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PubMedDate not supplied

Functional effect of contortrostatin, a snake venom disintegrin, on human glioma cell invasion in vitro.

The metastatic spread of cancer is a complex process that involves the combination of different cellular actions including cell adhesion to the extracellular matrix (ECM), breakdown of the ECM by specific matrix-degrading proteinases, and active cell locomotion. Contortrostatin (CN), a homodimeric snake venom disintegrin, has previously been demonstrated to be effective in blocking vitronectin/fibronectin-dependent adhesion and invasion of T98G human glioblastoma cells through Matrigel using in vitro studies. However, it is not known at what step of the invasion process CN exerts its inhibitory effect. In the present report, CN is shown to decrease invasion of various glioma cell lines through Matrigel affecting neither cell adhesion, nor cell viability. While CN had no effect on cell binding to laminin and type IV collagen, it blocked adhesion of alphav beta3-positive, but not alphav beta3-negative cells, to vitronectin and fibronectin. Furthermore, members of the matrix metalloproteinase (MMP) family and their physiological inhibitors, and of the plasminogen activator (PA)/plasmin system were demonstrated not to be involved in CN-induced loss of glioma cell invasiveness. Instead, CN inhibited active locomotion of cells on Matrigel. These data suggest that CN-mediated inhibition of glioma cell invasion through Matrigel is a direct result of impaired cell motility. Moreover, use of several glioma cell lines and integrin antibodies strongly indicates the versatility of CN in inhibiting the invasion process based on the ability of CN to interact with different integrins, including alphav beta3, alphav beta5, and alpha5beta1.

Blotting, Western↗
PubMed1988

Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3).

Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion-promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+-binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins.

Amino Acid Sequence↗
PubMed1990

Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.

Animals↗
PubMed1991

An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3-mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation.

The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well-defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required.

Amino Acid Sequence↗
PubMed1994

Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

Animals↗
PubMed1996

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release?

Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.

Amino Acids↗
PubMed1999

Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Antibodies↗
PubMed1999

Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.

Migration, proliferation, and tube formation of endothelial cells are regulated by a protein kinase C isoenzyme PKCtheta. A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20. Overexpression of TAP20 decreased cell adhesion and enhanced migration on vitronectin and tube formation in three-dimensional culture. An antiintegrin alphavbeta5 antibody prevented these TAP20 effects. Overexpression of TAP20 also decreased focal adhesion formation in alphavbeta3-deficient cells. The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting. Thus, the discovery of TAP20, which interacts with integrin beta5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKCtheta.

Amino Acid Sequence↗
PubMed2000

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.

Adult↗
PubMed2002

The relationship between force and focal complex development.

To adhere and migrate, cells must be capable of applying cytoskeletal force to the extracellular matrix (ECM) through integrin receptors. However, it is unclear if connections between integrins and the ECM are immediately capable of transducing cytoskeletal contraction into migration force, or whether engagement of force transmission requires maturation of the adhesion. Here, we show that initial integrin-ECM adhesions become capable of exerting migration force with the recruitment of vinculin, a marker for focal complexes, which are precursors of focal adhesions. We are able to induce the development of focal complexes by the application of mechanical force to fibronectin receptors from inside or outside the cell, and we are able to extend focal complex formation to vitronectin receptors by the removal of c-Src. These results indicate that cells use mechanical force as a signal to strengthen initial integrin-ECM adhesions into focal complexes and regulate the amount of migration force applied to individual adhesions at localized regions of the advancing lamella.

3T3 Cells↗
PubMed2003

Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins.

The binding of urokinase plaminogen activator (uPA) to its cell surface receptor (uPAR; CD87) promotes cell adhesion by increasing the affinity of the receptor for both vitronectin (VN) and integrins. We provide evidence that plasminogen activator inhibitor (PAI)-1 can detach cells by disrupting uPAR-VN and integrin-VN interactions and that it does so by binding to the uPA present in uPA-uPAR-integrin complexes on the cell surface. The detached cells cannot reattach to VN unless their surface integrins are first activated by treatment with MnCl2. Immunoprecipitation and subcellular fractionation experiments reveal that PAI-1 treatment triggers deactivation and disengagement of uPA-uPAR-integrin complexes and their endocytic clearance by the low density lipoprotein receptor-related protein. Transfection experiments demonstrate that efficient cell detachment by PAI-1 requires an excess of matrix-engaged uPA-uPAR-integrin complexes over free engaged integrins and that changes in this ratio alter the efficacy of PAI-1. Together, these results suggest a VN-independent, uPA-uPAR-dependent mechanism by which PAI-1 induces cell detachment. This pathway may represent a general mechanism, since PAI-1 also can detach cells from fibronectin and type-1 collagen. This novel "deadhesive" activity of PAI-1 toward a variety of cells growing on different extracellular matrices may begin to explain why high PAI-1 levels often are associated with a poor prognosis in human metastatic disease.

Animals↗
PubMed2005

Anti-CD63 antibodies suppress IgE-dependent allergic reactions in vitro and in vivo.

High-affinity IgE receptor (FcepsilonRI) cross-linking on mast cells (MCs) induces secretion of preformed allergy mediators (degranulation) and synthesis of lipid mediators and cytokines. Degranulation produces many symptoms of immediate-type allergic reactions and is modulated by adhesion to surfaces coated with specific extracellular matrix (ECM) proteins. The signals involved in this modulation are mostly unknown and their contribution to allergic reactions in vivo is unclear. Here we report the generation of monoclonal antibodies that potently suppress FcepsilonRI-induced degranulation, but not leukotriene synthesis. We identified the antibody target as the tetraspanin CD63. Tetraspanins are membrane molecules that form multimolecular complexes with a broad array of molecules including ECM protein-binding beta integrins. We found that anti-CD63 inhibits MC adhesion to fibronectin and vitronectin. Furthermore, anti-CD63 inhibits FcepsilonRI-mediated degranulation in cells adherent to those ECM proteins but not in nonadherent cells. Thus the inhibition of degranulation by anti-CD63 correlates with its effect on adhesion. In support of a mechanistic linkage between the two types of inhibition, anti-CD63 had no effect on FcepsilonRI-induced global tyrosine phosphorylation and calcium mobilization but impaired the Gab2-PI3K pathway that is known to be essential for both degranulation and adhesion. Finally, we showed that these antibodies inhibited FcepsilonRI-mediated allergic reactions in vivo. These properties raise the possibility that anti-CD63 could be used as therapeutic agents in MC-dependent diseases.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Cloning and characterization of a novel zinc finger protein that associates with nuclear matrix.

We have recently reported that the nuclei of B16 melanoma cells are intensely stained with anti-rat vitronectin (Vn) antibody, which reacts with both mouse and rat Vn. In the present study, we characterized the protein immunoreactive with the antibody using NIH3T3 cells, whose nuclei were also stained with the antibody. Western blot analysis showed that a protein with an approximate molecular weight of 75 kDa (p75), which was distinct from Vn, existed in the nuclear fraction, and, more specifically, in the nuclear matrix fraction, of NIH3T3 cells. Screening of an NIH3T3 cDNA library resulted in the isolation of a nearly full-length cDNA clone encoding p75. A database search revealed that the cDNA represents a novel gene. The deduced amino acid sequence showed that the protein is 580 amino acids long and contains two C2H2-type zinc finger motifs and glutamic acid-rich domains in the C-terminal region. When a fusion protein of green fluorescence protein and p75 was expressed in NIH3T3 cells, fluorescence was preferentially observed in the nuclei, demonstrating that the protein has a nuclear localization signal. The p75 protein, termed ZAN75, exhibited DNA-binding activity in a zinc-dependent manner. Southern blot analysis demonstrated that the ZAN75 gene exists in a single copy in the mouse genome and that a closely related gene is also present in chicken, rat, and human. Northern blot analysis showed that the ZAN75 gene is ubiquitously expressed in adult mouse tissues. In the cell cycle of NIH3T3 cells, expression was low in the G0/G1 phase, increased during the G1 phase, and persisted during the S and G2/M phases, suggesting that ZAN75 plays a role in regulating cell growth.

3T3 Cells↗
PubMed2005

Induction and detection of human mesenchymal stem cell migration in the 48-well reusable transwell assay.

The induction and detection of chemotactic migration in mesenchymal stem cells (MSC) are complicated by their adherent nature. We describe here the experimental details for inducing the transmigration of MSC seeded in microchemotaxis chambers and reliably identifying the translocated cells. A combination of 8-microm pore-sized membranes and a two-step staining procedure resulted in the detection of MSC migration against a gradient of human plasma which plateaued after 4 h. Microscopic enumeration of the transmigrated cells clearly distinguished contaminating nonmigrated cells from transmigrated cells. Chemokinetic and chemotactic effects could be separated and were influenced by precoated fibronectin or vitronectin. Thus, the technique described allows rapid and reliable induction and determination of migration in adherent MSC.

Bone Marrow Cells↗
PubMedDate not supplied

Mediation of biomaterial-cell interactions by adsorbed proteins: a review.

An appropriate cellular response to implanted surfaces is essential for tissue regeneration and integration. It is well described that implanted materials are immediately coated with proteins from blood and interstitial fluids, and it is through this adsorbed layer that cells sense foreign surfaces. Hence, it is the adsorbed proteins, rather than the surface itself, to which cells initially respond. Diverse studies using a range of materials have demonstrated the pivotal role of extracellular adhesion proteins--fibronectin and vitronectin in particular--in cell adhesion, morphology, and migration. These events underlie the subsequent responses required for tissue repair, with the nature of cell surface interactions contributing to survival, growth, and differentiation. The pattern in which adhesion proteins and other bioactive molecules adsorb thus elicits cellular reactions specific to the underlying physicochemical properties of the material. Accordingly, in vitro studies generally demonstrate favorable cell responses to charged, hydrophilic surfaces, corresponding to superior adsorption and bioactivity of adhesion proteins. This review illustrates the mediation of cell responses to biomaterials by adsorbed proteins, in the context of osteoblasts and selected materials used in orthopedic implants and bone tissue engineering. It is recognized, however, that the periimplant environment in vivo will differ substantially from the cell-biomaterial interface in vitro. Hence, one of the key issues yet to be resolved is that of the interface composition actually encountered by osteoblasts within the sequence of inflammation and bone regeneration.

Adsorption↗
PubMed1993

Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.

3T3 Cells↗
PubMed1994

The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts.

Tenascin (TN) is a large oligomeric glycoprotein that is present transiently in the extracellular matrix (ECM) of cells and is involved in morphogenetic movements, tissue patterning, and tissue repair. It has multiple domains, both adhesive and anti-adhesive, that interact with cells and with fibronectin (FN) and other ECM macromolecules. We have studied the consequences of the interaction of TN with a FN matrix on gene expression in rabbit synovial fibroblasts. Fibroblasts plated on a mixed substrate of FN and TN, but not on FN alone, upregulated synthesis of four genes: collagenase, stromelysin, the 92-kDa gelatinase, and c-fos. Although the fibroblasts spread well on both FN and FN/TN substrates, nuclear c-Fos increased within 1 h only in cells that were plated on FN/TN. TN did not induce the expression of collagenase in cells plated on substrates of type I collagen or vitronectin (VN). Moreover, soluble TN added to cells adhering to a FN substrate or to serum proteins had no effect, suggesting that TN has an effect only in the context of mixed substrates of FN and TN. Collagenase increased within 4 h of plating on a FN/TN substrate and exhibited kinetics similar to those for induction of collagenase gene expression by signaling through the integrin FN receptor. Arg-Gly-Asp peptide ligands that recognize either the FN receptor or the VN receptor and function-perturbing anti-integrin monoclonal antibodies diminished the interaction of fibroblasts with a mixed substrate of FN, TN, and VN, but had no effect on the adhesion of fibroblasts to a substrate of FN and VN, suggesting that both receptors recognize the complex. Anti-TN68, an antibody that recognizes an epitope in the carboxyl-terminal type III repeats involved in the interaction of TN with both FN and cells, blocked the inductive effect of the FN/TN substrate, whereas anti-TNM1, an antibody that recognizes an epitope in the amino-terminal anti-adhesive region of epidermal growth factor-like repeats, had no effect. These data suggest that transient alteration of the composition of ECM by addition of proteins like TN may regulate the expression of genes involved in cell migration, tissue remodeling, and tissue invasion, in regions of tissue undergoing phenotypic changes.

Amino Acid Sequence↗
PubMed1993

Comparison of integrin expression and terminal differentiation capacity in cell lines derived from oral squamous cell carcinomas.

Receptors of the integrin family regulate adhesion and terminal differentiation of keratinocytes. In order to investigate the significance of changes in integrin expression associated with malignant transformation we have examined normal human oral keratinocytes and seven oral squamous cell carcinoma (SCC) lines. Cell surface levels of the alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1 and beta 4 integrin subunits were determined by flow cytometry and the distribution of the beta 1 subunit was examined by immunohistochemistry. In normal keratinocytes and one SCC line the beta 1 subunit was most abundant in the basal cell layer, but in other lines anti-beta 1 antibodies stained basal and suprabasal layers uniformly. All lines had reduced surface levels of at least one integrin subunit and in some cell lines distinct subpopulations could be distinguished on the basis of differences in integrin expression. Reduced integrin expression was not, however, generally reflected in reduced adhesion to laminin, fibronectin, type IV collagen or vitronectin in three cell lines examined. Those cell lines with the lowest capacity for terminal differentiation, as measured by involucrin expression, had the lowest levels of the alpha 6 and beta 4 subunits or were completely lacking alpha v. Oral SCC show considerable variation in integrin expression, but focal or extensive loss of the alpha 6 and beta 4 subunits is a common feature of poorly differentiated tumours. The cell lines we have examined therefore provide a relevant experimental model with which to explore the relationship between aberrant integrin expression and impaired terminal differentiation capacity.

Carcinoma, Squamous Cell↗