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Molecular Cancer Dec 2014Loss of cell-cell adhesion is important for the development of cancer invasion and metastasis. Vinculin, a key adhesion-related protein, can affect metastasis and...
BACKGROUND
Loss of cell-cell adhesion is important for the development of cancer invasion and metastasis. Vinculin, a key adhesion-related protein, can affect metastasis and prognosis in several tumours. Here, we determined the biological roles of vinculin in the metastasis of colorectal cancer (CRC) and evaluated its clinical significance as a potential disease biomarker.
METHODS
The expression level of vinculin in CRC cell lines and tissues was measured using Real-Time PCR and western blotting. Moreover, vinculin function was analysed using Transwell assays and in vivo metastasis assays in gain- and loss-of-function experiments. Furthermore, the impact of vinculin together with membrane-bound β-catenin on the prognosis of 228 CRC patients was investigated by immunohistochemistry. Additionally, the expression of epithelial-mesenchymal transition (EMT) indicators was verified by immunohistochemistry in CRC tissues obtained from these patients.
RESULT
Vinculin expression was found to be significantly downregulated in highly metastatic CRC cell lines and metastatic tissues. Both in vitro and in vivo experiments showed that vinculin suppressed invasion, migration and metastasis in CRC cells and that this suppression could be attenuated by silencing β-catenin. Moreover, the expression of vinculin and membrane-bound β-catenin were positively correlated in CRC tissues, and lack of vinculin expression emerged as an independent prognostic factor in patients with CRC. Finally, the loss of vinculin and membrane-bound β-catenin was associated with node metastasis, organ metastasis and expression of EMT indicators.
CONCLUSION
Our results suggest that vinculin may play specific roles in the EMT and metastasis of CRC and that loss of vinculin could be used as a prognostic factor for CRC.
Topics: Animals; Caco-2 Cells; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Down-Regulation; Epithelial-Mesenchymal Transition; Female; HCT116 Cells; HT29 Cells; Humans; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Prognosis; Vinculin; beta Catenin
PubMed: 25496021
DOI: 10.1186/1476-4598-13-263 -
ELife Sep 2020Cell-cell and cell-matrix junctions transmit mechanical forces during tissue morphogenesis and homeostasis. α-Catenin links cell-cell adhesion complexes to the actin...
Cell-cell and cell-matrix junctions transmit mechanical forces during tissue morphogenesis and homeostasis. α-Catenin links cell-cell adhesion complexes to the actin cytoskeleton, and mechanical load strengthens its binding to F-actin in a direction-sensitive manner. Specifically, optical trap experiments revealed that force promotes a transition between weak and strong actin-bound states. Here, we describe the cryo-electron microscopy structure of the F-actin-bound αE-catenin actin-binding domain, which in solution forms a five-helix bundle. In the actin-bound structure, the first helix of the bundle dissociates and the remaining four helices and connecting loops rearrange to form the interface with actin. Deletion of the first helix produces strong actin binding in the absence of force, suggesting that the actin-bound structure corresponds to the strong state. Our analysis explains how mechanical force applied to αE-catenin or its homolog vinculin favors the strongly bound state, and the dependence of catch bond strength on the direction of applied force.
Topics: Actins; Cryoelectron Microscopy; Protein Binding; alpha Catenin
PubMed: 32915141
DOI: 10.7554/eLife.60878 -
International Journal of Molecular... Jan 2021Focal adhesions (FAs) serve as dynamic signaling hubs within the cell. They connect intracellular actin to the extracellular matrix (ECM) and respond to environmental... (Review)
Review
Focal adhesions (FAs) serve as dynamic signaling hubs within the cell. They connect intracellular actin to the extracellular matrix (ECM) and respond to environmental cues. In doing so, these structures facilitate important processes such as cell-ECM adhesion and migration. Pathogenic microbes often modify the host cell actin cytoskeleton in their pursuit of an ideal replicative niche or during invasion to facilitate uptake. As actin-interfacing structures, FA dynamics are also intimately tied to actin cytoskeletal organization. Indeed, exploitation of FAs is another avenue by which pathogenic microbes ensure their uptake, survival and dissemination. This is often achieved through the secretion of effector proteins which target specific protein components within the FA. Molecular mimicry of the leucine-aspartic acid (LD) motif or vinculin-binding domains (VBDs) commonly found within FA proteins is a common microbial strategy. Other effectors may induce post-translational modifications to FA proteins through the regulation of phosphorylation sites or proteolytic cleavage. In this review, we present an overview of the regulatory mechanisms governing host cell FAs, and provide examples of how pathogenic microbes have evolved to co-opt them to their own advantage. Recent technological advances pose exciting opportunities for delving deeper into the mechanistic details by which pathogenic microbes modify FAs.
Topics: Animals; Bacteria; Bacterial Infections; Bacterial Physiological Phenomena; Bacterial Proteins; Extracellular Matrix; Focal Adhesions; Host-Pathogen Interactions; Humans; Integrins; Signal Transduction
PubMed: 33572997
DOI: 10.3390/ijms22031358 -
Experimental Eye Research Apr 2019Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell...
Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.
Topics: Analysis of Variance; Biomechanical Phenomena; Cell Adhesion; Cell Movement; Cornea; Epithelial Cells; Epithelium; Humans; Limbus Corneae; Phosphorylation; Vinculin
PubMed: 30653966
DOI: 10.1016/j.exer.2019.01.014 -
PLoS Computational Biology Oct 2023Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which...
Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.
Topics: Focal Adhesions; Vinculin; Actins; Integrins; Extracellular Matrix; Cell Adhesion; Talin
PubMed: 37801464
DOI: 10.1371/journal.pcbi.1011500 -
Anatomical Record (Hoboken, N.J. : 2007) Sep 2014Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines,... (Review)
Review
Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines, etc.). SM contractile function can be regulated via expression and distribution of the contractile and cytoskeletal proteins, and activation of any of the second messenger pathways that regulate them. Spatial-temporal changes in the contractile, cytoskeletal or regulatory components of SM cells (SMCs) have been proposed to alter SM contractile activity. Ca(2+) sensitization/desensitization can occur as a result of changes at any of these levels, and specific pathways have been identified at all of these levels. Understanding when and how proteins can translocate within the cytoplasm, or to-and-from the plasmalemma and the cytoplasm to alter contractile activity is critical. Numerous studies have reported translocation of proteins associated with the adherens junction and G protein-coupled receptor activation pathways in isolated SMC systems. Specific examples of translocation of vinculin to and from the adherens junction and protein kinase C (PKC) and 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) to and from the plasmalemma in isolated SMC systems but not in intact SM tissues are discussed. Using both isolated SMC systems and SM tissues in parallel to pursue these studies will advance our understanding of both the role and mechanism of these pathways as well as their possible significance for Ca(2+) sensitization in intact SM tissues and organ systems.
Topics: Adherens Junctions; Animals; Calcium; Excitation Contraction Coupling; Humans; Intracellular Signaling Peptides and Proteins; Muscle Contraction; Muscle Proteins; Muscle, Smooth; Myocytes, Smooth Muscle; Phosphoprotein Phosphatases; Protein Kinase C; Protein Transport; Receptors, G-Protein-Coupled; Signal Transduction; Time Factors; Vinculin
PubMed: 25125185
DOI: 10.1002/ar.22970 -
The Korean Journal of Physiology &... Mar 2020Interstitial cells of Cajal (ICC) are known as the pacemaker cells of gastrointestinal tract, and it has been reported that acute gastroenteritis induces intestinal...
Interstitial cells of Cajal (ICC) are known as the pacemaker cells of gastrointestinal tract, and it has been reported that acute gastroenteritis induces intestinal dysmotility through antibody to vinculin, a cytoskeletal protein in gut, resulting in small intestinal bacterial overgrowth, so that anti-vinculin antibody can be used as a biomarker for irritable bowel syndrome. This study aimed to determine correlation between serum anti-vinculin antibody and ICC density in human stomach. Gastric specimens from 45 patients with gastric cancer who received gastric surgery at Kangwon National University Hospital from 2013 to 2017 were used. ICC in inner circular muscle, and myenteric plexus were counted. Corresponding patient's blood samples were used to determine the amount of anti-vinculin antibody by enzyme-linked immunosorbent assay. Analysis was done to determine correlation between anti-vinculin antibody and ICC numbers. Patients with elevated anti-vinculin antibody titer (above median value) had significantly lower number of ICC in inner circular muscle (71.0 240.5, p = 0.047), and myenteric plexus (12.0 68.5, p < 0.01) compared to patients with lower anti-vinculin antibody titer. Level of serum anti-vinculin antibody correlated significantly with density of ICC in myenteric plexus (r = -0.379, p = 0.01; Spearman correlation). Increased level of circulating anti-vinculin antibody was significantly correlated with decreased density of ICC in myenteric plexus of human stomach.
PubMed: 32140042
DOI: 10.4196/kjpp.2020.24.2.185 -
Histochemistry and Cell Biology Oct 2022Ongoing liver injury leads to fibrosis and ultimately cirrhosis, a leading cause of death worldwide. The primary mechanism underlying the fibrogenic response is the...
Ongoing liver injury leads to fibrosis and ultimately cirrhosis, a leading cause of death worldwide. The primary mechanism underlying the fibrogenic response is the activation of cells known as hepatic stellate cells (HSCs) which are "quiescent" in the normal liver but become "activated" after injury by transdifferentiating into extracellular matrix-secreting myofibroblasts. Since integrins (extracellular matrix binding receptors) are important mediators of HSC activation and fibrogenesis, we hypothesized that focal adhesion (FA) proteins, which link integrins to the intracellular protein machinery, may be important in the activation process. Therefore, using both an in vitro model of activation in primary rat HSCs and an in vivo model of liver injury, we examined three FA proteins: vinculin, FAK, and talin. All three proteins were significantly upregulated during HSC activation at both the messenger RNA (mRNA) and protein levels. Confocal microscopy demonstrated that the proteins had a widespread expression throughout HSCs with prominent localization at the end of actin filaments. Finally, we stimulated HSCs with the profibrotic ligands endothelin-1 (ET-1) and transforming growth factor beta (TGF-β) and observed an increase in the size of vinculin-containing FAs and the cell area occupied by them. The data indicate that HSCs possess a broad array of FA proteins, and given their upregulation during activation, this raises the possibility that they play a role in the fibrogenic response to injury.
Topics: Animals; Cells, Cultured; Endothelin-1; Focal Adhesions; Hepatic Stellate Cells; Integrins; Ligands; Liver; RNA, Messenger; Rats; Rodentia; Talin; Transforming Growth Factor beta; Vinculin
PubMed: 35960334
DOI: 10.1007/s00418-022-02123-y -
International Journal of Molecular... Sep 2022The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor...
The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of , , , , , and mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both knockout and TGF-β treatment and were validated by RT-qPCR of , , and . Since PGE is a product of arachidonic acid metabolism, its lowered concentration in media from -knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.
Topics: Arachidonic Acid; Cell Line, Tumor; Cell Movement; Cyclooxygenase 2; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Prostaglandins E; Transforming Growth Factor beta; Vinculin
PubMed: 36142758
DOI: 10.3390/ijms231810845 -
Aging Feb 2021In the process of epithelial-mesenchymal transition (EMT), epithelial cancer cells transdifferentiate into mesenchymal-like cells with high motility and aggressiveness,...
High expression of vinculin predicts poor prognosis and distant metastasis and associates with influencing tumor-associated NK cell infiltration and epithelial-mesenchymal transition in gastric cancer.
In the process of epithelial-mesenchymal transition (EMT), epithelial cancer cells transdifferentiate into mesenchymal-like cells with high motility and aggressiveness, resulting in the spread of tumor cells. Immune cells and inflammation in the tumor microenvironment are the driving factors of EMT, but few studies have explored the core targets of the interaction between EMT and tumor immune cells. We analyzed thousands of cases of gastric cancer and gastric tissue specimens of TCGA, CPTAC, GTEx and analyzing QPCR and IHC data of 56 gastric cancer patients in SYSU Gastric Cancer Research Center. It was known that EMT has an important connection with the infiltration of NK cells, and that the expression of vinculin may be the target of the phenomenon. The increased expression of vinculin is closely related to the aggressiveness and distant metastasis of cancer, which affects the survival prognosis of the patient. Moreover, through experiments under 3D conditions, we found that vinculin, cell invasion and metastasis are clearly linked. VCL can affect EMT and tumor immunity by regulating EPCAM gene expression. The role and mechanism of action of vinculin have been controversial, but this molecule may downregulate EpCAM (epithelial cellular adhesion molecule) and its own role in gastric cancer through DNA methylation, causing NK cells to enrich into tumor cells and kill tumor cells. At the same time, it promotes the occurrence of EMT, which in turn causes tumor metastasis and thus poorer prognosis.
Topics: Adenocarcinoma; Aged; Epithelial Cell Adhesion Molecule; Epithelial-Mesenchymal Transition; Female; Humans; Killer Cells, Natural; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Stomach Neoplasms; Vinculin
PubMed: 33535187
DOI: 10.18632/aging.202440