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Asian Pacific Journal of Cancer... Jul 2022Glioblastoma is the most aggressive and lethal brain tumor in adults with highly invasive properties. In this present study, we explored the effects of Phyllanthus...
OBJECTIVE
Glioblastoma is the most aggressive and lethal brain tumor in adults with highly invasive properties. In this present study, we explored the effects of Phyllanthus taxodiifolius Beille extract on molecules known to be hallmarks of aggressive glioblastoma including N-cadherin and vimentin, mesenchymal markers, as well as paxillin, a major adaptor protein that regulates the linking of focal adhesions to the actin cytoskeleton.
METHODS
P. taxodiifolius were air-dried, powdered and percolated with methanol, filtered, concentrated and lyophilized to yield a crude methanol extract. C6 glioblastoma cell line was used in this study. The expression of N-cadherin and vimentin, as well as the activation of paxillin was determined using Western blot analysis. The effect of the extract on focal adhesions and actin cytoskeleton were investigated using immunofluorescence staining and confocal imaging.
RESULTS
In the presence of 40 µg/ml Phyllanthus taxodiifolius Beille extract, the expression of N-cadherin and vimentin were significantly decreased (p<0.001 and p<0.05, respectively). Activation of paxillin was also diminished as indicated by a reduction of phosphorylated-paxillin (p<0.01). Consequently, actin stress fibers in glioblastoma cells were abolished as evidenced by the decrease in focal adhesion (p<0.001) and stress fibers numbers (p<0.001).
CONCLUSION
Our study demonstrates for the first time that P. taxodiifolius interferes with multiple key molecules related to pathological hallmarks of glioblastoma. These molecules are involved with cell contacts, focal adhesions, and the formation and stabilization of actin stress fibers, which are required for glioblastoma metastatic behavior. These results provide further evidence supporting the potential of P. taxodiifolius and its bioactive compounds as anti-cancer agents.
Topics: Actins; Cadherins; Cell Adhesion; Focal Adhesion Protein-Tyrosine Kinases; Glioblastoma; Humans; Methanol; Paxillin; Phosphoproteins; Phosphorylation; Phyllanthus; Plant Extracts; Stress Fibers; Vimentin
PubMed: 35901345
DOI: 10.31557/APJCP.2022.23.7.2379 -
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 -
Molecular Biology of the Cell Aug 2019Binding of angiopoietin-1 (Ang-1) to its receptor Tie2 on endothelial cells (ECs) promotes vessel barrier integrity and angiogenesis. Here, we identify PAK2 and paxillin...
Binding of angiopoietin-1 (Ang-1) to its receptor Tie2 on endothelial cells (ECs) promotes vessel barrier integrity and angiogenesis. Here, we identify PAK2 and paxillin as critical targets of Ang-1 responsible for EC migration, polarization, and sprouting. We found that Ang-1 increases PAK2-dependent paxillin phosphorylation and remodeling of focal adhesions and that PAK2 and paxillin are required for EC polarization, migration, and angiogenic sprouting in response to Ang-1. Our findings show that Ang-1 triggers Cdc42 activation at the leading edges of migrating ECs, which is dependent on PAK2 and paxillin expression. We also established that the polarity protein Par3 interacts with Cdc42 in response to Ang-1 in a PAK2- and paxillin-dependent manner. Par3 is recruited at the leading edges of migrating cells and in focal adhesion, where it forms a signaling complex with PAK2 and paxillin in response to Ang-1. These results show that Ang-1 triggers EC polarization and angiogenic sprouting through PAK2-dependent paxillin activation and remodeling of focal adhesions, which are necessary for local activation of Cdc42 and the associated polarity complex. We have shown that PAK2 controls a signaling pathway important for angiogenic sprouting that links focal adhesions to polarity signaling in ECs.
Topics: Angiopoietin-1; Animals; Aorta; Cattle; Cell Adhesion; Cell Movement; Cell Polarity; Endothelial Cells; Focal Adhesions; Neovascularization, Physiologic; Paxillin; Phosphorylation; Receptor, TIE-2; Signal Transduction; cdc42 GTP-Binding Protein; p21-Activated Kinases
PubMed: 31141452
DOI: 10.1091/mbc.E18-08-0486 -
Scientific Reports Aug 2018Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound...
Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12. Cell-free interaction assay with immobilized paxillin shows that TG-2 bind to paxillin directly. JNK was the strongest kinase for paxillin phosphorylation in the in-vitro kinase screen, but TG-2 could not phosphorylate paxillin directly. Increasing TG-2 concentrations did not increase the amount of JNK in the TG-2/paxillin complex. Immunofluoresent staining shows that TG-2 colocalises with vinculin and paxillin in FA of migrating cells. TG-2 binds to paxillin and JNK-containing FA but does not recruit JNK directly. Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate paxillin, these processes critically regulate corneal epithelial adhesion and migration.
Topics: Blotting, Western; Cell Line; Cell Movement; Focal Adhesions; GTP-Binding Proteins; Humans; Immunoprecipitation; MAP Kinase Signaling System; Mass Spectrometry; Paxillin; Phosphorylation; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; RNA, Small Interfering; Transglutaminases
PubMed: 30120307
DOI: 10.1038/s41598-018-30172-8 -
International Review of Cell and... 2020The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are...
The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are important in cell adhesion and migration of both normal and cancer cells. Historically, the role of these proteins in regulating the actin cytoskeleton through focal adhesion-mediated signaling has been well documented. However, studies in recent years have revealed additional functions in modulating the microtubule and intermediate filament cytoskeletons to affect diverse processes including cell polarization, vesicle trafficking and mechanosignaling. Expression of paxillin family proteins in stromal cells is also important in regulating tumor cell migration and invasion through non-cell autonomous effects on the extracellular matrix. Both paxillin and Hic-5 can also influence gene expression through a variety of mechanisms, while their own expression is frequently dysregulated in various cancers. Accordingly, these proteins may serve as valuable targets for novel diagnostic and treatment approaches in cancer.
Topics: Focal Adhesions; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Neoplasm Invasiveness; Neoplasms; Paxillin; Signal Transduction
PubMed: 32859368
DOI: 10.1016/bs.ircmb.2020.05.003 -
BMC Cancer Mar 2023Osteosarcoma (OS) is the most common malignant bone tumor with a high incidence in children and adolescents. Frequent tumor metastasis and high postoperative recurrence...
BACKGROUND
Osteosarcoma (OS) is the most common malignant bone tumor with a high incidence in children and adolescents. Frequent tumor metastasis and high postoperative recurrence are the most common challenges in OS. However, detailed mechanism is largely unknown.
METHODS
We examined the expression of CD248 in OS tissue microarrays by immunohistochemistry (IHC) staining. We studied the biological function of CD248 in cell proliferation, invasion and migration of OS cells by CCK8 assay, transwell and wound healing assay. We also studied its function in the metastasis of OS in vivo. At last, we explored the potential mechanism how CD248 promotes OS metastasis by using RNA-seq, western blot, immunofluorescence staining and co-immunoprecipitation using CD248 knockdown OS cells.
RESULTS
CD248 was highly expressed in OS tissues and its high expression was correlated with pulmonary metastasis of OS. Knockdown of CD248 in OS cells significantly inhibited cell migration, invasion and metastasis, while had no obvious effect on cell proliferation. Lung metastasis in nude mice was significantly inhibited when CD248 was knocked down. Mechanistically, we found that CD248 could promote the interaction between ITGB1 and extracellular matrix (ECM) proteins like CYR61 and FN, which activated the FAK-paxillin pathway to promote the formation of focal adhesion and metastasis of OS.
CONCLUSION
Our data showed that high CD248 expression is correlated with the metastatic potential of OS. CD248 may promote migration and metastasis through enhancing the interaction between ITGB1 and certain ECM proteins. Therefore, CD248 is a potential marker for diagnosis and effective target for the treatment of metastatic OS.
Topics: Animals; Mice; Antigens, CD; Antigens, Neoplasm; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Mice, Nude; Osteosarcoma; Paxillin; Integrin beta1
PubMed: 36997926
DOI: 10.1186/s12885-023-10731-7 -
International Journal of Oral Science Aug 2023Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular...
Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling.
Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.
Topics: Humans; Paxillin; Protein-Lysine 6-Oxidase; Carcinoma, Squamous Cell; Epithelial-Mesenchymal Transition; Integrin alpha2beta1; Mouth Neoplasms; Collagen; Fibroblasts; Extracellular Vesicles; Cell Line, Tumor; Tumor Microenvironment
PubMed: 37532712
DOI: 10.1038/s41368-023-00236-1 -
Proceedings of the National Academy of... Aug 2023Integrin adhesion complexes are essential membrane-associated cellular compartments for metazoan life. The formation of initial integrin adhesion complexes is a dynamic...
Integrin adhesion complexes are essential membrane-associated cellular compartments for metazoan life. The formation of initial integrin adhesion complexes is a dynamic process involving focal adhesion proteins assembled at the integrin cytoplasmic tails and the inner leaflet of the plasma membrane. The weak multivalent protein interactions within the complex and with the plasma membrane suggest that liquid-liquid phase separation could play a role in the nascent adhesion assembly. Here, we report that solid-supported lipid membranes supplemented with phosphoinositides induce the phase separation of minimal integrin adhesion condensates composed of integrin 1 tails, kindlin, talin, paxillin, and FAK at physiological ionic strengths and protein concentrations. We show that the presence of phosphoinositides is key to enriching kindlin and talin on the lipid membrane, which is necessary to further induce the phase separation of paxillin and FAK at the membrane. Our data demonstrate that lipid membrane surfaces set the local solvent conditions for steering the membrane-localized phase separation even in a regime where no condensate formation of proteins occurs in bulk solution.
Topics: Animals; Integrins; Paxillin; Talin; Cell Membrane; Integrin beta1; Phosphatidylinositols; Cell Adhesion
PubMed: 37494400
DOI: 10.1073/pnas.2301881120 -
Molecular Biology of the Cell Dec 2017Cell polarization and directed migration play pivotal roles in diverse physiological and pathological processes. Herein, we identify new roles for paxillin-mediated...
Cell polarization and directed migration play pivotal roles in diverse physiological and pathological processes. Herein, we identify new roles for paxillin-mediated HDAC6 inhibition in regulating key aspects of cell polarization in both two-dimensional and one-dimensional matrix environments. Paxillin, by modulating microtubule acetylation through HDAC6 regulation, was shown to control centrosome and Golgi reorientation toward the leading edge, a hallmark of cell polarization to ensure directed trafficking of promigratory factors. Paxillin was also required for pericentrosomal Golgi localization and centrosome cohesion, independent of its localization to, and role in, focal adhesion signaling. In addition, we provide evidence of an accumulation of paxillin at the centrosome that is dependent on focal adhesion kinase (FAK) and identify an important collaboration between paxillin and FAK signaling in the modulation of microtubule acetylation, as well as centrosome and Golgi organization and polarization. Finally, paxillin was also shown to be required for optimal anterograde vesicular trafficking to the plasma membrane.
Topics: Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Polarity; Centrosome; Cytoskeletal Proteins; Extracellular Matrix; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Golgi Apparatus; Histone Deacetylase 6; Humans; Paxillin; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction
PubMed: 29046398
DOI: 10.1091/mbc.E17-08-0488 -
PloS One 2012Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases...
Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases Rac1 and RhoA are indispensable for cell migration as they regulate cell protrusion, cell-extracellular matrix (ECM) interactions and force transduction. However, the consequences of their activity at a molecular level within the cell remain undetermined. Using a combination of FRET, FRAP and biochemical analyses we show that the interactions between the focal adhesion proteins vinculin and paxillin, as well as the closely related family member Hic-5 are spatially and reciprocally regulated by the activity of Rac1 and RhoA. Vinculin in its active conformation interacts with either paxillin or Hic-5 in adhesions in response to Rac1 and RhoA activation respectively, while inactive vinculin interacts with paxillin in the membrane following Rac1 inhibition. Additionally, Rac1 specifically regulates the dynamics of paxillin as well as its binding partner and F-actin interacting protein actopaxin (α-parvin) in adhesions. Furthermore, FRET analysis of protein:protein interactions within cell adhesions formed in 3D matrices revealed that, in contrast to 2D systems vinculin interacts preferentially with Hic-5. This study provides new insight into the complexity of cell-ECM adhesions in both 2D and 3D matrices by providing the first description of RhoGTPase-coordinated protein:protein interactions in a cellular microenvironment. These data identify discrete roles for paxillin and Hic-5 in Rac1 and RhoA-dependent cell adhesion formation and maturation; processes essential for productive cell migration.
Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Movement; Cells, Cultured; Cytoskeletal Proteins; DNA-Binding Proteins; Focal Adhesions; LIM Domain Proteins; Mice; Paxillin; Signal Transduction; Vinculin; rac1 GTP-Binding Protein; rhoA GTP-Binding Protein
PubMed: 22629471
DOI: 10.1371/journal.pone.0037990