGF-beta1) is a secreted protein that promotes differentiation of synovial fibroblasts to alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts to repair the damaged joints. Synovial fluid from patients with RA (RA-SF) induced expression of alpha-SMA in human adipose tissue-derived mesenchymal stem cells (hASCs). RA-SF-induced alpha-SMA expression was abrogated by immunodepletion of TGF-beta1 from RA-SF with anti-TGF-beta1 antibody. Furthermore, pretreatment of hASCs with the TGF-beta type I receptor inhibitor SB431542 or lentiviral small hairpin RNA-mediated silencing of TGF-beta type I receptor expression in hASCs blocked RA-SF-induced alpha-SMA expression. Small interfering RNA-mediated silencing of Smad2 or adenoviral overexpression of Smad7 (an inhibitory Smad isoform) completely inhibited RA-SF-stimulated alpha-SMA expression. These results suggest that TGF-beta1 plays a pivotal role in RA-SF-induced differentiation of hASCs to alpha-SMA-positive cells.

The Prognostic Significance of Fascin Expression in Colorectal Carcinoma

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.

Gene Expression Changes Associated with Sustained p16 Expression in Hepatocellular Carcinoma Cells

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.

Ultrastructural characteristics of tensional regions in tendons from rats of different ages

The calcaneal tendon and the deep digital flexor tendon are collagen-rich structures which are adapted to resist tensile stress. Since during aging tendons undergo modifications in their mechanical properties and in collagen aggregation, an understanding of the structural changes involved is important. In this work, the structural organization of the tensile region of the calcaneal and deep difital flexor tendons was studied in male Wistar rats 30, 180 and 730 days old. Large quantities of rough endoplasmic reticulum and peripheral secretory microvesicles were observed in the calcaneal tendon of 30-day-old rats. In the case of the deep digital flexor tendon, this organelle remained well-developed up to 180 days. A marked decrease in rough endoplasmic reticulum was observed in both tendons in 730-day-old rats. Proteoglycans associated with collagen fibrils were visible in the two tendons of all age groups. The reduced amount of rough endoplasmic reticulum and secretory microvesicles may be correlated with the known lower turnover of extracellular matrix components during aging.

The Effect of Hydrogen Peroxide on the Migration and FAK Activation in Lens Epithelial Cells

PURPOSE: Hydrogen peroxide has been implicated as a causative factor of various cellular dysfunction, cell death, transformation. In addition, oxidative stress has been suggested as a crucial inducer of cataract formation not only the nuclear-type cataract but also anterior polar type cataract. Transformation of lens epithelial cells accompanying accumulation of extracellular matrix molecules, and cell migration is observed in the cataract forming area of lens. In the present study, we investigated in the migration of lens epithelial cells and the activation of focal adhesion kinase(FAK), because there have been many reports showing that activation of FAK increased cell migration. METHOD: After treatment hygrogen peroxide in a dose-dependent on HLE B-3 cell line, we have performed immuno fluorenscence staing of actin stress fiber and migration assay, and then isolated total RNA to identify expression of MMP-2 from the cell line. To examine FAK activity, we are performed Phosphotyrosine Immunoblot analysis. RESULT: We observed the increased cell migration in response to hydrogen peroxide in a dose dependent manner. In addition, we observed that the phosphorylation of focal adhesion kinase was increased with the treatment of hydrogen peroxide. Also, we examined the expression of the matrix metalloproteinases-2(MMP-2) which disintegrates extracellular matrix and participates in cell migration. The result showed that the mRNA level of MMP-2 did not increase by hydrogen peroxide. CONCLUSIONS: The results suggest that hydrogen peroxide enhanced migration of lens epithelial cell, and that FAK may play a role in the process of cell migration. In conclusion, migration of lens epithelial cell and phosphorylation of focal adhesion kinase was increased by treatment of hydrogen peroxide. Thus oxidative stress plays a crucial role in the transformation of lens epithelial cells and anterior polar type cataract formation.

An experimental study on the effect of cytoskeletal changes on osteoblastic cell activities / 대한치과교정학회지

The cytoskeleton has been shown to form a network, connecting the extracelluar matrix via integrin with the nucleus and the cytoplasmic constituents of the cell It is therefore assumed that the cytoskeleton may mediate signals generated by perturbations originating in the matrix. The purpose of this study is to examine the effect of cytoskeletal change on osteoblastic cell activities. The author cultured osteohinstic cells obtained from neonatal mouse calvaria The cells were treated with cytochalasin B(CB) or colchicine (COL) at four concentrations for 3 hours and after another 24 hours the conditioned media was collected and assayed for prostaglandin E(2) (PGE(2)), interleukin-6(IL-6), tumor necrosis factors-alpha(TNF-alpha) and matrix metalloproteinase-1(MMP-1). In addition, the cytoskeletal protein actin were observed by immunofluorescence. The results were as follows: 1. The production of PGE(2) showed the tendency to be increased in CB-treated group. PGE(2) was increased in COL-treated group dose-dependantly. 2. IL-6 Production, in CB-treated group, was increased, except at 1.0 microgram/ml. IL-6 was induced in COL-treated group. 3. TNF-alpha production was increased in CB-treated group, except at 1.0 microgram/ml, and in COL-treated group, that was increased. 4. The MMP-1 production was decreased in CB-treated goup and was not changed in COL-treated group, which could be selectively visualized by immunoblotting with monospecific antibody. 5. The cytoskeletal actin stress fibers were disappeared and the cells showed to be rounded in CB-treated group. These results indicated that there are a relationship between the cytoskeletal rearrangements and osteoblastic cell activities, especially in release of paracrine/autocrine factors, such as PGE(2), IL-6, and TNF-alpha.