The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells

We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins alpha 2 beta 1, and alpha 3 beta 1 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-alpha 3 beta 1 mAb, P1F2, localized alpha 3 beta 1 in FAs on purified laminin greater than fibronectin/collagen, indicating that laminin was the best exogeneous ligand for alpha 3 beta 1. However, in long term culture, alpha 3 beta 1 preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-alpha 3 beta 1, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of alpha 3 beta 1 with the secreted laminin-containing ECM was primarily responsible for HFK adhesion in long term culture. (b) In FA assays, alpha 2 beta 1 localized in FAs conincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-alpha 2 beta 1 inhibited initial HFK adhesion to both laminin and collagen. Thus, alpha 2 beta 1 contributes to initial HFK adhesion to laminin but alpha 3 beta 1 is primarily responsible for long-term HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2(+)-induced aggregation of HFKs resulted in relocation of alpha 2 beta 1 and alpha 3 beta 1 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-alpha 3 beta 1 (P1B5) and a new anti-beta 1 mAb (P4C10). Thus, interaction of alpha 3 beta 1 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2(+)-induced HFK aggregation. (d) It is suggested that interaction of alpha 3 beta 1 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of alpha 3 beta 1 in basal cell adhesion to the BMZ in skin. Further, relocation of alpha 2 beta 1 and alpha 3 beta 1 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.     

Fibronectin fragmentation promotes alpha4beta1 integrin-mediated contraction of a fibrin-fibronectin provisional matrix

In injured tissues, the fibrin-fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin-FN provisional matrix model to determine the contributions of the FN-binding integrin receptors alpha5beta1 and alpha4beta1 to matrix contraction. CHOalpha5 cells expressing alpha5beta1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin-FN matrix. In contrast, CHOalpha4 cells expressing the alpha4beta1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin-FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOalpha4 cells were dramatically enhanced, to levels equivalent to CHOalpha5 cells, when proteolyzed FN was used in place of intact FN in the fibrin-FN matrix. Similar enhancement was observed when ligand binding by alpha4beta1 integrins was activated by treatment with Mn(++), but not by stimulation of actin organization with LPA. Therefore, alpha4beta1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.     

The interplay between integrins alphaMbeta2 and alpha5beta1 during cell migration to fibronectin

A directed migration of leukocytes through the extracellular matrix requires the regulated engagement of integrin cell adhesion receptors. The integrin alpha(M)beta(2) (CD11b/CD18, Mac-1) is progressively upregulated to high levels on migrating phagocytic leukocytes in response to inflammatory stimuli and is able to bind numerous ligands in the interstitial matrix. The role of alpha(M)beta(2) in migration of leukocytes through the extracellular matrix and its cooperation with other leukocyte integrins during migration are not understood. Using a model system consisting of cells that express different levels of alpha(M)beta(2) and an invariable level of endogenous integrin alpha(5)beta(1), we have explored a situation relevant to migrating neutrophils when alpha(M)beta(2) and alpha(5)beta(1) engage the same ligand, fibronectin. We show that fibronectin is a ligand for alpha(M)beta(2) and that both alpha(M)beta(2) and alpha(5)beta(1) on the alpha(M)beta(2)-expressing cells contribute to adhesion to fibronectin. However, migration of these cells to fibronectin is mediated by alpha(5)beta(1), whereas alpha(M)beta(2) retards migration. The decrease in migration correlates directly with the increased alpha(M)beta(2) density. Ligation of alpha(M)beta(2) with function-blocking antibodies can reverse this effect. The restorative effects of antibodies are caused by the removal of restraint imposed by the excess of alpha(M)beta(2)-fibronectin adhesive bonds. These findings indicate that alpha(M)beta(2) can increase general cell adhesiveness which results in braking of cell migration mediated by integrin alpha(5)beta(1). Because alpha(M)beta(2) binds numerous proteins in the extracellular matrix with a specificity overlapping that of the beta(1) integrins, the results suggest that alpha(M)beta(2) can affect the beta(1) integrin-mediated cell migration.     

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.     

Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes

Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin alpha 3 beta 1, in focal adhesions (FAs), of cultured human keratinocytes (HFKs) as a mediator of HFK adhesion to secreted BMZ-like extracellular matrix (ECM; Carter, W.G., E.A. Wayner, T.S. Bouchard, and P. Kaur. 1990. J. Cell Biol. 110: 1387-1404). Here, we have examined the relation of integrins alpha 6 beta 4 and alpha 3 beta 1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that alpha 6 beta 4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with alpha 3 beta 1- FAs to mediate adhesion to ECM, based on the following. (a) Comparison of secreted ECM, with exogenous laminin, fibronectin and collagen identified ECM as the preferred ligand for HFK adhesion and spreading and for formation of both alpha 6 beta 4-SACs and alpha 3 beta 1-FAs. (b) Inhibition of HFK adhesion with combined anti-alpha 3 beta 1 (P1B5) and anti-alpha 6 beta 4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while alpha 3 beta 1 played a dominant role in spreading. (c) alpha 6 beta 4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both alpha 6 beta 4- SACs and alpha 3 beta 1-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to alpha 3 beta 1-FAs, alpha 6 beta 4-SACs were present only in nonmotile cells, not associated with stress fibers, and were relatively stable to detergents and urea, suggesting a nonmotile, or anchoring function for SACs and motility functions for alpha 3 beta 1-FAs. (e) alpha 6 beta 4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with alpha 6 beta 4. (f) We suggest that alpha 6 beta 4/BPA-SACs in culture restrict migration of HFKs on ECM while alpha 3 beta 1-FAs form dynamic adhesions in spreading and migrating cells. alpha 6 beta 4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.     

Interaction of integrins alpha 3 beta 1 and alpha 2 beta 1: potential role in keratinocyte intercellular adhesion

The colocalization of integrins alpha 2 beta 1 and alpha 3 beta 1 at intercellular contact sites of keratinocytes in culture and in epidermis suggests that these integrins may mediate intercellular adhesion (ICA). P1B5, an anti-alpha 3 beta 1 mAb previously reported to inhibit keratinocyte adhesion to epiligrin, was also found to induce ICA. Evidence that P1B5-induced ICA was mediated by alpha 2 beta 1 and alpha 3 beta 1 was obtained using both ICA assays and assays with purified, mAb-immobilized integrins. Selective binding of alpha 2 beta 1-coated beads to epidermal cells or plate-bound alpha 3 beta 1 was observed. This binding was inhibited by mAbs to integrin alpha 3, alpha 2, or beta 1 subunits and could be stimulated by P1B5. We also demonstrate a selective and inhibitable interaction between affinity- purified integrins alpha 2 beta 1 and alpha 3 beta 1. Finally, we show that expression of alpha 2 beta 1 by CHO fibroblasts results in the acquisition of collagen and alpha 3 beta 1 binding. Binding to both of these ligands is inhibited by P1H5, an anti-alpha 2 beta 1 specific mAb. Results of these in vitro experiments suggest that integrins alpha 2 beta 1 and alpha 3 beta 1 can interact and may do so to mediate ICA in vivo. Thus, alpha 3 beta 1 mediates keratinocyte adhesion to epiligrin and plays a second role in ICA via alpha 2 beta 1.     

Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 +/- 0.7 microm/h and is independent of cell migration. It is induced by ligation of alpha(5)beta(1) integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied alpha(5)beta(1) integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating alpha(5)beta(1) integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.     

Accessibility to the fibronectin synergy site in a 3D matrix regulates engagement of alpha5beta1 versus alphavbeta3 integrin receptors

Cell adhesion and migration on fibronectin (FN) extracellular matrix are mediated by integrin receptors. Integrins alpha5beta1 and alphavbeta3 require the RGD cell-binding sequence in FN, but alpha5beta1 also requires the nearby synergy site for maximal binding. In this study, we investigated how differences in the numbers of RGD or synergy sites within a three-dimensional (3D) FN-rich matrix influence cell adhesion and migration. CHO cell adhesion, spreading, and migration were reduced on 3D chimeric matrix containing FN lacking RGD (FN(RGD-)). Incorporation of FN with mutation of the synergy site (FN(syn-)), however, resulted in selective usage of integrins. CHO cells expressing alpha5beta1 showed decreased interactions with FN(syn-) chimeric matrix. In contrast, the presence of FN(syn-) had no effect on CHOalphavbeta3 cell migration. Interestingly, CHOalpha5/alphavbeta3 cells expressing both integrins selectively used alpha5beta1 for migration on wild type FN matrix but preferred alphavbeta3 for migration on FN(syn-) chimeric matrix. Thus sequestration or exposure of the FN synergy site within a 3D matrix may represent a novel mechanism for regulating cell functions through differential usage of integrin receptors. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resource: a video recording shows migration of HT1080 cells on 3D matrix. HT1080 cells were allowed to attach to the matrix in serum-free DMEM for 2 h. FBS was then added to the medium to a final concentration of 10% and video recording was started. Images were taken every 5 min for 2 h. The video plays at 6 frames/s.].     

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.     

Inhibitory role of alpha 6 beta 4-associated erbB-2 and phosphoinositide 3-kinase in keratinocyte haptotactic migration dependent on alpha 3 beta 1 integrin

Keratinocytes and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins alpha 3 beta 1 and alpha 6 beta 4. While alpha 3 beta 1 mediates adhesion, spreading, and migration (Kreidberg, J.A. 2000. Curr. Opin. Cell Biol. 12:548--553), alpha 6 beta 4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 112:411--418). We investigated a possible regulatory interplay between alpha 3 beta 1 and alpha 6 beta 4 in cell motility using HaCaT keratinocytes as a model. We found that alpha 6 beta 4 antibodies inhibit alpha 3 beta 1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by alpha 3 beta 1 activation), and not when chemotactic (i.e., triggered by epidermal growth factor receptor). Inhibition of migration by alpha 6 beta 4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti-alpha 6 beta 4 antibodies, activation of PI3-K is mediated by alpha 6 beta 4-associated erbB-2, as indicated by erbB-2 autophosphorylation and erbB-2/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-2 abolishes inhibition of haptotaxis by anti-alpha 6 beta 4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of alpha 3beta 1 and alpha 6 beta 4 integrins, (b) alpha 6 beta 4-associated erbB-2 and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by alpha 6 beta 4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM.     

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.     

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.     

Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation

Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Environmental modulation of alpha(v), alpha(2) and beta(1) integrin subunit expression in human oesophageal squamous cell carcinomas

Integrins are cell adhesion molecules pivotal in regulating normal cell behaviour. Ectopic expression of integrins, characteristic of transformed cells, is instrumental in differentiation, proliferation, apoptosis, angiogenesis, matrix degradation and migration. Oesophageal squamous cell carcinoma (SCC) has a propensity to metastasize and hence an extremely poor prognosis. It is shown here that oesophageal SCCs express alpha(v)strongly and that normal oesophageal tissue does not express alpha(v). This makes alpha(v)a significant indicator of the transformed phenotype. alpha(2)and beta(1)integrin subunits are down-regulated in oesophageal SCCs compared to normal oesophagus. Dominance of the alpha(2)beta(1)heterodimer is symptomatic of potential loss of other beta(1)binding integrins in oesophageal SCCs. These results suggest a decrease in rigid cell adhesion possibly increasing migratory potential, whilst simultaneously permitting the adhesion and migration of SCC cells on a large repertoire of ligands due to de novo alpha(v)expression.     

Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.     

Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta 1 and alpha 2beta 1

Polymerization of the ECM proteins fibronectin and laminin has been shown to take place in close vicinity to the cell surface and be facilitated by beta(1) integrins (Lohikangas, L., Gullberg, D., and Johansson, S. (2001) Exp. Cell Res. 265, 135-144 and Wennerberg, K., Lohikangas, L., Gullberg, D., Pfaff, M., Johansson, S., and Fassler, R. (1996) J. Cell Biol. 132, 227-238). We have studied the role of collagen receptors, integrins alpha(11)beta(1) and alpha(2)beta(1), and fibronectin in collagen polymerization using fibronectin-deficient mouse embryonic fibroblast cell lines. In contrast to the earlier belief that collagen polymerization occurs via self-assembly of collagen molecules we show that a preformed fibronectin matrix is essential for collagen network formation and that collagen-binding integrins strongly enhance this process. Thus, collagen deposition is regulated by the cells, both indirectly through integrin alpha(5)beta(1)-dependent polymerization of fibronectin and directly through collagen-binding integrins.     

The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1- expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.     

Cross-talk of integrin alpha3beta1 and tissue factor in cell migration

In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility.     

Differences in phosphatase modulation of alpha4beta1 and alpha5beta1 integrin-mediated adhesion and migration of B16F1 cells.

It is well established that a biphasic relationship exists between the adhesive strength of beta1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in beta1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in alpha4beta1 and alpha5beta1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, alpha5beta1 functioned as the predominant receptor for cell movement; a role for alpha4beta1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of alpha4beta1 and alpha5beta1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of alpha5beta1, but not alpha4beta1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of beta1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various beta1 integrins exist.