Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Differentiation of skeletal tissues, such as bone, ligament and cartilage, is regulated by complex interaction between genetic and epigenetic factors. In the present study, we attempted to elucidate the possible role of cell-extracellular matrix (ECM) adhesion on the inhibitory regulation in chondrogenesis responding to the tension force. The midpalatal suture cartilages in rats were expanded by orthopedic force. In situ hybridization for type I and II collagens, immunohistochemical analysis for fibronectin, alpha5 and beta1 integrins, paxillin, and vinculin, and cytochemical staining for actin were used to demonstrate the phenotypic change of chondrocytes. Immunohistochemical analysis for phosphorylation and nuclear translocation of extracellular signal-regulated kinase (ERK)-1/2 was performed. The role of the cell-ECM adhesion in the response of the chondroprogenitor cells to mechanical stress and the regulation of gene expression of focal adhesion kinase (FAK) and integrins were analyzed by using an in vitro system. A fibrous suture tissue replaced the midpalatal suture cartilage by the expansive force application for 14 days. The active osteoblasts that line the surface of bone matrix in the newly formed suture tissue strongly expressed the type I collagen gene, whereas they did not express the type II collagen gene. Although the numbers of precartilaginous cells expressing alpha5 and beta1 integrin increased, the immunoreactivity of alpha5 integrin in each cell was maintained at the same level throughout the experimental period. During the early response of midpalatal suture cartilage cells to expansive stimulation, formation of stress fibers, reorganization of focal adhesion contacts immunoreactive to a vinculin-specific antibody, and phosphorylation and nuclear translocation of ERK-1/2 were observed. In vitro experiments were in agreement with the results from the in vivo study, i.e. the inhibited expression of type II collagen and upregulation in integrin expression. The arginine-glycine-aspartic acid-containing peptide completely rescued chondrogenesis from tension-mediated inhibition. Thus, we conclude that stretching activates gene expression of beta1 integrin and FAK and inhibits chondrogenesis through cell-ECM interactions of chondroprogenitor cells.
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PMID:Effect of stretching on gene expression of beta1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions. 1275 4

Integrin-mediated signalling has been implicated in asbestos-induced carcinogenesis. In studies here, we examined signal transduction events associated with integrin-directed cell reactions triggered by crocidolite asbestos in the pleural mesothelial cell line 4/4 RM-4. Crocidolite fibres induced a significant time- and dose-dependent activation of the extracellular-signal-regulated kinases ERK1 and ERK2. ERK activation was specifically inhibited by integrin-blocking agents, that are integrin-binding peptides containing the sequence arginine-glycine-aspartic acid (RGD), and monoclonal antibodies against the integrin beta1-chain. Integrin-dependent activation of ERK1/2 in response to asbestos appeared to be independent of focal adhesion kinase pp125FAK (FAK) since FAK autophosphorylation remained unaffected in crocidolite-exposed mesothelial cells. Instead, we observed striking similarities in the kinetics of asbestos-induced ERK1/2 responses and phosphorylation of protein kinase B (AKT) at serine 473, a possible target residue for integrin-linked kinase. As with ERK activation, asbestos-induced AKT stimulation was significantly blocked by both the RGD-peptide and the beta1-integrin antibodies. These studies are the first to establish that in mesothelial cells ERK1/2 and AKT are simultaneously phosphorylated upon asbestos exposure in a beta1-integrin-dependent manner.
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PMID:beta1-integrin mediates asbestos-induced phosphorylation of AKT and ERK1/2 in a rat pleural mesothelial cell line. 1462 93

Resistance to anoikis, or apoptosis triggered by detachment from the extracellular matrix (ECM), lengthens the survival of malignant cells, facilitating reattachment and colonization of secondary sites. To examine the molecular mechanisms underlying resistance to anoikis in human oral squamous cell carcinoma (SCC) cells, we cultured human squamous carcinoma (HSC-3) cells in suspension on plates coated with poly-2-hydroxyethyl methacrylate, which blocks access to the ECM. Cells in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. Aggregates, but not single cells, had high levels of fibronectin. Preincubation with a cyclic arginine-glycine-aspartic acid peptide or fibronectin-blocking antibody significantly increased anoikis. Single cells had markedly lower expression of the integrin alpha(v) receptor than aggregates. Blocking alpha(v) function with a blocking antibody or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, phosphorylation of the integrin-associated focal adhesion kinase (FAK) at tyrosine 397 was reduced, and p53 levels were increased. Apoptosis was increased by blocking FAK with an antisense oligonucleotide and reduced by blocking p53. These findings show that SCC cells escape suspension-induced anoikis by forming multicellular aggregates that avail themselves of fibronectin survival signals mediated by integrin alpha(v). Single cells in suspension that do not form aggregates undergo anoikis because of decreased FAK phosphorylation and increased p53 levels. Thus, SCC cells appear to use neighboring cells and the ECM molecule FN to promote the metastatic phenotype.
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PMID:Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis: fibronectin and integrin alphav mediate survival signals through focal adhesion kinase. 1533 8

The functional receptor for the flavivirus West Nile (WNV) infection has been characterized in this study with a combination of biochemical and molecular approaches. A 105-kDa protease-sensitive glycoprotein that binds WNV was isolated from the plasma membrane of cells permissive to WNV infection. The protein was subjected to peptide sequencing, and this glycoprotein was identified as a member of the integrin superfamily. Infection of WNV was shown to be markedly inhibited in Vero cells pretreated with blocking antibodies against alpha(v)beta(3) integrin and its subunits by receptor competition assay. It was also noted that cells pretreated with antibodies against alpha(v)beta(3) integrin can effectively inhibit flavivirus Japanese encephalitis but to a lesser extent flavivirus dengue infections. West Nile virus entry is independent of divalent cations and is not highly blocked by arginine-glycine-aspartic acid (RGD) peptides, suggesting that the interaction between the virus and alpha(v)beta(3) integrin is not highly dependent on the classical RGD binding motif. In addition, gene silencing of the beta(3) integrin subunit in cells has resulted in cells largely resistant to WNV infection. In contrast, expression of recombinant human beta(3) integrin substantially increased the permissiveness of CS-1 melanoma cells for WNV infection. Soluble alpha(v)beta(3) integrin can also effectively block WNV infection in a dose-dependent manner. Furthermore, WNV infection also triggered the outside-in signaling pathway via the activation of integrin-associated focal adhesion kinase. The identification of alpha(v)beta(3) integrin as a receptor for WNV provides insight into virus-receptor interaction, hence creating opportunities in the development of anti-viral strategies against WNV infection.
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PMID:Interaction of West Nile virus with alpha v beta 3 integrin mediates virus entry into cells. 1547 43

Adipogenesis plays a central role in obesity development. The processes of adipogenesis include migration, adhesion, proliferation and survival of preadipocytes and differentiation to mature adipocytes. Many of these biological functions are related to integrins. Here, we found that snake venom-derived arginine-glycine-aspartic acid (RGD)-containing disintegrin inhibited adipogenesis. Rhodostomin but not rhodostomin RGD mutants (RGE-Rn and AKGDWN-Rn) caused the detachment of primary cultured preadipocyte. Furthermore, rhodostomin also inhibited focal adhesion of preadipocyte, including the inhibition of the expression of focal adhesion kinase (FAK) and FAK phosphorylation, assembly of vinculin and reorganization of actin cytoskeleton. Cell viability of preadipocytes was decreased after rhodostomin treatment in a concentration-dependent manner. The results of flow cytometric analysis showed that rhodostomin induced cell apoptosis. In addition, chromatin condensation was observed in DAPI staining. The increase of Bax expression and activation of capsase-3 was detected following rhodostomin treatment. Addition of dexamethasone, IBMX and insulin induced differentiation of preadipocytes into mature adipocytes and treatment of cells with rhodostomin during the initial 3 days showed less mature adipocytes following 9-10 days of differentiating period. The triglyceride content and gene expression of peroxisome proliferators-activated receptor gamma (PPARgamma) and leptin also decreased in response to the treatment of rhodostomin. These results suggest that disintegrin inhibits processes of adipogenesis and may be developed to treat obesity.
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PMID:Inhibition of adipogenesis by RGD-dependent disintegrin. 1619 20

Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine-glycine-aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7-10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD-PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7-10-functionalized surfaces displayed specificity for alpha5beta1 integrin, while cell adhesion to supports presenting RGD or RGD-PHSRN was primarily mediated by alphavbeta3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.
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PMID:Integrin specificity and enhanced cellular activities associated with surfaces presenting a recombinant fibronectin fragment compared to RGD supports. 1684 40

Multiple stimuli promote the tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which ultimately facilitates migration. Little is known about the effect of adhesion-dependent signals and cytoskeleton organization on the regulation of FAK phosphorylation at serine sites, or about the role of FAK serine phosphorylation in cell migration. Here, we show that FAK phosphorylation at Ser-843 is strikingly increased when adherent cells are removed from the substratum and held in suspension or by treatment of adherent cells with cytochalasin D, conditions that disrupt the F-actin cytoskeleton and promote focal adhesion disassembly. Notably, the increase in Ser-843 phosphorylation was accompanied by a concomitant sharp decrease in Tyr-397 phosphorylation. To further examine the cause-effect relationship between these two phosphorylation sites we generated Ser-843 phosphorylation-deficient and phosphorylation-mimicking FAK mutants. We found that mutation of Ser-843 to aspartic acid (FAK[S843D]) markedly decreased FAK Tyr-397 phosphorylation in integrin-stimulated cells. While the migratory defect of FAK-deficient fibroblasts was rescued by stable re-expression of WT FAK or FAK[S843A], stable re-expression of FAK[S843D] failed to restore the ability of the cells to migrate into the denuded area of a wound. Our results indicate that increased FAK phosphorylation at Ser-843 represses FAK phosphorylation at Tyr-397, thus suggesting a mechanism of cross-talk between these phosphorylation sites that could regulate FAK-mediated cell shape and migration.
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PMID:FAK phosphorylation at Ser-843 inhibits Tyr-397 phosphorylation, cell spreading and migration. 1709 71

Upon ligand binding the 1alpha,25-dihydroxy Vitamin D3 receptor (VDR) undergoes a conformational change that allows interaction with coactivator proteins including p160/SRC family members and the multimeric DRIP complex through the DRIP205 subunit. Casein kinase II (CKII) phosphorylates VDR both in vitro and in vivo at serine 208 within the hinge domain. This phosphorylation does not affect the ability of VDR to bind DNA, but increases its ability to transactivate target promoters. Here, we have analyzed whether phosphorylation of VDR by CKII modulates the ability of VDR to interact with coactivators in vitro. We find that both mutation of serine 208 to aspartic acid (VDRS208D) or phosphorylation of VDR by CKII enhance the interaction of VDR with DRIP205 in the presence of 1alpha,25-dihydroxy Vitamin D3. We also find that the mutation VDRS208D neither affects the ability of this protein to bind DNA nor to interact with SRC-1 and RXRalpha. Together, our results indicate that phosphorylation of VDR at serine 208 contributes to modulate the affinity of VDR for the DRIP complex and therefore may have a role in vivo regulating VDR-mediated transcriptional enhancement.
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PMID:Phosphorylation at serine 208 of the 1alpha,25-dihydroxy Vitamin D3 receptor modulates the interaction with transcriptional coactivators. 1736 82

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.
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PMID:Identification and characterization of a novel prespheroid 3-dimensional hepatocyte monolayer on galactosylated substratum. 1751 43

The adhesion ligand arginine-glycine-aspartic acid (RGD) has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters key cell behaviors. Previous studies have failed, however, to conclusively decouple the effects of RGD bulk density and individual pattern parameters (i.e. RGDs/island and island distribution) on these altered cell responses. Using a nanopatterned RGD-coupled alginate hydrogel matrix, this work combines computational, statistical and experimental approaches to elucidate the effects of RGD patterns on four key cell responses. This study shows that in MC3T3 preosteoblasts focal adhesion kinase (FAK) Y397 phosphorylation, cell spreading, and osteogenic differentiation can be controlled by RGD nanopatterning, with the distribution of islands throughout the hydrogel (i.e. how closely spaced the islands are) being the most significant pattern parameter. More closely spaced islands favor FAK Y397 phosphorylation and cell spreading, while more widely spaced islands favor differentiation. Proliferation, in contrast, is primarily a function of RGD bulk density. Nanopatterning of cell adhesion ligands has tremendous potential as a simple tool to gain significant control over multiple cell behaviors in engineered extracellular matrix (ECM).
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PMID:Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: a combined computational and experimental approach. 1761 56


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