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American Journal of Respiratory and... Oct 2022The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. To relate functional abnormalities in...
The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated β1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.
Topics: Animals; Antiviral Agents; Elafin; Endogenous Retroviruses; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Integrins; Leukocyte Elastase; Mice; Neutrophils; Proteomics; Pulmonary Arterial Hypertension; Vinculin
PubMed: 35696338
DOI: 10.1164/rccm.202102-0446OC -
International Journal of Molecular... May 2022The mechanical signals within the extracellular matrix (ECM) regulate cell growth, proliferation and differentiation, and integrins function as the hub between the ECM... (Review)
Review
The mechanical signals within the extracellular matrix (ECM) regulate cell growth, proliferation and differentiation, and integrins function as the hub between the ECM and cellular actin. Focal adhesions (FAs) are multi‑protein, integrin‑containing complexes, acting as tension‑sensing anchoring points that bond cells to the extracellular microenvironment. Talin‑1 serves as the central protein of FAs that participates in the activation of integrins and connects them with the actin cytoskeleton. As a cytoplasmic protein, Talin‑1 consists of a globular head domain and a long rod comprised of a series of α‑helical bundles. The unique structure of the Talin‑1 rod domain permits folding and unfolding in response to the mechanical stress, revealing various binding sites. Thus, conformation changes of the Talin‑1 rod domain enable the cell to convert mechanical signals into chemical through multiple signaling pathways. The present review discusses the binding partners of Talin‑1, their interactions, effects on the cellular processes, and their possible roles in diseases.
Topics: Cell Adhesion; Focal Adhesions; Integrins; Mechanotransduction, Cellular; Protein Binding; Talin
PubMed: 35266014
DOI: 10.3892/ijmm.2022.5116 -
Cancers Sep 2023Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is...
Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody-drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients' data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies.
PubMed: 37686651
DOI: 10.3390/cancers15174374 -
The Journal of Biological Chemistry Feb 2016The cytoskeletal protein vinculin is a major regulator of cell adhesion and attaches to the cell surface by binding to specific phospholipids. Structural, biochemical,... (Review)
Review
The cytoskeletal protein vinculin is a major regulator of cell adhesion and attaches to the cell surface by binding to specific phospholipids. Structural, biochemical, and biological studies provided much insight into how vinculin binds to membranes, what components it recognizes, and how lipid binding is regulated. Here we discuss the roles and mechanisms of phospholipids in regulating the structure and function of vinculin and of its muscle-specific metavinculin splice variant. A full appreciation of these processes is necessary for understanding how vinculin regulates cell motility, migration, and wound healing, and for understanding of its role in cancer and cardiovascular diseases.
Topics: Animals; Cardiovascular Diseases; Cell Adhesion; Cell Membrane; Cell Movement; Humans; Neoplasms; Phospholipids; Vinculin
PubMed: 26728462
DOI: 10.1074/jbc.R115.686493 -
Experimental Cell Research Apr 2016External forces play a key role in shaping development and normal physiology. Aberrant responses to forces, or changes in the nature of such forces, are implicated in a... (Review)
Review
External forces play a key role in shaping development and normal physiology. Aberrant responses to forces, or changes in the nature of such forces, are implicated in a variety of diseases. Cells contain several types of adhesions, linking them to their external environment. It is through these adhesions that forces are both sensed (from the outside inwards) and applied (from inside to out). Furthermore, several adhesion-based proteins are sensitive to changes in intracellular forces, utilising them for activation and regulation. Here, we outline how vinculin, a key component of integrin-mediated adhesions linking the actin cytoskeleton to the extracellular matrix (ECM), is regulated by force and acts as force transducing protein. We discuss the role of vinculin in vivo and its place in health and disease; summarise the proposed mechanisms by which vinculin is recruited to and activated at integrin-ECM adhesions; and discuss recent findings that place vinculin as the major force sensing and transmitting component of cell-matrix adhesion complexes. Finally, we discuss the role of vinculin in regulating the cellular responses to both the physical properties of the external environment and to externally applied physical stimuli.
Topics: Cell Adhesion; Extracellular Matrix; Humans; Integrins; Models, Biological; Stress, Mechanical; Vinculin
PubMed: 26607713
DOI: 10.1016/j.yexcr.2015.11.017 -
Oncotarget Oct 2015
Topics: Animals; Cell Communication; Cell Membrane; Cell Movement; Focal Adhesions; Humans; Mice; Vinculin
PubMed: 26431280
DOI: 10.18632/oncotarget.5868 -
Journal of the American Society of... Jun 2018Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex...
Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein. We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin. Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and -actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well. Palladin has an important role in podocyte function and .
Topics: Actins; Animals; Cytoskeletal Proteins; Cytoskeleton; Female; Focal Adhesions; Gene Expression; Gene Silencing; Humans; Kidney Glomerulus; Male; Mice, Knockout; Microfilament Proteins; Morpholinos; Phosphoproteins; Podocytes; RNA, Messenger; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 29720549
DOI: 10.1681/ASN.2017091039 -
ACS Applied Materials & Interfaces Feb 2024The complex interplay between cells and materials is a key focus of this research, aiming to develop optimal scaffolds for regenerative medicine. The need for tissue...
The complex interplay between cells and materials is a key focus of this research, aiming to develop optimal scaffolds for regenerative medicine. The need for tissue regeneration underscores understanding cellular behavior on scaffolds, especially cell adhesion to polymer fibers forming focal adhesions. Key proteins, paxillin and vinculin, regulate cell signaling, migration, and mechanotransduction in response to the extracellular environment. This study utilizes advanced microscopy, specifically the AiryScan technique, along with advanced image analysis employing the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) cluster algorithm, to investigate protein distribution during osteoblast cell adhesion to polymer fibers and glass substrates. During cell attachment to both glass and polymer fibers, a noticeable shift in the local maxima of paxillin and vinculin signals is observed at the adhesion sites. The focal adhesion sites on polymer fibers are smaller and elliptical but exhibit higher protein density than on the typical glass surface. The characteristics of focal adhesions, influenced by paxillin and vinculin, such as size and density, can potentially reflect the strength and stability of cell adhesion. Efficient adhesion correlates with well-organized, larger focal adhesions characterized by increased accumulation of paxillin and vinculin. These findings offer promising implications for enhancing scaffold design, evaluating adhesion to various substrates, and refining cellular interactions in biomedical applications.
Topics: Paxillin; Vinculin; Focal Adhesions; Mechanotransduction, Cellular; Cell Adhesion; Polymers; Phosphoproteins; Focal Adhesion Protein-Tyrosine Kinases
PubMed: 38354103
DOI: 10.1021/acsami.3c19035 -
Frontiers in Cell and Developmental... 2022Cellular adhesion and migration are key functions that are disrupted in numerous diseases. We report that desmin, a type-III muscle-specific intermediate filament, is a...
Cellular adhesion and migration are key functions that are disrupted in numerous diseases. We report that desmin, a type-III muscle-specific intermediate filament, is a novel cell adhesion regulator. Expression of p.R406W mutant desmin, identified in patients with desmin-related myopathy, modified focal adhesion area and expression of adhesion-signaling genes in myogenic C2C12 cells. Satellite cells extracted from desmin-knock-out (DesKO) and desmin-knock-in-p.R405W (DesKI-R405W) mice were less adhesive and migrated faster than those from wild-type mice. Moreover, we observed mislocalized and aggregated vinculin, a key component of cell adhesion, in DesKO and DesKI-R405W muscles. Vinculin expression was also increased in desmin-related myopathy patient muscles. Together, our results establish a novel role for desmin in cell-matrix adhesion, an essential process for strength transmission, satellite cell migration and muscle regeneration. Our study links the patho-physiological mechanisms of desminopathies to adhesion/migration defects, and may lead to new cellular targets for novel therapeutic approaches.
PubMed: 35350386
DOI: 10.3389/fcell.2022.783724 -
Medical Sciences (Basel, Switzerland) Aug 2022Vinculin and talin-1, which are cytoskeletal proteins affecting focal adhesions, were reported to be down-expressed in atherosclerotic lesions. Recently, we reported...
Vinculin and talin-1, which are cytoskeletal proteins affecting focal adhesions, were reported to be down-expressed in atherosclerotic lesions. Recently, we reported high concentrations of plasma talin-1 in patients with coronary artery disease (CAD). However, blood vinculin concentrations in CAD patients have not been clarified. Plasma vinculin concentrations as well as talin-1 were studied in 327 patients in whom coronary angiography was performed. CAD was proven in 177 patients (1-vessel, = 79; 2-vessel, = 57; 3-vessel disease, = 41). However, vinculin concentrations were not markedly different between the CAD(-) and CAD groups (median 122.5 vs. 119.6 pg/mL, = 0.325) or among patients with CAD(-), 1-, 2-, and 3-vessel diseases (122.5, 112.8, 107.9, and 137.2 pg/mL, = 0.202). In contrast, talin-1 concentrations were higher in CAD than the CAD(-) group (0.29 vs. 0.23 ng/mL, = 0.006) and increased stepwise in the number of stenotic vessels: 0.23 in CAD(-), 0.28 in 1-vessel, 0.29 in 2-vessel, and 0.33 ng/mL in 3-vessel disease ( = 0.043). No correlation was observed between vinculin and talin-1 concentrations. In multivariate analysis, vinculin concentrations were not a factor for CAD. In conclusion, plasma vinculin concentrations in patients with CAD were not high and were not associated with the presence or severity of CAD.
Topics: Coronary Angiography; Coronary Artery Disease; Focal Adhesions; Humans; Talin; Vinculin
PubMed: 36135831
DOI: 10.3390/medsci10030046