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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 -
The Journal of Cell Biology Jan 2020Talin, vinculin, and paxillin are core components of the dynamic link between integrins and actomyosin. Here, we study the mechanisms that mediate their activation and...
Talin, vinculin, and paxillin are core components of the dynamic link between integrins and actomyosin. Here, we study the mechanisms that mediate their activation and association using a mitochondrial-targeting assay, structure-based mutants, and advanced microscopy. As expected, full-length vinculin and talin are autoinhibited and do not interact with each other. However, contrary to previous models that propose a critical role for forces driving talin-vinculin association, our data show that force-independent relief of autoinhibition is sufficient to mediate their tight interaction. We also found that paxillin can bind to both talin and vinculin when either is inactive. Further experiments demonstrated that adhesions containing paxillin and vinculin can form without talin following integrin activation. However, these are largely deficient in exerting traction forces to the matrix. Our observations lead to a model whereby paxillin contributes to talin and vinculin recruitment into nascent adhesions. Activation of the talin-vinculin axis subsequently leads to the engagement with the traction force machinery and focal adhesion maturation.
Topics: Actin Cytoskeleton; Animals; Cells, Cultured; Fibroblasts; Focal Adhesions; Mice; Mice, Inbred C57BL; Mice, Knockout; Paxillin; Protein Binding; Stress, Mechanical; Talin; Vinculin
PubMed: 31816055
DOI: 10.1083/jcb.201903134 -
Frontiers in Nutrition 2022Increasing translational evidence suggests that intestinal permeability may be a contributing factor to systemic inflammatory events and numerous pathologies. While...
Increasing translational evidence suggests that intestinal permeability may be a contributing factor to systemic inflammatory events and numerous pathologies. While associations between IgE-mediated food allergies and increased intestinal permeability have been well-characterized, the relationship between IgG-mediated food sensitivities and intestinal permeability is not well-described in the literature. Thus, we tested for associations between intestinal permeability biomarkers and food-specific IgG antibodies in 111 adults, with and without gastrointestinal symptoms. All biomarkers and food-specific IgG antibodies were measured ELISA. The intestinal permeability biomarkers anti-lipopolysaccharide (LPS) and anti-occludin IgG and IgA antibodies, but not anti-vinculin or anti-CdtB IgG antibodies, were significantly and positively associated with IgG-mediated food sensitivities. These significant relationships were attenuated by adjusting for the severity of wheat, dairy, and egg reactions. The results of this study support strong associations between titers of food-specific IgG antibodies and intestinal permeability biomarkers in adults, to the extent that the presence of multiple IgG antibodies to food, and increasing IgG food titers, can be considered indicative of increased antibodies to LPS and occludin. Notably, neither IgG titers to wheat, eggs, and dairy, nor permeability biomarkers, were increased in symptomatic participants compared to those without symptoms.
PubMed: 36147305
DOI: 10.3389/fnut.2022.962093 -
Cell Proliferation Oct 2017Contemporarily, a highly increasing attention was paid to nanoconstructs, particularly DNA nanostructures possessing precise organization, functional manipulation,...
OBJECTIVES
Contemporarily, a highly increasing attention was paid to nanoconstructs, particularly DNA nanostructures possessing precise organization, functional manipulation, biocompatibility and biodegradability. Amongst these DNA nanomaterials, tetrahedral DNA nanostructures (TDNs) are a significantly ideal bionanomaterials with focusing on the property that can be internalized into cytoplasm in the absence of transfection. Therefore, the focus of this study was on investigating the influence of TDNs on the chondrocytes locomotion.
MATERIALS AND METHODS
Tetrahedral DNA nanostructures was confirmed by 6% polyacrylamide gel electrophoresis (PAGE) and dynamic light scattering (DLS). Subsequently, the effect of TDNs on chondrocyte locomotion was investigated by real-time cell analysis (RTCA) and wound healing assay. The variation of relevant genes and proteins was detected by quantitative polymerase chain reaction (qPCR), western blotting and immunofluorescence respectively.
RESULTS
We demonstrated that tetrahedral DNA nanostructures have positive influence on chondrocytes locomotion and promoted the expression of RhoA, ROCK2 and vinculin. Additionally, upon exposure to TDNs with the concentration of 250 nmol L , the chondrocytes were showed the highest motility via both RTCA and wound healing assay. Meanwhile, the mRNA and protein expression of RhoA, ROCK2 and vinculin were also significantly enhanced with the same concentration.
CONCLUSIONS
It can be concluded that the TDNs with the optimal concentration of 250 nmol L could extremely promoted the chondrocytes locomotion through facilitating the expression of RhoA, ROCK2 and vinculin. These results seemed to reveal that this special three-dimensional DNA tetrahedral nanostructures may be applied to cartilage repair and treatment in the future.
Topics: Animals; Biocompatible Materials; Cell Movement; Cells, Cultured; Chondrocytes; DNA; Humans; Nanostructures; Rats, Sprague-Dawley; Up-Regulation; Vinculin; rho-Associated Kinases; rhoA GTP-Binding Protein
PubMed: 28792637
DOI: 10.1111/cpr.12368 -
International Journal of Molecular... Aug 2021Understanding the biological and morphological reactions of human cells towards different dentinal derivate grafting materials is fundamental for choosing the type of...
Understanding the biological and morphological reactions of human cells towards different dentinal derivate grafting materials is fundamental for choosing the type of dentin for specific clinical situations. This study aimed to evaluate human periodontal ligament fibroblasts (hPLF) cells exposed to different dentinal derivates particles. The study design included the in vitro evaluation of mineralized dentine (SG), deproteinized and demineralized dentine (DDP), and demineralized dentine (TT) as test materials and of deproteinized bovine bone (BIOS) as the positive control material. The materials were kept with the hPLF cell line, and the evaluations were made after 24 h, 72 h, and 7 days of in vitro culture. The evaluated outcomes were proliferation by using XTT assays, the morphological characteristics by light microscopy (LM) and by the use of scanning electron microscopy (SEM), and adhesion by using confocal microscopy (CLSM). Overall, the experimental materials induced a positive response of the hPLFs in terms of proliferation and adhesion. The XTT assay showed the TT, and the SG induced significant growth compared to the negative control at 7 days follow-up. The morphological data supported the XTT assay: the LM observations showed the presence of densely packed cells with a modified shape; the SEM observations allowed the assessment of how fibroblasts exposed to DDP and TT presented cytoplasmatic extensions; and SG and BIOS also presented the thickening of the cellular membrane. The CLMS observations showed the expression of the proliferative marker, as well as and the expression of cytoskeletal elements involved in the adhesion process. In particular, the vinculin and integrin signals were stronger at 72 h, while the actin signal remained constantly expressed in all the follow-up of the sample exposed to SG material. The integrin signal was stronger at 72 h, and the vinculin and actin signals were stronger at 7 days follow-up in the sample exposed to DDP material. The vinculin and integrin signals were stronger at 72 h follow-up in the sample exposed to TT material; vinculin and integrin signals appear stronger at 24 h follow-up in the sample exposed to BIOS material. These data confirmed how dentinal derivates present satisfying biocompatibility and high conductivity and inductivity properties fundamental in the regenerative processes. Furthermore, the knowledge of the effects of the dentin's degree of mineralization on cellular behavior will help clinicians choose the type of dentine derivates material according to the required clinical situation.
Topics: Animals; Biomarkers; Bone Substitutes; Cattle; Cell Proliferation; Cells, Cultured; Dentin; Fibroblasts; Humans; Integrins; Materials Testing; Microscopy, Confocal; Microscopy, Electron, Scanning; Periodontal Ligament; Vinculin
PubMed: 34445386
DOI: 10.3390/ijms22168681 -
Methods (San Diego, Calif.) Feb 2016Mechanosensing of the micro-environments has been shown to be essential for cell survival, growth, differentiation and migration. The mechanosensing pathways are... (Review)
Review
Mechanosensing of the micro-environments has been shown to be essential for cell survival, growth, differentiation and migration. The mechanosensing pathways are mediated by a set of mechanosensitive proteins located at focal adhesion and cell-cell adherens junctions as well as in the cytoskeleton network. Here we review the applications of magnetic tweezers on elucidating the molecular mechanisms of the mechanosensing proteins. The scope of this review includes the principles of the magnetic tweezers technology, theoretical analysis of force-dependent stability and interaction of mechanosensing proteins, and recent findings obtained using magnetic tweezers.
Topics: Cell Adhesion Molecules; Electron Spin Resonance Spectroscopy; Extracellular Matrix; Focal Adhesions; Magnetic Phenomena; Mechanotransduction, Cellular; Protein Stability; Talin; Vinculin; alpha Catenin
PubMed: 26318089
DOI: 10.1016/j.ymeth.2015.08.020 -
International Journal of Molecular... May 2023Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events,...
Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins ( ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins ( < 0.001), whose differential expression was confirmed by ELISA ( = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.
Topics: Humans; Crohn Disease; Tumor Necrosis Factor Inhibitors; Vinculin; Tumor Necrosis Factor-alpha; Antineoplastic Agents; Remission Induction; Infliximab
PubMed: 37240037
DOI: 10.3390/ijms24108695 -
Scientific Reports Apr 2019This study utilized a Förster resonance energy transfer (FRET)-based molecular tension sensor and live cell imaging to evaluate the effect of osteocytes, a...
This study utilized a Förster resonance energy transfer (FRET)-based molecular tension sensor and live cell imaging to evaluate the effect of osteocytes, a mechanosensitive bone cell, on the migratory behavior of tumor cells. Two cell lines derived from MDA-MB-231 breast cancer cells were transfected with the vinculin tension sensor to quantitatively evaluate the force in focal adhesions of the tumor cell. Tumor cells treated with MLO-A5 osteocyte-conditioned media (CM) decreased the tensile forces in their focal adhesions and decreased their migratory potential. Tumor cells treated with media derived from MLO-A5 cells exposed to fluid flow-driven shear stress (FFCM) increased the tensile forces and increased migratory potential. Focal adhesion tension in tumor cells was also affected by distance from MLO-A5 cells when the two cells were co-cultured, where tumor cells close to MLO-A5 cells exhibited lower tension and decreased cell motility. Overall, this study demonstrates that focal adhesion tension is involved in altered migratory potential of tumor cells, and tumor-osteocyte interactions decrease the tension and motility of tumor cells.
Topics: Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Female; Fluorescence Resonance Energy Transfer; Focal Adhesions; Humans; Neoplasms; Osteoblasts; Osteocytes; Stress, Mechanical; Vinculin
PubMed: 30948840
DOI: 10.1038/s41598-019-42132-x