Integrins induce expression of monocyte chemoattractant protein-1 via focal adhesion kinase in mesangial cells
Yujiro WatanabeMasahito TamuraAkihiko OsajimaHirofumi AnaiNarutoshi KabashimaRyota SerinoYasuhide Nakashima
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Integrins play crucial roles in cell adhesion, migration, and signaling by providing transmembrane links between the extracellular matrix and the cytoskeleton. Integrins cluster in macromolecular complexes to generate cell-matrix adhesions such as focal adhesions. In this mini-review, we compare certain integrin-based biological responses and signaling during cell interactions with standard 2D cell culture versus 3D matrices. Besides responding to the composition of the matrix, cells sense and react to physical properties that include three-dimensionality and rigidity. In routine cell culture, fibroblasts and mesenchymal cells appear to use focal adhesions as anchors. They then use intracellular actomyosin contractility and dynamic, directional integrin movements to stretch cell-surface fibronectin and to generate characteristic long fibrils of fibronectin in "fibrillar adhesions". Some cells in culture proceed to produce dense, three-dimensional matrices similar to in vivo matrix, as opposed to the flat, rigid, two-dimensional surfaces habitually used for cell culture. Cells within such more natural 3D matrices form a distinctive class of adhesion termed "3D-matrix adhesions". These 3D adhesions show distinctive morphology and molecular composition. Their formation is heavily dependent on interactions between integrin alpha5ß1 and fibronectin. Cells adhere much more rapidly to 3D matrices. They also show more rapid morphological changes, migration, and proliferation compared to most 2D matrices or 3D collagen gels. Particularly notable are low levels of tyrosine phosphorylation of focal adhesion kinase and moderate increases in activated mitogen-activated protein kinase. These findings underscore the importance of the dimensionality and dynamics of matrix substrates in cellular responses to the extracellular matrix.
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Abstract Investigating stages of maturation of cellular adhesions to the extracellular matrix from the initial binding events to the formation of small focal complexes has been challenging because of the difficulty in fabricating the necessary nanopatterned substrates with controlled biochemical functionality. We present the fabrication and characterization of surfaces presenting fibronectin nanopatterns of controlled size and pitch that provide well‐defined cellular adhesion sites against a nonadhesive polyethylene glycol background. The nanopatterned surfaces allow us to control the number of fibronectin proteins within each adhesion site from 9 to 250, thereby limiting the number of integrins involved in each cell–substrate adhesion. We demonstrate the presence of fibronectin on the nanoislands, while no protein was observed on the passivated background. We show that the cell adheres to the nanopatterns with adhesions that are much smaller and more evenly distributed than on a glass control. The nanopattern influences cellular proliferation only at longer times, but influences spreading at both early and later times, indicating adhesion size and adhesion density play a role in controlling cell adhesion and signaling. However, the overall density of fibronectin on all patterns is far lower than on homogeneously coated control surfaces, showing that the local density of adhesion ligands, not the average density, is the important parameter for cell proliferation and spreading. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 87A: 176–195, 2008
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Normal cellular growth control reflects a carefully orchestrated series of signal transduction events that culminate in changes in gene expression. The proliferative response, in multicellular organisms, is initiated by environmental cues, contributed largely by growth factors, and adhesive influences, provided by the extracellular matrix (ECM). The integrin family of heterodimeric receptors mediates adhesion of cells to the ECM. Engagement of integrin receptors with extracellular ligands gives rise to the formation of complex multiprotein structures, termed focal adhesions, which link the ECM to the cytoplasmic actin cytoskeleton. In addition to providing a structural link between the cell and its underlying matrix, focal adhesions contain protein tyrosine kinases, which become activated as a result of cell interaction with the substrate and initiate adhesion-dependent signal transduction cascades culminating in changes in gene expression. In this review, I will consider the role of integrin-mediated signal transduction in regulating genetic changes necessary for controlled cellular proliferation.
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To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin.Adhesion and migration of cultured SMCs were stimulated by different concentrations of fibronectin (FN), FAK and its phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. SMCs adhesion and migration were also measured by morphological enumeration and modified Boyden Chambers, respectively.FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 microg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5 - 60 microg/ml FN groups was reduced by 17.89% - 27.67%. Cell migration stimulated by FN at 10, 20, 40 and 60 microg/ml were reduced by 23.26%, 21.63%, 19.31% and 17.88%, respectively (P < 0.05).FAK phosphorylation and FAK-mediated signal transduction play important roles in SMCs adhesion and migration stimulated by ECM. The process can be inhibited effectively by FAK antisense ODNs.
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Abstract Cell adhesion to extracellular matrix (ECM) components through cell‐surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non‐adhesive surfaces presenting well‐defined, independent densities of two integrin‐specific engineered ligands for the type I collagen (COL‐I) receptor α 2 β 1 and the fibronectin (FN) receptor α 5 β 1 to evaluate the effects of integrin cross‐talk on adhesive responses. Engineered surfaces displayed ligand density‐dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL‐I ligand surfaces. Moreover, surfaces presenting mixed COL‐I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling. J. Cell. Physiol. 217: 450–458, 2008. © 2008 Wiley‐Liss, Inc.
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Cells mechanical behaviour in physiological environments is mediated by interactions with the extracellular matrix (ECM). In particular, cells can adapt their shape according to the availability of ECM proteins, e.g., fibronectin (FN). Several in vitro experiments usually simulate the ECM by functionalizing the surfaces on which cells grow with FN. However, the mechanisms underlying cell spreading on non-uniformly FN-coated two-dimensional substrates are not clarified yet. In this work, we studied cell spreading on variously functionalized substrates: FN was either uniformly distributed or selectively patterned on flat surfaces, to show that A549, BRL, B16 and NIH 3T3 cell lines are able to sense the overall FN binding sites independently of their spatial arrangement. Instead, only the total amount of available FN influences cells spreading area, which positively correlates to the FN density. Immunocytochemical analysis showed that β1 integrin subunits are mainly responsible for this behaviour, as further confirmed by spreading experiments with β1-deficient cells. In the latter case, indeed, cells areas do not show a dependency on the amount of available FN on the substrates. Therefore, we envision for β1 a predominant role in cells for sensing the number of ECM ligands with respect to other focal adhesion proteins.
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Fibronectins
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Fibroblasts adhesion, spreading, and proliferation was investigated in this study using glass and octadecyl glass (ODS) as models for hydrophobic substrata in the absence or presence of preadsorbed fibronectin (FN). To learn more about the underlying mechanism of the biocompatibility of materials, the organization of the β1, integrin and the phosphorylation of tyrosine residues in focal contacts was investigated by immunofluorescence microscopy. The diminished adhesion and spreading of fibroblasts on hydrophobic ODS in comparison to clean glass was indicated by a diffuse presence of actin and by the absence of focal contacts and phosphotyrosine activity, In contrast, on hydrophilic glass, initial stress fibres and focal adhesions appeared accompanied by a moderate phosphotyrosine activity. The preadsorption of FN improved the interaction of fibroblast with both surfaces as indicated by the formation of prominent actin stress fibres and the clusterization of β1 integrins in the focal contacts which was co-localized with an increased phosphotyrosine activity. The proliferation of fibroblasts measured after 72 h was inhibited on ODS in comparison to glass. Preadsorption of FN, however, increased the cell proliferation index on both surfaces, whch was higher than on pure glass. The improved cell adhesion, spreading, and proliferation of fibroblasts run in parallel with an increased total tyrosine phosphorylation activity measured by an enzyme immuno assay (EIA). It was concluded that the signalling via integrins might be a decisive event during the cell-biomaterial interaction.
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