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PubMed2000

Association of beta 1 integrin with focal adhesion kinase and paxillin in differentiating Schwann cells.

Schwann cells (SCs) differentiate into a myelinating cell when simultaneously adhering to an axon destined for myelination and basal lamina. We are interested in defining the signaling pathway activated by basal lamina. Using SC/sensory neuron (N) cocultures, we identified beta1 integrin and F-actin as components of a pathway leading to myelin gene expression and myelination (Fernandez-Valle et al., 1994, 1997). Here, we show that focal adhesion kinase (FAK) and paxillin are constitutively expressed by SCs contacting axons in the absence of basal lamina. Tyrosine phosphorylation of FAK and paxillin increases as SCs form basal lamina and differentiate. FAK and paxillin specifically coimmunoprecipitate with beta1 integrin in differentiating SC/N cocultures but not SC-only cultures. Paxillin coimmunoprecipitates with FAK and fyn kinase in differentiating SC/N cocultures. A subset of tyrosine-phosphorylated beta1 integrin, FAK, and paxillin molecules reside in the insoluble, F-actin-rich fraction of differentiating cocultures. Cytochalasin D, an actin depolymerizing agent, decreases tyrosine phosphorylation of FAK and paxillin and their association with beta1 integrin and causes a dose-dependent increase in the abundance of insoluble FAK and paxillin complexes. Collectively, our work indicates that beta1 integrin, FAK, paxillin, and fyn kinase form an actin-associated complex in SCs adhering to basal lamina in the presence of axons. This complex may be important for initiating the process of SC differentiation into a myelinating cell.

Animals↗

PubMed1999

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling.

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.

Caveolin 1↗

PubMed1997

Relationships between phosphatidylcholine-specific phospholipase C and integrins in cell-substratum adhesion and apoptosis in vascular endothelial cells.

In order to understand the mechanism by which VEC control cell-substratum adhesion and apoptosis, we investigated relationships between PC-PLC and the integrins that are normally expressed in VEC. We found that promotion of cell-substratum adhesion by suppression of PC-PLC was almost completely blocked by a monoclonal antibody (mAb) against integrin beta1, and was partially blocked by a mAb against intergrin beta3. The production of diacylglycerol (DAG) which was inhibited by suppression of PC-PLC activity, was increased by mAbs against intergrin beta1 and beta3. When the mAb against integrin beta4 was added to the seeding medium, cell-substratum adhesion and spreading of cells were triggered, but the activity of PC-PLC was unaffected by this mAb. Furthermore, when both the mAb against integrin beta4 and a specific inhibitor (D609) of PC-PLC were present in the seeding medium, cell-substratum adhesion and spreading were promoted to a greater extent than when either of these agents was present alone. These data suggest that integrins beta1 and beta3 might regulate cell-substratum adhesion and apoptosis via a PC-PLC-dependent pathway, while integrin beta4 might regulate these phenomena via PC-PLC-independent pathway. These findings provide the first evidence of relationships between PC-PLC and integrins in cell-substratum adhesion and apoptosis.

Antibodies, Monoclonal↗

PubMed2004

The major outer sheath protein of Treponema denticola inhibits the binding step of collagen phagocytosis in fibroblasts.

Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.

Actins↗

PubMed2003

Src kinase has a central role in in vitro cellular internalization of Staphylococcus aureus.

Traditionally recognized as an extracellular pathogen, the Gram-positive bacterium Staphylococcus aureus can also be internalized by a variety of cell types in vitro. Internalization is known to involve binding of the host extracellular protein fibronectin to the bacterium, recognition of the fibronectin-coated bacterium by the fibronectin-binding integrin alpha5beta1 on the host cell surface, and integrin-mediated internalization. Here we examine elements of mammalian cell signalling pathways involved in S. aureus internalization. The mouse fibroblast cell line GD25, in which the gene encoding the beta1 integrin subunit is inactivated, has been complemented with a beta1 integrin cDNA encoding a tyrosine (Y) to phenylalanine (F) mutation in each of the two beta1 integrin intracellular NPXY motifs. This cell line, GD25beta1 A Y783/795F, is defective in migration on fibronectin coated surfaces and intracellular signalling activities involving the tyrosine kinase Src. GD25beta1 A Y783/795F cells have a decreased ability to internalize S. aureus compared to GD25beta1 A cells expressing wild-type beta1 integrins. Furthermore, using mouse embryo fibroblasts in which different members of the Src family kinases are genetically inactivated, we demonstrate that optimal internalization is dependent on expression of Src kinase. Interferon, which has been implicated in repression of the effects of the viral homologue of Src inhibits internalization of S. aureus indicating that internalization may be blocked by inhibitors of Src kinase function. We then demonstrate that Src family kinase specific inhibitors effectively block S. aureus internalization into HeLa cells leading to the conclusion that a function unique to Src is required for optimal internalization of S. aureus in vitro.

Animals↗

PubMed2004

Integrin-dependent protein tyrosine phosphorylation is a key regulatory event in collagen-IV-mediated adhesion and proliferation of human lung tumor cell line, Calu-1.

BACKGROUND: The clinical phenomenon of lung cancer metastasis to specific target organs is believed to be a direct interaction between tumor cells and extracellular matrix components. During invasion, tumor cells attach to the basement membrane protein, collagen type IV, degrade it, migrate through blood vessels, and adhere to extracellular matrix proteins. METHODS: Four nonsmall-cell lung cancer cells were tested for adhesion, proliferation, migration and morphologic alterations on collagen type IV matrix by immunoprecipitation, Western blotting, phase contrast and time lapse video microscopy. RESULTS: Collagen type IV promoted Calu-1 cell adhesion (< 15 minutes) and motility (< 6 hours) without any significant effect on proliferation. beta(1)-integrin is essential for collagen type IV adhesion and 8 to 10 fold higher expression of beta1-integrin on the surface of Calu-1 cells was identified. A protein tyrosine phosphatase inhibitor, peroxyvanadate, caused 50% inhibition in the adhesion process within 5 minutes but exposure to 60 micromol/L genistein for 72 hours, a protein tyrosine kinase inhibitor, drastically inhibits Calu-1 cell proliferation (> 70%). We examined the influence of beta1-integrin, peroxyvanadate and genistein on the spreading morphogenesis of Calu-1 cells on Collagen type IV. Use of either 1 microg of anti beta1-integrin inhibitory antibody (P5D2), 100 micromol/L peroxyvanadate or 60 micromol/L genistein had dramatic influence on the spreading of Calu-1 cells. Finally, Focal adhesion kinase was identified as a phosphoprotein target. CONCLUSIONS: We have characterized an in vitro model of matrix-specific binding of a lung cancer cell line, Calu-1 to Coll IV. Calu-1 cells use primarily a beta1-integrin mediated intracellular tyrosine phosphorylation phenomenon as the key regulatory mechanism for its binding advantage to Coll IV matrix.

Carcinoma, Non-Small-Cell Lung↗

PubMed1998

The effect of interferon-alpha on beta-1 integrin mediated adhesion and growth regulation in chronic myelogenous leukemia.

There is considerable interest in understanding the mechanisms by which interferon-alpha can induce hematological and cytogenetic remissions and prolong survival in patients with chronic myelogenous leukemia (CML). There is evidence that the selective expansion and growth advantage of malignant hematopoietic progenitors in CML may be related, at least in part, to their deficient responsiveness to normal negative regulation by the marrow stromal microenvironment and that interferon-alpha may restore normal hematopoiesis in CML by restoring normal microenvironmental regulation of malignant CML progenitor proliferation. In normal hematopoiesis, beta1 integrin receptors mediate progenitor adhesion to stroma. Stimulation of beta1 integrin receptors on normal progenitors results in transmission of proliferation inhibitory signals. CML progenitors demonstrate defective beta1 integrin receptor-mediated adhesion and regulation of proliferation. We have shown that interferon-alpha can restore both beta1 integrin mediated adhesion as well as integrin-mediated proliferation inhibition of CML progenitors. Interferon-alpha enhances CML integrin function through direct effects on CML progenitors as well as indirect effects on stroma. Restored beta1 integrin-mediated regulation of malignant CML progenitor proliferation may result in restoration of normal hematopoiesis in CML patients treated with interferon-alpha. There is evidence that abnormal integrin mediated signaling in CML progenitors may result from abnormalities in integrin-cytoskeletal interactions induced by the p210BCR/ABL tyrosine kinase. Although the exact mechanisms by which interferon-alpha restores normal integrin signaling in CML are not known, preliminary studies indicate that this may at least partly be related to the restoration of normal integrin-cytoskeletal interactions. The above studies offer some insights into the mechanisms of abnormal hematopoietic regulation in CML and the mechanisms by which interferon-alpha may restore normal hematopoiesis, in this disease.

Antineoplastic Agents↗

PubMed2001

Alphav integrin expression is a novel marker of poor prognosis in advanced-stage ovarian carcinoma.

PURPOSE: To analyze the possible correlation between expression of the alphav and beta1 integrin chains and survival in advanced-stage ovarian carcinomas, studying two patient groups with extremely different disease outcome. EXPERIMENTAL DESIGN: Sections from 56 primary ovarian carcinomas and metastatic lesions from 34 patients diagnosed with advanced-stage ovarian carcinoma (Fédération Internationale des Gynaecologistes et Obstetristes stages III-IV), divided into long-term (16) and short-term (18) survivors, were evaluated for expression of alphav and beta1 integrin chains using mRNA in situ hybridization. Protein expression was additionally studied in 52 specimens using immunohistochemistry. RESULTS: The mean values for disease-free survival and overall survival were 115 and 132 months for long-term survivors, as compared with 4 and 23 months for short-term survivors, respectively. Expression of alphav integrin mRNA was observed in carcinoma (18 of 56; 32%) and stromal (17 of 56; 30%) cells. beta1 integrin mRNA was similarly detected in carcinoma (25 of 56; 47%) and stromal (19 of 56; 34%) cells. No significant differences were observed when primary and metastatic lesions were compared (P > 0.05). Alphav integrin mRNA was present more often in carcinoma cells in tumors of short-term survivors (P = 0.017 for carcinoma cells). In univariate survival analysis for all cases, alphav integrin mRNA expression in tumor cells correlated with poor survival (P = 0.012). This finding retained its predictive power in a multivariate survival analysis, in which all of the molecules studied previously in this patient cohort were included (P = 0.031). Immunohistochemistry confirmed the differences in alphav integrin expression in tumor cells of short-term as compared with long-term survivors, whereas beta1 integrin protein expression was comparable in the two groups. CONCLUSIONS: To our best knowledge, this is the first evidence associating integrin expression with poor survival in ovarian carcinoma. Alphav integrin is, thus, a novel prognostic marker in advanced-stage ovarian carcinoma.

Adult↗

PubMed2000

Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan.

N-cadherin and beta1-integrins play decisive roles in morphogenesis and neurite extension and are often present on the same cell. Therefore, the function of these two types of adhesion systems must be coordinated in time and space to achieve the appropriate cell and tissue organization. We now show that interaction of the chondroitin sulfate proteoglycan neurocan with its GalNAcPTase receptor coordinately inhibits both N-cadherin- and beta1-integrin-mediated adhesion and neurite outgrowth. Furthermore, the inhibitory activity is localized to an NH(2)-terminal fragment of neurocan containing an Ig loop and an HA-binding domain. The effect of neurocan on beta1-integrin function is dependent on a signal originating from the cadherin cytoplasmic domain, possibly mediated by the nonreceptor protein tyrosine kinase Fer, indicating that cadherin and integrin engage in direct cross-talk. In the developing chick, neural retina neurocan is present in the inner plexiform layer from day 7 on, and the GalNAcPTase receptor becomes restricted to the inner nuclear layer and the ganglion cell layer (as well as the fiber layer), the two forming a sandwich. These data suggest that the coordinate inhibition of cadherin and integrin function on interaction of neurocan with its receptor may prevent cell and neurite migration across boundaries.

Amino Acid Sequence↗

PubMed2002

Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner.

Motility of nerve growth cones (GCs) is regulated by region-specific activities of cell adhesion molecules (CAMs). CAM activities could be modified by their localization to detergent-resistant membranes (DRMs), specialized microdomains enriched in signaling molecules. This paper deals with a question of whether DRMs are involved in GC migration stimulated by three CAMs; L1, N-cadherin (Ncad), and beta1 integrin. We demonstrate that L1 and Ncad are present in DRMs, whereas beta1 integrin is exclusively detected in non-DRMs of neurons and that localization of L1 and Ncad to DRMs is developmentally regulated. GC migration mediated by L1 and Ncad but not by beta1 integrin is inhibited after DRM disruption by micro-scale chromophore-assisted laser inactivation (micro-CALI) of GM1 gangliosides or by pharmacological treatments that deplete cellular cholesterol or sphingolipids, essential components for DRMs. Characteristic morphology of GCs induced by L1 and Ncad is also affected by micro-CALI-mediated DRM disruption. Micro-CALI within the peripheral domain of GCs, or even within smaller areas such as the filopodia and the lamellipodia, is sufficient to impair their migration. However, micro-CALI within the central domain does not affect GC migration. These results demonstrate the region-specific involvement of DRMs in CAM-dependent GC behavior.

3T3 Cells↗

PubMed2003

Coordinated expression of integrin subunits, matrix metalloproteinases (MMP), angiogenic genes and Ets transcription factors in advanced-stage ovarian carcinoma: a possible activation pathway?

The events that mediate tumor progression in ovarian carcinoma are poorly understood to date. This review summarizes our results studying metastasis-associated molecules in advanced-stage ovarian carcinomas, details the co-expression of mRNA of these genes, and discusses their prognostic role. Fifty-five primary and metastatic FIGO stage III-IV ovarian carcinomas were analyzed for the expression of alpha v and beta1 integrin subunits, the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP, the MMP inhibitor TIMP-2, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), PEA3 and Ets-1 using mRNA in situ hybridization. Tumor and adjacent stromal cell expression was scored. The association between integrin subunit expression and the expression of MMP, TIMP-2, angiogenic genes, PEA3 and Ets-1 was statistically analyzed. Alpha v integrin subunit mRNA expression in carcinoma cells showed significant association with that of MMP-2 and IL-8 in this cellular compartment, while the presence of beta1 integrin subunit mRNA showed similar association with that of PEA3, Ets-1, IL-8, bFGF and MMP-2. Expression of beta1 integrin subunit mRNA in stromal cells was associated with that of TIMP-2 and Ets-1 in this compartment. In addition, significant intercellular associations were found between alpha v integrin subunit mRNA expression in carcinoma cells and stromal cell expression of Ets-1, as well as between stromal cell expression of alpha v integrin subunit and labeling for IL-8 in carcinoma cells. The presence of beta1 integrin subunit mRNA in carcinoma cells showed a significant association with that of Ets-1, IL-8 and bFGF in stromal cells, while the presence of beta1 integrin subunit mRNA in stromal cells was associated with tumor PEA3 mRNA expression. To the best of our knowledge, this is the first evidence for coordinated autocrine and paracrine expression of members of these four families of metastasis-associated genes in human cancer. The results of this analysis support experimental data regarding cross-talk between carcinoma cells and peritumoral fibroblasts. They also suggest the existence of a putative activation sequence of metastatic genes, involving the beta1 (and possibly alpha v) integrin subunits, IL-8, PEA3, Ets-1 and MMP in ovarian carcinoma.

Adenocarcinoma, Mucinous↗

PubMed2000

Expression of integrin subunit beta1B in integrin beta1-deficient GD25 cells does not interfere with alphaVbeta3 functions.

We have expressed the beta1B integrin subunit in beta1-deficient GD25 cells to examine beta1B functions without the interference of endogenous beta1A expression. As previously reported [Retta et al., 1998, Mol. Biol. Cell 9, 715-731], the beta1B integrins did not mediate cell adhesion under normal culture conditions, while the presence of 0.3 mM Mn(2+) allowed beta1B integrins to support adhesion. Mn(2+), as well as the small soluble peptide GRGDS, induced a beta1B conformation, which was recognized by the mAb 9EG7, a marker for active or ligand-bound integrins. beta1B integrins were found to localize to a subset of focal contacts in a ligand-independent manner on fibronectin, but not on vitronectin. However, clustering of beta1B did not induce tyrosine phosphorylation of FAK, p130(Cas), or paxillin, as studied by beta1B-mediated adhesion, to fibronectin in the presence of Mn(2+) or to anti-beta1 antibody in DMEM. Induction of ligand-occupied conformation by the GRGDS peptide during the adhesion to anti-beta1 antibody also failed to trigger FAK phosphorylation. Stimulation of tyrosine phosphorylation on FAK, p130(Cas), and paxillin by adhesion via integrin alphaVbeta3 to fibronectin or vitronectin was not disturbed in GD25-beta1B cells compared to the untransfected GD25 cells, nor were any negative effects of beta1B observed on alphaVbeta3-mediated cell attachment, spreading, and actin organization, or on the cell proliferation rate. These results show that the reported negative effects of beta1B on adhesive events do not apply to alphaVbeta3-dependent interactions and suggest that they may specifically act on beta1 integrins.

Actins↗

PubMed2001

PTP1B regulates neurite extension mediated by cell-cell and cell-matrix adhesion molecules.

N-cadherin and beta1-integrin adhesion and signaling play important roles in growth cone adhesion and guidance. Each of these adhesion receptor systems is composed of multiprotein complexes, and both adhesion and downstream signaling events are regulated through the interaction of protein tyrosine kinases and phosphatases with many of the proteins that make up these complex systems. Work from our laboratory reported that the nonreceptor protein tyrosine phosphatase PTP1B is localized to adherens junctions and focal adhesion complexes and regulates both N-cadherin- and beta1-integrin-mediated adhesion. PTP1B appears to modulate integrin-mediated adhesion through regulation of src activation and cadherin-mediated adhesion through dephosphorylation of beta-catenin. We have continued these studies and report that PTP1B is localized to the tips of growing neurites and that introduction of a noncatalytic mutant of PTP1B into PC12 cells results in inhibition of N-cadherin- and beta1-integrin-mediated neurite outgrowth but is without effect on neurite outgrowth on poly-L-lysine. Moreover, suppressing the level of PTP1B in primary embryonic chick neural retina cells using antisense oligonucleotides also inhibits N-cadherin- and beta1-integrin-mediated neurite outgrowth. Neither of these techniques reduces the levels of expression of either adhesion receptor. We conclude that PTP1B is a regulatory component of the molecular complex required for both N-cadherin and beta1-integrin-mediated axon growth.

Animals↗

PubMed1998

Identification of the ligand binding site for the integrin alpha9 beta1 in the third fibronectin type III repeat of tenascin-C.

The integrin alpha9 subunit forms a single heterodimer, alpha9 beta1 that mediates cell adhesion to a site within the third fibronectin type III repeat of tenascin-C (TNfn3). In contrast to at least 3 other integrins that bind to this region of tenascin-C, alpha9 beta1 does not recognize the common integrin recognition motif, Arg-Gly-Asp (RGD). In this report, we have used substitution mutagenesis to identify a unique ligand recognition sequence in TNfn3. We introduced mutations substituting alanine for each of the acidic residues in or adjacent to each of the exposed loops predicted from the solved crystal structure. Most of these mutations had little or no effect on adhesion of alpha9-transfected SW480 colon carcinoma cells, but mutations of either of two acidic residues in the B-C loop region markedly reduced attachment of these cells. In contrast, cells expressing the integrin alphav beta3, previously reported to bind to the RGD sequence in the adjacent F-G loop, attached to all mutant fragments except one in which the RGD site was mutated to RAA. The peptide, AEIDGIEL, based on the sequence of human tenascin-C in this region blocked the binding of alpha9-transfected cells, but not beta3-transfected cells to wild type TNfn3. This sequence contains a tripeptide, IDG, homologous to the sequences LDV, IDA, and LDA in fibronectin and IDS in VCAM-1 recognized by the closely related integrin alpha4 beta1. These findings support the idea that this tripeptide motif serves as a ligand binding site for the alpha4/alpha9 subfamily of integrins.

Amino Acid Sequence↗

PubMed2006

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein.

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin beta1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin beta1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin beta1, and consequently reversed TIMP-1-mediated integrin beta1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.

Antigens, CD↗

PubMed1998

Cell migration strategies in 3-D extracellular matrix: differences in morphology, cell matrix interactions, and integrin function.

Cell migration in extracellular matrix is a complex process of adhesion and deadhesion events combined with cellular strategies to overcome the biophysical resistance imposed by three-dimensionally interconnected matrix ligands. Using a 3-D collagen matrix migration model in combination with computer-assisted cell tracking for reconstruction of migration paths and confocal microscopy, we investigated molecular principles governing cell-matrix interactions and migration of different cell types. Highly invasive MV3 melanoma cells and fibroblasts are large and highly polarized cells migrating at low speed (0.1-0.5 microm/min) and at high directional persistence. MV3 melanoma cells utilize adhesive migration strategies as characterized by high beta1 integrin surface expression, beta1 integrin clustering at interactions with matrix fibers, and beta1 integrin-mediated adhesion for force generation and migration. In contrast, T lymphocytes and dendritic cells are highly mobile cells of lower beta1 integrin expression migrating at 10- to 40-fold higher velocities, and directionally unpredictable path profiles. This migration occurs in the absence of focal adhesions and largely independent of beta1 integrin-mediated adhesion. Whereas cell-matrix interactions of migrating tumor cells result in traction and reorientation of collagen fibers, partial matrix degradation, and pore formation, leukocytes form transient and short-lived interactions with the collagen lacking structural proteolysis and matrix remodeling. In conclusion, the 3-D extracellular matrix provides a spatially complex and biomechanically demanding substrate for cell migration, thereby differing from cell migration across planar ligands. Highly adhesive and integrin-dependent migration strategies detected in morphologically large and slowly migrating cells may result in reorganization of the extracellular matrix, whereas leukocytes favor largely integrin-independent, rapid, and flexible migration strategies lacking typical focal adhesions and structural matrix remodeling.

Cell Adhesion↗

PubMed2004

Renal ischemia/reperfusion injury: functional tissue preservation by anti-activated {beta}1 integrin therapy.

Renal ischemia/reperfusion injury (IRI) is an important cause of acute renal failure. Cellular and molecular responses of the kidney to IRI are complex and not fully understood. beta1 integrins localize to the basal surface of tubular epithelium interacting with extracellular matrix components of the basal membrane, including collagen IV. Whether preservation of tubular epithelium integrity could be a therapeutic approach for IRI was assessed. The effects of HUTS-21 mAb administration, which recognizes an activation-dependent epitope of beta1 integrins, in a rat model of IRI were investigated. Preischemic HUTS-21 administration resulted in the preservation of renal functional and histopathologic parameters. Analyses of activated beta1 integrins expression and focal adhesion kinase phosphorylation suggest that its deactivation after IRI was prevented by HUTS-21 treatment. Moreover, HUTS-21 impaired the inflammatory response in vivo, as indicated by inhibition of proinflammatory mediators and the absence of infiltrating cells. Ex vivo adhesion assays using reperfused kidneys revealed that HUTS-21 induced a significant increase of epithelial cell attachment to collagen IV. In conclusion, the data provide evidence that HUTS-21 has a protective effect in renal IRI, preventing tubular epithelial cell detachment by preserving activated beta1 integrins functions.

Acute Kidney Injury↗

PubMed2000

The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation.

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit beta1 (Y783 and Y795) to phenylalanines markedly reduces the capability of beta1A integrins to mediate directed cell migration. In this study, beta1-dependent cell spreading was found to be delayed in GD25 cells expressing beta1A(Y783/795F) compared to that in wild-type GD25-beta1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to beta1-dependent adhesion in GD25-beta1A(Y783/795F) cells compared to that in wild-type GD25-beta1A or mutants in which only a single tyrosine was altered (beta1A(Y783F) or beta1A(Y795F)). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via beta1A(Y783/795F) lies at the level of the initial autophosphorylation step. Indeed, beta1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the beta1A(Y783/795F) cells, consistent with the impairment in FAK activation. In contrast, p130(CAS) overall tyrosine phosphorylation was unaffected by the beta1 mutations. Despite the defect in beta1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-beta1A(Y783/795F) cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit beta1A are critical mediators of FAK activation and cell spreading in GD25 cells.

Amino Acid Substitution↗