Different integrins mediate cell spreading,haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (α5β1, αvβ1 and αvβ6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of αvβ6 integrin was strongly and specifically upregulated by transforming growth factor-β1 (TGFβ1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFβ1. Based on antibody blocking experiments, both untreated and TGFβ1-treated HaCaT cells used αvβ6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFβ1-treated cells, the untreated cells also needed α5β1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFβ1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on αvβ6 integrin, while αvβ1 and α5β1 integrins played a lesser role both in untreated and TGFβ1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by β1 integrins, and αvβ6 integrin showed a minor role. The migration process appeared to involve a number of β1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Platelet-derived growth factor and inflammatory cytokines have differential effects on the expression of integrins α1β1 and α5β1 by human dermal fibroblasts in vitro

Dermal fibroblasts are essential for the repair of cutaneous wounds. Fibroblasts presumably use cell surface receptors of the integrin family during migration into a wound from the adjacent uninjured tissue and for the subsequent matrix repairs. We have investigated the possible roles of platelet-derived growth factor and inflammatory cytokines in the regulation of integrin expression on wound fibroblasts using a porcine cutaneous wound model and cultured human cells. Tissue specimens collected from 4-day pig wounds were stained with antibodies specific for the α1 and α5 integrin subunits. Staining for α1 was markedly decreased on fibroblasts adjacent to the wound and in the granulation tissue, while staining for α5 was clearly enhanced in both locations. Normal adult human dermal fibroblasts in culture express the integrins α1β1, a collagen receptor, and α5β1, a fibronectin receptor. Quantitative flow cytometry was used to measure cell surface integrin expression after treatment with platelet-derived growth factor (PDGF)-AA, PDGF-AB, or PDGF-BB. Each isoform of PDGF produced a significant decrease in the level of α1 present on the cell surface and an increase in the level of α5. Furthermore, PDGF-BB produced a corresponding decrease in α1 mRNA and an increase in α5 mRNA. In contrast, treatment with three inflammatory cytokines, IL-1β, TNF-α, and IFN-γ, produced clear increases in the levels of α1 and α5 present on the cell surface. Our observations suggest that the differential effects of PDGF and inflammatory cytokines may be part of the mechanism regulating the expression of α1 and α5 integrins by dermal fibroblasts during wound repair. © 1996 Wiley-Liss, Inc.     

Stem Cell Factor Regulates Human Melanocyte-Matrix Interactions

Stem cell factor (SCF) is hypothesized to play a critical role in the migration of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, c-kit, result in defects in coat pigmentation in mice and in skin pigmentation in humans. In this report we directly show that SCF alters the adhesion and migration of human melanocytes to extracellular matrix (ECM) ligands and regulates integrin expression at the protein level. SCF decreased adhesion of neonatal and fetal cells to collagen IV, and increased attachment of fetal cells to laminin. Attachment of fetal cells to fibronectin was decreased, but was unchanged in neonatal cells. Flow cytometry analysis of neonatal melanocytes showed that SCF down-regulated the expression of the α2 receptor, and up-regulated the expression of the α3, α5 and β1 integrin receptors. SCF down-regulated expression of α2, α5 and β1 integrins by fetal melanocytes, and up-regulated expression of the αv and α3 integrin receptors. Analysis of melanocyte migration using time-lapse videomicroscopy showed that SCF significantly increased migration of neonatal, but not fetal, melanocytes on fibronectin (FN). We conclude that SCF regulates integrin expression at the protein level and that SCF has pleiotropic effects on melanocyte attachment and migration on ECM ligands. We suggest that this may be one mechanism by which SCF regulates melanocyte migration during development of the skin.     

Fibronectin-mediated upregulation of α5β1 integrin and cell adhesion during differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells have a broad potential application in regenerative medicine and can be differentiated into cells of all three germ layers. Adhesion of ES cells to extracellular matrix (ECM) proteins is essential for the differentiation pathway; Cell-ECM adhesion is mediated by integrins that have the ability to activate many intracellular signaling pathways. Therefore, we hypothesize that the expression and function of integrin receptors is a critical step in ES differentiation. Using functional cell adhesion assays, our study demonstrates that α5β1 is a major functional integrin receptor expressed on the cell surface of undifferentiated mouse ES-D3 cells, which showed significantly higher binding to fibronectin as compared to collagens. This adhesion was specific mediated by integrin α5β1 as evident from the inhibition with a disintegrin selective for this particular integrin. Differentiation of ES-D3 cells on fibronectin or on a collagen type1/fibronectin matrix, caused further selective up-regulation of the α5β1 integrin. Differentiation of the cells, as evaluated by immunofluorescence, FACS analysis and quantitative RT-PCR, was accompanied by the upregulation of mesenchymal (Flk1, isolectin B4, α-SMA, vimentin) and endodermal markers (FoxA2, SOX 17, cytokeratin) in parallel to increased expression of α5β1 integrin. Taken together, the data indicate that fibronectin-mediated, upregulation of α5β1 integrin and adhesion of ES-D3 cells to specific ECM molecules are linked to early stages of mouse embryonic stem cells commitment to meso-endodermal differentiation.     

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5β1 or αvβ3/β5 integrin antagonists in U87MG glioma cells

BackgroundIntegrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5β1 and αvβ3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5β1 and αvβ3/β5 purified integrins.MethodsSmall antagonists were either selective for α5β1 integrin, for αvβ3/β5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5β1 integrin in the biological assays.ResultsU87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5β1 or αvβ3/β5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5β1 integrin and was inhibited selectively by α5β1 integrin antagonists but increased by αvβ3/β5 integrin antagonists.ConclusionsWe provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions.General significanceOur data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5β1 integrin-dependent cell migration.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.     

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

Brain‐derived neurotrophic factor induces migration of endothelial cells through a TrkB–ERK–integrin αVβ3–FAK cascade

Brain‐derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal‐regulated kinase (ERK), integrin α_Vβ₃, and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α_Vβ₃ and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti‐integrin α_Vβ₃ antibody suppressed the BDNF‐induced migration. BDNF increased the levels of integrin α_Vβ₃ and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α_Vβ₃ and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α_Vβ₃/FAK, and this may help to enhance the regeneration of periodontal tissue. J. Cell. Physiol. 227: 2123–2129, 2012. © 2011 Wiley Periodicals, Inc.     

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.     

Effect of hypoxia on integrin-mediated adhesion of endothelial progenitor cells

Homing of endothelial progenitor cells (EPCs) is crucial for neoangiogenesis, which might be negatively affected by hypoxia. We investigated the influence of hypoxia on fibronectin binding integrins for migration and cell‐matrix‐adhesion. AMP‐activated kinase (AMPK) and integrin‐linked kinase (ILK) were examined as possible effectors of hypoxia.Human EPCs were expanded on fibronectin (FN) and integrin expression was profiled by flow cytometry. Cell‐matrix‐adhesion‐ and migration‐assays on FN were performed to examine the influence of hypoxia and AMPK‐activation. Regulation of AMPK and ILK was shown by Western blot analysis. We demonstrate the presence of integrin β₁, β₂ and α₅ on EPCs. Adhesion to FN is reduced by blocking β₁ and α₅ (49% and 2% of control, P < 0.05) whereas α₄‐blockade has no effect. Corresponding effects were shown for migration. Hypoxia and AMPK‐activation decrease adhesion on FN. Although total AMPK‐expression remains unchanged, phospho‐AMPK increases eightfold.The EPCs require α₅ for adhesion on FN. Hypoxia and AMPK‐activation decrease adhesion. As α₅ is the major adhesive factor for EPCs on FN, this suggests a link between AMPK and α₅‐integrins. We found novel evidence for a connection between hypoxia, AMPK‐activity and integrin activity. This might affect the fate of EPCs in ischaemic tissue.     

Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells

The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 μM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin αv and α5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that αvβ1 and αvβ3 integrins were involved in adhesion of cells to Vn, and αvβ3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, α5β1 and αvβ1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.     

Laminin 5 Deposition Promotes Keratinocyte Motility

We examined the role of individual integrins in promoting human keratinocyte migration. In short-term assays on collagen type I- or fibronectin-coated substrates, migration was blocked by antibody to the α2 integrin and the α5 integrin, respectively. Unexpectedly, antibodies to integrin α3 also significantly inhibited cell locomotion on both ligands. Time-course immunofluorescence staining revealed that keratinocyte migration was accompanied by deposition of endogenous laminin 5. Since α3β1 is a known receptor for this ligand, this observation suggested that migrating keratinocytes use freshly deposited laminin 5 in locomotion. Indeed, further investigation showed that anti-laminin 5 blocking antibodies effectively inhibited keratinocyte motility on both collagen and fibronectin substrates. Furthermore, cell migration on laminin 5-coated substrates was blocked by both anti-α3 and anti-laminin 5 antibodies. Laminin 5 did not appear important in the initial attachment of keratinocytes, since adhesion of cells to collagen type I- or fibronectin-coated surfaces was not blocked by antibody to α3 integrin or to laminin 5, but could be inhibited by antibody to α2 or α5, respectively. Using anin vitrowound assay, blocking antibodies to α3 integrin and to laminin 5 also blocked reepithelization of the denuded monolayer. These results show that α3β1 integrin plays an important role in the migration of keratinocytes via their interaction with laminin 5. Furthermore, they suggest that cell migration is dependent not only on exogenous ligands but, importantly, on endogenously secreted laminin 5. Finally, the data are consistent with our earlier finding that laminin 5 is the first extracellular matrix component to be expressed and deposited by migrating keratinocytes during wound healingin vivo[1].     

Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation

Background information. The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient‐matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). Results. Both the HOF and HDF cell types underwent TGF‐β1 (transforming growth factor‐β1)‐induced myofibroblastic differentiation [upregulation of the expression of α‐sma (α‐smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of α‐sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits α5 (fibronectin) or αv (vitronectin) were used to determine whether the effects of TGF‐β1 were regulated via integrin signalling pathways. α‐sma expression in both HOFs and HDFs was down‐regulated by antibodies against both α5 and αv. Functionally, TGF‐β1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF‐β1 (P<0.05). When TGF‐β1‐stimulated cells were incubated with blocking antibodies against α5 and αv, gel contraction was decreased to that of non‐stimulated cells; however, blocking αv or α5 could not restore cellular migration in both HOFs and HDFs. Conclusions. Despite intrinsic differences in their basal state, the cellular events associated with TGF‐β1‐induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up‐regulation of α‐sma expression and increases in collagen gel contraction are vitronectin‐ and fibronectin‐receptor‐dependent processes, whereas wound re‐population is not.     

A novel role of ERK5 in integrin‐mediated cell adhesion and motility in cancer cells via Fak signaling

In metastatic cancer, high expression levels of vitronectin (VN) receptors (integrins), FAK, and ERK5 are reported. We hypothesized that integrin‐mediated ERK5 activation via FAK may play a pivotal role in cell adhesion, motility, and metastasis. ERK5 and FAK phosphorylation when metastatic MDA‐MB‐231 and PC‐3 cells were plated on VN was enhanced. Further experiments showed co‐immunoprecipitation of integrins β1, αVβ3, or αVβ5 with ERK5 and FAK. To gain better insight into the mechanism of ERK5, FAK, and VN receptors in cell adhesion and motility, we performed loss‐of‐function experiments using integrin blocking antibodies, and specific mutants of FAK and ERK5. Ectopic expression of dominant negative ERK5/AEF decreased ERK5 and FAK (Y397) phosphorylation, cell adhesion, and haptotactic motility (micromotion) on VN. Additionally, DN FAK expression attenuated ERK5 phosphorylation, cell adhesion, and motility. This study documents the novel finding that in breast and prostate cancer cells, ERK5 is a critical target of FAK in cell adhesion signaling. Using different cancer cells, our experiments unveil a novel mechanism by which VN receptors and FAK could promote cancer metastasis via ERK5 activation. J. Cell. Physiol. 219: 152–161, 2009. © 2008 Wiley‐Liss, Inc.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen Matrices

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.     

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.     

Negative Cooperativity between α3β1and α2β1Integrins in Human Mammary Carcinoma MDA MB 231 Cells

The α₃β₁integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of α₃β₁seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell–cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of α₃β₁integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two α₃integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory α₃integrin-specific antibodies was not observed in the α₃β₁integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of α₃β₁integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the α₂integrin subunit. Function blocking anti-α₅integrin subunit mAb was without effect while anti-β₁-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of α₂β₁function by α₃β₁in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.