Adaptor protein-2 exhibits alpha 1 beta 1 or alpha 6 beta 1 integrin-dependent redistribution in rhabdomyosarcoma cells

Downregulation of several signaling pathways, such as those stimulated by growth factor receptors, occurs by internalization of signaling receptors through clathrin-coated pits. The first step in internalization or endocytosis is interaction with AP-2, which results in coated pit formation by assembly of clathrin to AP-2. Changes in endocytosis are reflected in the distribution of AP-2 molecules at the cell surface. Integrins are receptors which mediate attachment to the extracellular matrix and also stimulate numerous intracellular signaling pathways; however, it is not known how signaling through integrins is terminated or downregulated. Endocytosis through clathrin-coated pits offers an attractive mechanism for this. This work explores the relationship between AP-2 and beta(1) integrins. RD cells grown for 24 h on collagen or laminin exhibit a redistribution of AP-2 to the cell periphery relative to those grown on fibronectin or polylysine. The total AP-2 protein levels in the cells are unaffected. Blocking alpha(1)beta(1) integrin ligand binding on collagen prevents this redistribution fully. On laminin where alpha(1)beta(1) and alpha(6)beta(1) integrins are engaged, both receptors must be simultaneously blocked to prevent AP-2 redistribution, confirming that the redistribution depends on the specific engagement of the receptors. Immunofluorescence reveals that the majority of alpha(1)beta(1) integrins colocalize with alpha(6)beta(1) integrins in linear structures identified as focal adhesions. A separate fraction of alpha(1)beta(1) integrins colocalize with AP-2 in coated pits. Interestingly, alpha(6)beta(1) integrins are not located in coated pits, demonstrating that integrin colocalization with AP-2 is not necessary to induce redistribution of AP-2.     

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.     

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

Characterization of the anti-angiogenic properties of arresten, an alpha1beta1 integrin-dependent collagen-derived tumor suppressor

Physiological and pathological turnover of basement membranes liberates biologically active cryptic molecules. Several collagen-derived fragments possess anti-angiogenic activity. Arresten is the 26-kDa non-collagenous domain of type IV collagen α1 chain. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models, but its anti-angiogenic mechanism is not completely known. Here we show that arresten significantly increases apoptosis of endothelial cells in vitro by decreasing the amount of anti-apoptotic molecules of the Bcl-family; Bcl-2 and Bcl-xL. Although the pro-apoptotic effect of arresten is endothelial cell specific in vitro, in mouse tumors arresten induced apoptosis both in endothelial and tumor cells. The tumor cell apoptosis is likely an indirect effect due to the inhibition of blood vessel growth into the tumor. The active site of arresten was localized by deletion mutagenesis within the C-terminal half of the molecule. We have previously shown that arresten binds to α1β1 integrin on human umbilical vein endothelial cells. However, the microvascular endothelial cells (MLECs) are more important in the context of tumor vasculature. We show here that arresten binds also to the microvascular endothelial cells via α1β1 integrin. Furthermore, it has no effect on Matrigel neovascularization or the viability of integrin α1 null MLECs. Tumors implanted on integrin α1 deficient mice show no integrin α1 expression in the host-derived vascular endothelium, and thus arresten does not inhibit the tumor growth. Collectively, this data sheds more light into the anti-angiogenic mechanism of arresten.     

A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include alpha V beta 3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-alpha. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to alpha V beta 3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to beta 1 integrin. These results define a novel pathway for the recognition of apoptotic cells.     

Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity

Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation.     

Chemokines fail to up-regulate beta 1 integrin-dependent adhesion in human Th2 T lymphocytes

Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of P- and E-selectin ligands and selected chemokine receptors. Here we investigated the expression and function of beta 1 integrins in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands laminin 1 and fibronectin in response to chemokines such as RANTES and stromal cell-derived factor-1, and this process is paralleled by the activation of the Rac1 GTPase and by a rapid burst of actin polymerization. Selective inhibitors of phosphoinositide-3 kinase prevent efficiently all of the above processes, whereas the protein kinase C inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting Rac1 activation and actin polymerization. Notably, chemokine-induced adhesion to beta 1 integrin ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving phosphoinositide-3 kinase, Rac1, and actin turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced beta 1 integrin-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.     

Cytosolic phospholipase A2 activation is essential for beta 1 and beta 2 integrin-dependent adhesion of human eosinophils.

We examined the role of cytosolic phospholipase A2 (cPLA2) during human eosinophil adherence to ICAM-1- or VCAM-1-coated plates. IL-5-stimulated eosinophils adhered to ICAM-1 through the beta 2 integrin CD11b/CD18, while nonstimulated eosinophils did not. By contrast, nonstimulated eosinophils adhered to VCAM-1 through the beta 1-integrin VLA-4/CD29. Both IL-5-induced adhesion to ICAM-1 and spontaneous adhesion to VCAM-1 corresponded temporally to cPLA2 phosphorylation, which accompanied enhanced catalytic activity of cPLA2. The structurally unrelated cPLA2 inhibitors, arachidonyl trifluoromethylketone and surfactin, significantly inhibited eosinophil adhesion to ICAM-1 and VCAM-1 in a concentration-dependent manner. Inhibition of secretory PLA2, 5-lipoxygenase, or cyclooxygenase did not affect eosinophil adhesion. Addition of arachidonic acid to eosinophils after cPLA2 inhibition with arachidonyl trifluoromethylketone or surfactin did not reverse the blockade of adhesion to ICAM-1 or VCAM-1. However, CV-6209, a receptor-specific antagonist of platelet-activating factor, inhibited all integrin-mediated adhesion. The activated conformation of CD11b as identified by the mAb, CBRM1/5, as well as quantitative surface CD11b expression were up-regulated after IL-5 stimulation. However, cPLA2 inhibition neither prevented CBRM1/5 expression nor blocked surface Mac-1 up-regulation caused by IL-5. Our data suggest that cPLA2 activation and its catalytic product platelet-activating factor play an essential role in regulating beta 1 and beta 2 integrin-dependent adhesion of eosinophils. This blockade occurs even in the presence of up-regulated eosinophil surface integrin.     

A novel binding site in collagen type III for integrins alpha1beta1 and alpha2beta1

Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins alpha(1) and alpha(2) (alpha(1)I and alpha(2)I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by alpha(1)I and alpha(2)I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both alpha(1)I and alpha(2)I with high affinity and effectively inhibit the binding of alpha(1)I and alpha(2)I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circulating integrins: alpha 5 beta 1, alpha 6 beta 4 and Mac-1, but not alpha 3 beta 1, alpha 4 beta 1 or LFA-1.

The alpha 5 beta 1, alpha 6 beta 4 and Mac-1 integrins all participate in the endocytotic cycle. By contrast, alpha 3 beta 1, alpha 4 beta 1 and LFA-1 do so much more slowly, or not at all, in the cell lines examined. This indicates that the alpha-chains appear to determine whether an integrin cycles or not, and that alpha 5 beta 1, alpha 6 beta 4 and Mac-1 can be brought to the leading edge of a moving cell by endocytosis and recycling.     

Pro-inflammatory secretory phospholipase A2 type IIA binds to integrins alphavbeta3 and alpha4beta1 and induces proliferation of monocytic cells in an integrin-dependent manner

Secretory phospholipase A2 group IIA (sPLA2-IIA) plays an important role in the pathogenesis of inflammatory diseases. Catalytic activity of this enzyme that generates arachidonic acid is a major target for development of anti-inflammatory agents. Independent of its catalytic activity, sPLA2-IIA induces pro-inflammatory signals in a receptor-mediated mechanism (e.g. through the M-type receptor). However, the M-type receptor is species-specific: sPLA2-IIA binds to the M-type receptor in rodents and rabbits, but not in human. Thus sPLA2-IIA receptors in human have not been established. Here we demonstrated that sPLA2-IIA bound to integrin alphavbeta3 at a high affinity (K(D) = 2 x 10(-7) m). We identified amino acid residues in sPLA2-IIA (Arg-74 and Arg-100) that are critical for integrin binding using docking simulation and mutagenesis. The integrin-binding site did not include the catalytic center or the M-type receptor-binding site. sPLA2-IIA also bound to alpha4beta1. We showed that sPLA2-IIA competed with VCAM-1 for binding to alpha4beta1, and bound to a site close to those for VCAM-1 and CS-1 in the alpha4 subunit. Wild type and the catalytically inactive H47Q mutant of sPLA2-IIA induced cell proliferation and ERK1/2 activation in monocytic cells, but the integrin binding-defective R74E/R100E mutant did not. This indicates that integrin binding is required, but catalytic activity is not required, for sPLA2-IIA-induced proliferative signaling. These results suggest that integrins alphavbeta3 and alpha4beta1 may serve as receptors for sPLA2-IIA and mediate pro-inflammatory action of sPLA2-IIA, and that integrin-sPLA2-IIA interaction is a novel therapeutic target.     

Cross-talk between integrins alpha1beta1 and alpha2beta1 in renal epithelial cells

The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function.     

Kinetic studies on partially liganded species of carboxyhemoglobin: (alpha 1CO-beta 1CO)alpha 2 beta 2 or (alpha 2CO beta 2CO)alpha 1 beta 1

Kinetics of CO combination with and dissociation from isomer III, (alpha 1CO beta 1CO)alpha 2 beta 2 or alpha 1 beta 1 (alpha 2CO beta 2CO), and Hb Rothschild have been studied using the double mixing and microperoxidase methods. Isomer III was prepared in a manner so that it was the only reactive species in the reaction mixture. The biphasic reaction time course in both the "on" and "off" reactions of isomer III and the CO combination reaction of Hb Rothschild are attributed to slow relaxation between the fast and slow CO-reacting species in the two proteins: isomer III: l'f = 6 x 10(6) M-1 s-1, l'dimer = 1.7 x 10(6) M-1 s-1, l's = 2.2 x 10(5) M-1 s-1, lf = 0.15 s-1, ls = 0.01 s-1; Hb Rothschild: l'f = 2.8 x 10(6) M-1 s-1; l's = 2.7 x 10(5) M-1 s-1.     

A new hemoglobin variant altering the alpha 1 beta 2 contact: Hb Chemilly alpha 2 beta 2 99(G1)Asp leads to Val

The contribution of hepatic glycogen to lipogenesis was studied in isolated, intact rat hepatocytes. To establish its importance as a substrate for lipogenesis, the glycogen of isolated hepatocytes was prelabelled with 14C from glucose. Evidence is presented that neither glucose nor glycogen constitute major sources of carbon for de novo synthesis of fatty acids and that less than 1% of glycogen is converted into fatty acids.     

Expression and functional importance of collagen-binding integrins,alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1)beta(1) parallels that of viral-specific effector CD8(+) T cells (defined by tetramer and IFN-gamma staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of alpha(1)beta(1) blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.     

Contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.     

Chondrocyte aggregation in suspension culture is GFOGER-GPP- and beta1 integrin-dependent

Isolated chondrocytes form aggregates in suspension culture that maintain chondrocyte phenotype in a physiological pericellular environment. The molecular mechanisms involved in chondrocyte aggregation have not been previously identified. Using this novel suspension culture system, we performed mRNA and protein expression analysis along with immunohistochemistry for potential cell adhesion molecules and extracellular matrix integrin ligands. Inhibition of aggregation assays were performed using specific blocking agents. We found that: (i) direct cell-cell interactions were not involved in chondrocyte aggregation, (ii) chondrocytes in aggregates were surrounded by a matrix rich in collagen II and cartilage oligomeric protein (COMP), (iii) aggregation depends on a beta1-integrin, which binds a triple helical GFOGER sequence found in collagens, (iv) integrin alpha10-subunit is the most highly expressed alpha-subunit among those tested, including alpha5, in aggregating chondrocytes. Taken together, this body of evidence suggests that the main molecular interaction involved in aggregation of phenotypically stable chondrocytes is the alpha10beta1-collagen II interaction.