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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 -
Life (Basel, Switzerland) Mar 2022Endometrial cancer (EC) is the most common form of gynecological cancer. Type 2 diabetes mellitus is associated with an increased risk of EC. Currently, no proteomic...
Endometrial cancer (EC) is the most common form of gynecological cancer. Type 2 diabetes mellitus is associated with an increased risk of EC. Currently, no proteomic studies have investigated the role of diabetes in endometrial cancers from clinical samples. The present study aims to elucidate the molecular link between diabetes and EC using a proteomic approach. Endometrial tissue samples were obtained from age-matched patients (EC Diabetic and EC Non-Diabetic) during surgery. Untargeted proteomic analysis of the endometrial tissues was carried out using a two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF). A total of 53 proteins were identified, with a significant difference in abundance (analysis of variance (ANOVA) test, ≤ 0.05; fold-change ≥ 1.5) between the two groups, among which 30 were upregulated and 23 downregulated in the EC Diabetic group compared to EC Non-Diabetic. The significantly upregulated proteins included peroxiredoxin-1, vinculin, endoplasmin, annexin A5, calreticulin, and serotransferrin. The significantly downregulated proteins were myosin regulatory light polypeptide 9, Retinol dehydrogenase 12, protein WWC3, intraflagellar transport protein 88 homolog, superoxide dismutase [Cu-Zn], and retinal dehydrogenase 1. The network pathway was related to connective tissue disorder, developmental disorder, and hereditary disorder, with the identified proteins centered around dysregulation of ERK1/2 and F Actin signaling pathways. Cancer-associated protein alterations such as upregulation of peroxiredoxin-1, annexin 5, and iNOS, and downregulation of RDH12, retinaldehyde dehydrogenase 1, SOD1, and MYL 9, were found in the EC tissues of the diabetic group. Differential expression of proteins linked to cancer metastasis, such as the upregulation of vinculin and endoplasmin and downregulation of WWC3 and IFT88, was seen in the patients with diabetes. Calreticulin and alpha-enolase, which might have a role in the interplay between diabetes and EC, need further investigation.
PubMed: 35454982
DOI: 10.3390/life12040491 -
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 -
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 -
ELife Jul 2020Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting...
Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.
Topics: Actin Cytoskeleton; Actins; Membranes, Artificial; Phosphatidylinositols; Talin; Vinculin
PubMed: 32657269
DOI: 10.7554/eLife.56110 -
Structure (London, England : 1993) Oct 2019Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin...
Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin in its ability to bundle filamentous actin (F-actin). To elucidate the molecular basis for these differences, we employed computational and experimental approaches. Results from these analyses suggest that the C terminus of both vinculin and metavinculin form stable interactions with the F-actin surface. However, the metavinculin tail (MVt) domain contains a 68 amino acid insert, with helix 1 (H1) sequestered into a globular subdomain, which protrudes from the F-actin surface and prevents actin bundling by sterically occluding actin filaments. Consistent with our model, deletion and selective point mutations within the MVt H1 disrupt this protruding structure, and facilitate actin bundling similar to vinculin tail (Vt) domain.
Topics: Actins; Alternative Splicing; Animals; Binding Sites; Cryoelectron Microscopy; Models, Molecular; Mutation; Protein Binding; Protein Domains; Protein Structure, Secondary; Vinculin
PubMed: 31422909
DOI: 10.1016/j.str.2019.07.013 -
Biologics : Targets & Therapy 2023The stiffness of the extracellular matrix (ECM) controls many cellular processes, such as migration and differentiation. Cells detect stiffness through adhesion...
BACKGROUND
The stiffness of the extracellular matrix (ECM) controls many cellular processes, such as migration and differentiation. Cells detect stiffness through adhesion structures termed focal adhesions (FAs). Vinculin, an actin-binding FA protein, plays a pivotal role in FA-mediated mechanotransduction.
AIM
This study aimed to explore the role of vinculin in the development of HBV/HCV-induced hepatocellular carcinoma (HCC).
METHODS
Vinculin levels in a total number of 100 serum samples from patients with HBV/HCV-induced liver cirrhosis and HCC, as well as healthy controls, were analyzed using an enzyme-linked immunosorbent assay (ELISA).
RESULTS
In patients with HCC and liver cirrhosis, the serum vinculin levels were significantly greater than in controls (503.8±242.2 and 728.4±1044.8 vs 77.7±36.1 respectively, p<0.001). However, results showed no link between serum vinculin and the clinicopathological features of HCC.
CONCLUSION
Patients with HBVor HCV-induced liver cirrhosis and HCC have significantly higher serum levels of vinculin than do controls. This might point to a potential role for vinculin in the development of HCC. More research into how this protein affects the development of HCC at the molecular level could lead to better clinical treatments and the development of new molecular therapies.
PubMed: 36969330
DOI: 10.2147/BTT.S405500 -
FEBS Letters Feb 2022Lysophosphatidylcholine (LPC), the active metabolite of palmitate, triggers hepatocyte death by activating endoplasmic reticulum stress and JNK signalling-mediated...
Lysophosphatidylcholine (LPC), the active metabolite of palmitate, triggers hepatocyte death by activating endoplasmic reticulum stress and JNK signalling-mediated lipoapoptosis. However, LPC-induced cytotoxicity in hepatocytes is not well understood. Here, we found for the first time that LPC-induced cell rounding occurred prior to apoptosis. LPC-induced rounding of cells reduced both cell-extracellular matrix (ECM) adhesion and cell-cell junctions, which promoted detachment-induced apoptosis (defined as anoikis) in hepatocytes. Further study revealed that LPC altered cellular morphology and cell adhesion by inhibiting integrin and cadherin signalling-mediated microfilament polymerization. We also found that ECM supplementation and microfilament cytoskeletal stabilization inhibited LPC-induced hepatocyte death by attenuating anoikis. Our data indicate a novel cytotoxic process and signalling pathway induced by LPC.
Topics: Actin Cytoskeleton; Anoikis; Apoptosis Regulatory Proteins; Cadherins; Caspase 8; Cell Adhesion; Cell Line, Tumor; Endoplasmic Reticulum Stress; Extracellular Matrix; Gene Expression Regulation; Hep G2 Cells; Hepatocytes; Humans; Integrins; Intercellular Junctions; Lysophosphatidylcholines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Vinculin
PubMed: 35043979
DOI: 10.1002/1873-3468.14291