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Effect of fibronectin phosphorylation on cell adhesion, spreading and metabolic activity on flat substrates. A: Schematic of the experimental procedure. Fibronectin-coated surfaces were treated by either alkaline phosphatase (Fn-AP), kept native (Fn-native), or by casein kinase II (Fn-CKII). Fn(-/-) mouse fibroblasts were seeded and analyzed. B: Number of adherent cells and C cell spreading area and representative DIC micrographs taken 30 min after cell seeding. Depicted are the mean and standard deviation of 200 cells from duplicates in two independent experiments. D: Metabolic activity of the fibroblasts 24 h after seeding using the standard cell proliferation reagent WST-1. Statistical comparison done with Student's t-test between pair of samples connected with lines on the column plots: ��� , p-value<0.005; �� , p-value<0.01; � , p-value<0.05; not significant (ns).
Source publication
A large number of extracellular matrix proteins have been found in phosphorylated states, yet little is known about how the phosphorylation of extracellular matrix proteins might affect cell functions. We thus tested the hypothesis whether the phosphorylation of fibronectin, a major adhesion protein, affects cell behavior. Controlled in vitro phosp...
Contexts in source publication
Context 1
... ask the principle question whether the phosphorylation of human plasma fibronectin can indeed alter basic mechanical cell functions in cell culture, human fibronectin was first adsorbed to glass bottom dishes and subsequently either phosphorylated in vitro by CKII (Fn-CKII), or dephosphorylated by alkaline phosphatase (AP; Fn-AP), or left as-is (Fn-native) ( Fig 1A). This sequence of sample preparation was chosen to ensure that all surfaces, treated and untreated, had the same amount of surface adsorbed fibronectin. ...
Context 2
... assess various cell functions, we first analyzed the number of adherent cells 30 min after seeding, washing and fixing the cells. Compared to the untreated fibronectin substrates, CKII phosphorylation significantly increased the number (average cell number: 30) of adherent fibroblasts by ~60% for the same seeding densities, whereas dephosphorylation (average cell number: 18) had no pronounced effect compared to the native fibronectin control (average cell number: 20) (Fig 1B). Simultaneously, the cell spreading area was significantly increased by ~35% on the Fn-CKII substrates (average area: 1480 μm 2 ), but unaltered on the Fn-AP samples (average area: 1100 μm 2 ) (Fig 1C, Figure A in S1 File). ...
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... to the untreated fibronectin substrates, CKII phosphorylation significantly increased the number (average cell number: 30) of adherent fibroblasts by ~60% for the same seeding densities, whereas dephosphorylation (average cell number: 18) had no pronounced effect compared to the native fibronectin control (average cell number: 20) (Fig 1B). Simultaneously, the cell spreading area was significantly increased by ~35% on the Fn-CKII substrates (average area: 1480 μm 2 ), but unaltered on the Fn-AP samples (average area: 1100 μm 2 ) (Fig 1C, Figure A in S1 File). In a control, phosphorylated Fn-CKII was subsequently dephosphorylated with alkaline phosphatase (Fn-CKII-AP) which significantly reduced the spreading area by roughly 20% compared to the Fn-CKII samples down to a level that was comparable to the Fn-AP surfaces ( Fig 1C). ...
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... the cell spreading area was significantly increased by ~35% on the Fn-CKII substrates (average area: 1480 μm 2 ), but unaltered on the Fn-AP samples (average area: 1100 μm 2 ) (Fig 1C, Figure A in S1 File). In a control, phosphorylated Fn-CKII was subsequently dephosphorylated with alkaline phosphatase (Fn-CKII-AP) which significantly reduced the spreading area by roughly 20% compared to the Fn-CKII samples down to a level that was comparable to the Fn-AP surfaces ( Fig 1C). ...
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... ask whether the increased cell spreading is accompanied by a change of cell metabolic activity, we cultured the fibroblasts for 24 h and then quantified their metabolic activity by the WST-1 cell proliferation assay (see Methods). A significant enhancement of metabolic activity on CKII-phosphorylated fibronectin (Fn-CKII) was observed, whereas no difference could be detected between native fibronectin (Fn-native) and dephosphorylated fibronectin (Fn-AP) samples ( Fig 1D). In contrast, we did not find a major effect of fibronectin phosphorylation on cell proliferation and migration ( Figure B in S1 File). ...
Context 6
... absolute cell spreading areas on nanopillars for the different conditions, Fn-AP, Fnnative and Fn-CKII, were significantly smaller than those on flat substrates (Fig 1C), as observed before [12,13]. Our data further show that enhanced cell spreading correlates with enhanced total strain energy per cell (Fig 2D and 2E), as well as with the total strain energy normalized per unit area (Fig 2F). ...
Context 7
... is also well known that fibronectin undergoes major conformational changes towards more extended states upon adsorption to surfaces or Effect of fibronectin phosphorylation on cell traction forces as probed by Fn-coated nanopillar arrays. Fn(-/-) mouse fibroblasts were cultured for 30 min after seeding on fibronectin-coated nanopillar arrays as described above ( Fig 1A). These nanopillar arrays were fabricated from SU-8 and the pillars had a spring constant of 79 nN/μm. ...
Context 8
... the phosphorylation of ECM proteins was reported already in 1883 [16] and in 1988 for fibronectin [8], we report here for the first time that the phosphorylation of fibronectin upregulates fundamental cell functions including cell spreading and proliferation (Fig 1) and leads to an increase of cell traction forces (Fig 2). Moreover, mass spectrometry was used to map the phosphorylated sites of human plasma fibronectin. ...
Citations
... CX-4945 can therapeutically activate IRF3 and downregulate extracellular matrix protein expression and therefore reduce glioblastoma invasiveness in vitro and in vivo [77]. Indeed, CK2 is capable of phosphorylating extracellular proteins as demonstrated in the case of fibronectin and consequently increasing cell adhesion forces [111]. In addition, CX-4945 administered with gefitinib exerted [104] [107] (TBB/TBBt) 4,5,6,7-tetrabromo-1H-benzotriazole CK2 Ki (0.40 µM) IC50 (0.50 μM) − cytotoxic activity against T98G glioma cells; ...
Protein kinase CK2 has become a target of experimental antiglioma therapies as laboratory data are almost uniformly favorable and the number of synthetized CK2 inhibitors is rapidly growing. The evidence of their use for other brain tumors is on the increase as well. Great expectations are entrusted in naturally occurring compounds capable of inhibiting CK2 kinase. These compounds are extracted and purified by means of biochemistry methods and are amenable for innovative drug delivery systems as well. CK2 kinase inhibitors have been proven suitable for combined therapies with other investigational antiglioma agents and treatment modalities. However a greater share of efforts should be undertaken towards inhibiting functions relatively specific for glial tumors including infiltrative growth and invasiveness or maintenance of glioma initiating cells. Many of these function appear to converge on mTOR and JAK/STAT pathways which are being meticulously studied in this respect. Protein kinase CK2 holds therapeutic promise especially when combined with other agents aimed at molecular signatures of gliomas.
... The inhibition of integrin glycosylation including that of αvβ6 integrin leads to the enhanced invasive behavior of cells involved in metastasis [205,206]. Demonstrating the importance of fibronectin as a component of the ECM, the phosphorylation of fibronectin leads to increased mechanical forces being needed for cell adhesion in various cancers [207][208][209]. Glycosaminoglycans can undergo sulphation in various cancers, and this impacts cell-matrix signaling [210][211][212]. ...
The extracellular matrix (ECM) is a ubiquitous member of the body and is key to the maintenance of tissue and organ integrity. Initially thought to be a bystander in many cellular processes, the extracellular matrix has been shown to have diverse components that regulate and activate many cellular processes and ultimately influence cell phenotype. Importantly, the ECM's composition, architecture, and stiffness/elasticity influence cellular phenotypes. Under normal conditions and during development, the synthesized ECM constantly undergoes degradation and remodeling processes via the action of matrix proteases that maintain tissue homeostasis. In many pathological conditions including fibrosis and cancer, ECM synthesis, remodeling, and degradation is dysregulated, causing its integrity to be altered. Both physical and chemical cues from the ECM are sensed via receptors including integrins and play key roles in driving cellular proliferation and differentiation and in the progression of various diseases such as cancers. Advances in 'omics' technologies have seen an increase in studies focusing on bidirectional cell-matrix interactions, and here, we highlight the emerging knowledge on the role played by the ECM during normal development and in pathological conditions. This review summarizes current ECM-targeted therapies that can modify ECM tumors to overcome drug resistance and better cancer treatment.
... Inhibition of integrins glycosylation including αvβ6 integrin leads to enhanced invasive behaviour of cells involved in metastasis [220,221]. Demonstrating the importance of fibronectin as a component of the ECM, the phosphorylation of fibronectin leads to increased mechanical forces needed for cell adhesion in various cancers [222][223][224]. Glycosaminoglycans can undergo sulphation in various cancers and this impact cell-matrix signaling [225][226][227]. ...
The extracellular matrix (ECM) is a ubiquitous member of the body and is key to the maintenance of tissues and organs integrity. Initially thought to be a bystander in many cellular processes, the extracellular matrix has been shown to have diverse components that regulate and activate many cellular processes and ultimately influence cell phenotype. Importantly, the ECM composition, architecture, and stiffness/elasticity influence cellular phenotypes. Under normal conditions and during development, the synthesized ECM is constantly undergoing degradation and remodeling processes via the action of matrix proteases that maintain tissue homeostasis. In many pathological conditions including fibrosis and cancer, the ECM synthesis, remodeling, and degradation is dysregulated causing its integrity to be altered. Both physical and chemical cues from the ECM are sensed via receptors including integrins and play key roles in driving cellular proliferation and differentiation and in various disease progression such as cancers. Advances in 'omics' technologies have seen an increase in studies focussing on bi-directional cell-matrix interactions and here we highlight emerging knowledge on the role played by the ECM during normal development and in pathological conditions. This review summarizes current ECM-targeted-therapies that can modify tumor ECM to overcome drug resistance and better cancer treatment.
... It is the phosphatase, that takes the primary responsibility of CNPs in cell swelling, bursting, and recovery, as phosphorylation and dephosphorylation of fibronectin regulates the cell attachment/detachment and perhaps meditates the relocation of water channel protein AQP5 (Yalak et al. 2019;Bork and Doolittle 1993;Bhalla et al. 1999). Fan et al. ...
Background
Quantum nanodots especially carbon nanoparticles (CNPs) have been widely studied in biomedicine in imaging, and drug delivery, but anti-cancer mechanisms remain elusive.
Methods
Here, we investigated a type of cell death induced by food (beet, soybean) derived CNPs in cancer cells and tested whether CNPs induced DNA damage and resistant to anti-cancer agent PARP inhibitor (PARPi) could be overcome by quantum calculations, TEM, AFM, FT-IR, soft agar assay, and cytotoxicity assay.
Results
At high doses, CNPs derived from beet lead to a pop-like apoptosis (Carbopoptosis) in cancer cells. Quantum mechanical calculations confirmed CNPs binding with phosphate groups as well as DNA bases. At low doses, CNPs develop PARPi drug resistance through interactions between CNPs and PARPi. A synergistic drug effect was achieved with the combination of phosphatase inhibitor (PPi), PARPi, and CNPs. This is corroborated by the fact that sulfur modulated CNPs which exhibit super high phosphatase nanozyme activity abrogated the CNPs induced colony formation in anchorage-independent cancer cell growth.
Conclusion
Thus, our data suggest the CNPs intrinsic nanozyme activity of phosphatase may crosstalk with drug resistance, which can be reversed upon modulations.
... It is the phosphatase, that takes the primary responsibility of cell swelling, bursting, and recovery, as phosphorylation and dephosphorylation of bronectin regulates the cell attachment/detachment and perhaps meditates the relocation of water channel protein AQP5 [43][44][45]. CNPs can induce cell death through the regulation of osmosis-related phenotype. The very nature of the reversibility of phosphatase may, therefore, determine the respectability of observed carbopopotosis. ...
Background Quantum nanodots especially carbon nanoparticles (CNPs) have been widely studied in biomedicine in imaging, drug delivery, but anti-cancer mechanisms remain elusive. Methods Here we studied on a type of cell death induced by food (beet, soybean) derived CNPs in cancer cells and tested whether CNPs induced DNA damage and resistant to anticancer agent PARP inhibitor (PARPi) could be overcome by quantum calculation, SEM, TEM, AFM, FT-IR, soft agar assay, and cytotoxicity assay. Results At high doses, CNPs derived from beet lead to a pop-like apoptosis of cells. Quantum mechanical calculation confirmed CNPs binding with phosphate groups as well as DNA bases. At low doses, CNPs develop PARPi drug resistance through interactions between CNPs and PARPi. A synergistic drug effect was achieved with the combination of phosphatase inhibitor (PPi), PARPi and CNPs. This is corroborated by the fact that a sulfur modulated CNPs which exhibit super high phosphatase nanozyme activity abrogated the CNPs induced colony formation in anchorage-independent cancer cell growth. Conclusion Thus, our data suggest the CNPs intrinsic nanozyme activity of phosphatase may crosstalk with drug resistance, which can be reversed upon modulations.
... Furthermore, pro-oncogenic reprogramming of the stroma is accompanied by an upregulation of ECM components, such as fibronectin [340]. Yalak et al. reported that CK2-mediated phosphorylation of fibronectin promoted cell spreading and metabolic activity in vitro [341]. ...
... Yalak et al. elucidated the role of CK2-mediated phosphorylation of ECM proteins. They demonstrated in vitro by using nanopillar arrays that CK2dependent phosphorylation of fibronectin significantly upregulated cell traction forces and total strain energy, as well as cell spreading and metabolic activity [341]. Fibronectin is a key component of the ECM and is highly upregulated in cancer [425]. ...
... Fibronectin is a key component of the ECM and is highly upregulated in cancer [425]. Published pro-teomic data demonstrated that fibronectin is heavily phosphorylated in domains responsible for growth factor signaling and fibrillogenesis in clinical cancer tissue samples [341,426]. ...
Cancer is a leading cause of death worldwide. Casein kinase 2 (CK2) is commonly dysregulated in cancer, impacting diverse molecular pathways. CK2 is a highly conserved serine/threonine kinase, constitutively active and ubiquitously expressed in eukaryotes. With over 500 known substrates and being estimated to be responsible for up to 10% of the human phosphoproteome, it is of significant importance. A broad spectrum of diverse types of cancer cells has been already shown to rely on disturbed CK2 levels for their survival. The hallmarks of cancer provide a rationale for understanding cancer’s common traits. They constitute the maintenance of proliferative signaling, evasion of growth suppressors, resisting cell death, enabling of replicative immortality, induction of angiogenesis, the activation of invasion and metastasis, as well as avoidance of immune destruction and dysregulation of cellular energetics. In this work, we have compiled evidence from the literature suggesting that CK2 modulates all hallmarks of cancer, thereby promoting oncogenesis and operating as a cancer driver by creating a cellular environment favorable to neoplasia.
... The phosphorylated sites included residues in serum albumin, Fibronectin, S100-A9, and in Kininogen. Phosphorylated Fibronectin has been shown to enhance cell attachment (Yalak et al. 2019), and fibronectin was reported to be elevated in the serum of pregnant ZIKV patients (Foo et al. 2017). Altered expression of the inflammatory protein S100-A9 is associated with recurrent pregnancy loss (Nair et al. 2013). ...
Zika virus (ZIKV) and dengue virus (DENV) are two closely related flaviviruses with similar symptoms; understanding differences in their molecular impact on the host is therefore of high interest. Viruses interact with the host's post-translational modifications, inducing changes visible in serum. As modifications are diverse and of low abundance, they typically require additional sample processing which is not feasible for large cohort studies. Therefore, we tested the potential of next-generation proteomics data in its ability to prioritize specific modifications for later targeted analysis. We re-mined published mass spectra from 122 unenriched serum samples from ZIKV and DENV patients for the presence of phosphorylated, methylated, oxidized, glycosylated/glycated, sulfated, and carboxylated peptides. We identified 272 modified peptides with significantly differential abundance in ZIKV and DENV patients. Amongst these, methionine-oxidized peptides from apolipoproteins and glycosylated peptides from immunoglobulin proteins were more abundant in ZIKV patient serum and generate hypotheses on the potential roles of the modification in the infection. The results demonstrate how data-independent acquisition techniques can help prioritize future analyses of peptide modifications.
... Collagenase is used by cancer cells to attach to laminin and destroy basement membrane collagen type IV [87]. Cells then attach to fibronectin in the extracellular matrix and spread locally [88]. Entrance into vascular or lymphatic spaces allows for distant spread [89]. ...
Simple Summary
Increasing evidence suggests that bacterial infection not only promotes carcinogenesis in primary colorectal cancer, but also affects metastatic progression and organ selectivity through modification of the microenvironment at primary and secondary tumor sites. The metastatic cascade is the process by which neoplastic tumors potentiate cancerous spread to distant organs, and evidence suggests that this process is provoked in the setting of bacterial infection. Biofilm formation, paired migration, and quorum sensing are processes by which bacteria self-signal, recruit, and effectively establish a pre-metastatic niche at distant sites, rendering a suitable environment for tumor cell survival and proliferation.
Abstract
While the gut microbiome is composed of numerous bacteria, specific bacteria within the gut may play a significant role in carcinogenesis, progression, and metastasis of colorectal carcinoma (CRC). Certain microbial species are known to be associated with specific cancers; however, the interrelationship between bacteria and metastasis is still enigmatic. Mounting evidence suggests that bacteria participate in cancer organotropism during solid tumor metastasis. A critical review of the literature was conducted to better characterize what is known about bacteria populating a distant site and whether a tumor depends upon the same microenvironment during or after metastasis. The processes of carcinogenesis, tumor growth and metastatic spread in the setting of bacterial infection were examined in detail. The literature was scrutinized to discover the role of the lymphatic and venous systems in tumor metastasis and how microbes affect these processes. Some bacteria have a potent ability to enhance epithelial–mesenchymal transition, a critical step in the metastatic cascade. Bacteria also can modify the microenvironment and the local immune profile at a metastatic site. Early targeted antibiotic therapy should be further investigated as a measure to prevent metastatic spread in the setting of bacterial infection.
... In addition to alternative splicing, fibronectin can also undergo multiple posttranslational modifications such as phosphorylation and glycosylation 85 . In mouse fibroblasts, phosphorylation of fibronectin participates in cell spreading, proliferation, and cell traction forces 86 . In human U2OS (human bone osteosarcoma) and HeLa (human cervical adenocarcinoma epithelial) cell lines, five N-glycosylation sites (N430, N528, N542, N1007, and N1244) were identified in the fibronectin protein. ...
Skeletal muscle requires a highly orchestrated coordination between multiple cell types and their microenvironment to exert its function and to maintain its homeostasis and regenerative capacity. Over the past decades, significant advances, including lineage tracing and single-cell RNA sequencing, have contributed to identifying multiple muscle resident cell populations participating in muscle maintenance and repair. Among these populations, muscle stem cells (MuSC), also known as satellite cells, in response to stress or injury, are able to proliferate, fuse, and form new myofibers to repair the damaged tissue. These cells reside adjacent to the myofiber and are surrounded by a specific and complex microenvironment, the stem cell niche. Major components of the niche are extracellular matrix (ECM) proteins, able to instruct MuSC behavior. However, during aging and muscle-associated diseases, muscle progressively loses its regenerative ability, in part due to a dysregulation of ECM components. This review provides an overview of the composition and importance of the MuSC microenvironment. We discuss relevant ECM proteins and how their mutations or dysregulation impact young and aged muscle tissue or contribute to diseases. Recent discoveries have improved our knowledge about the ECM composition of skeletal muscle, which has helped to mimic the architecture of the stem cell niche and improved the regenerative capacity of MuSC. Further understanding about extrinsic signals from the microenvironment controlling MuSC function and innovative technologies are still required to develop new therapies to improve muscle repair.
... Cell adhesion to the extracellular matrix (ECM) is key to a series of cellular events that govern the fate of cells, which plays a dominant role in mediating and regulating other cellular processes including but not limited to cell spreading, migration, bidirectional signaling, and wound healing [1,2]. In other words, cell adhesion to an ECM or a biomimetic material provides further cues that determine cell functionality and fate [3][4][5][6][7], and thus a comprehensive understanding of cell adhesion is of much importance [8][9][10][11][12]. ...
Cell adhesion to extracellular matrices (ECM) is critical to physiological and pathological processes as well as biomedical and biotechnological applications. It has been known that a cell can adhere on an adhesive microisland only over a critical size. But no publication has concerned critical adhesion areas of cells on microislands with nanoarray decoration. Herein, we fabricated a series of micro-nanopatterns with different microisland sizes and arginine-glycine-aspartate (RGD) nanospacings on a nonfouling poly(ethylene glycol) background. Besides reproducing that nanospacing of RGD, a ligand of its receptor integrin (a membrane protein), significantly influences specific cell adhesion on bioactive nanoarrays, we confirmed that the concept of critical adhesion area originally suggested in studies of cells on micropatterns was justified also on the micro-nanopatterns, yet the latter exhibited more characteristic behaviors of cell adhesion. We found increased critical adhesion areas of human mesenchymal stem cells (hMSCs) on nanoarrayed microislands with increased RGD nanospacings. However, the numbers of nanodots with respect to the critical adhesion areas were not a constant. A unified interpretation was then put forward after combining nonspecific background adhesion and specific cell adhesion. We further carried out the asymptotic analysis of a series of micro-nanopatterned surfaces to obtain the effective RGD nanospacing on unpatterned free surfaces with densely grafted RGD, which could be estimated nonzero but has never been revealed previously without the assistance of the micro-nanopatterning techniques and the corresponding analysis.
Electronic supplementary material:
Supplementary materials and methods (details of fabrication of micro-nanopatterns), and supplementary results (selective adhesion or localization of hMSCs on nanoarrayed microislands with non-fouling background, calculation of critical number of integrin-ligand binding N*, etc.) are available in the online version of this article at 10.1007/s12274-021-3711-6.
