Involvement of oligosaccharide changes in alpha5beta1 integrin in a cisplatin-resistant human squamous cell carcinoma cell line

Multiple mechanisms are involved in the resistance of cancer cells to cisplatin, including the expression of multidrug resistance-associated protein (MRP) and enhanced DNA repair. Here, we report findings to show that oligosaccharide changes in alpha5beta1 integrin are associated with cisplatin resistance in a head and neck squamous cell carcinoma cell line, HSC-2. Cisplatin-resistant HSC-2 (HSC-2/CR) cells were established by stepwise treatment with various concentrations of cisplatin. The oligosaccharides containing beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching, detected by leukoagglutinating phytohemagglutinin (L(4)-PHA) lectin blot, were found to be dramatically decreased in alpha5beta1 integrin immunoprecipitated from HSC-2/CR cells. To better understand the mechanisms underlying cisplatin resistance and oligosaccharide alteration, we analyzed the downstream signaling of alpha5beta1 integrin, one of the target glycoproteins of beta1-6GlcNAc transferase [UDP-GlcNAc:alpha-D-mannoside beta1, 6-N-acetylglucosaminyltransferase (GnT-V)]. Cell adhesion to fibronectin and phosphorylation of focal adhesion kinase (FAK), which are associated with alpha5beta1 integrin and involved in a cell survival signaling, were found to be increased in the cisplatin-resistant cells. Enhancement of the inhibition of cell adhesion and FAK phosphorylation also support the above data in GnT-V transfectants of HSC-2 cells. Interestingly, the differences in sensitivity to cisplatin and FAK phosphorylation between cisplatin-sensitive and -resistant cells were completely abolished by treatment with a neutral antibody of alpha5beta1 integrin. These results suggest that modification of oligosaccharides of alpha5beta1 integrin represents one of the possible mechanisms of drug resistance in head and neck cancer cells.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin- mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1- mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR- CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin- mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1- dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into mammalian cells

Shigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans. Bacteria reaching the colon invade intestinal epithelial cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor- mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigella flexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with alpha5beta1 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha5beta1 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125FAK, an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to alpha 5 beta 1 integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, alpha5beta1 integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha5beta1 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.     

Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex.

Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.     

SHPS-1 negatively regulates integrin alphaIIbbeta3 function through CD47 without disturbing FAK phosphorylation.

CD47 (integrin-associated protein) serves as a receptor for thrombospondin-1 (TSP-1) and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), and the TSP-1/CD47 interaction has been believed to augment integrin-mediated platelet function. Here, employing SHPS-1-immunoglobulin (Ig) as a ligand, we have newly demonstrated that CD47 acts as an inhibitory receptor for platelet function. The binding of SHPS-1-Ig was solely mediated by CD47, because CD47-deficient platelets failed to bind murine SHPS-1-Ig. The human SHPS-1/CD47 interaction inhibited the platelet aggregation induced by several kinds of agonists at a low concentration. Moreover, human SHPS-1 expressed on the cell surface as well as soluble SHPS-1-Ig markedly inhibited the platelet spreading on, but not initial adhesion to, immobilized fibrinogen. Again, neither murine SHPS-1 expressed on the cell surface nor murine SHPS-1-Ig inhibited the spreading of CD47-deficient platelets. We further investigated the tyrosine phosphorylation of signaling proteins during platelet spreading on immobilized fibrinogen. Unexpectedly, SHPS-1 inhibited alpha(IIb)beta(3)-mediated platelet spreading without disturbing focal adhesion kinase (FAK) tyrosine phosphorylation. Further examination revealed that SHPS-1 inhibited the tyrosine phosphorylation of alpha-actinin, a downstream effector of FAK, but not of cortactin. Thus, it is likely that the SHPS-1/CD47 interaction inhibits alpha(IIb)beta(3)-mediated outside-in signaling by interfering with the downstream pathway of FAK. Taken together, our data suggest that SHPS-1 negatively regulates platelet function via CD47, especially alpha(IIb)beta(3)-mediated outside-in signaling.     

Inhibition of endothelial cell survival and angiogenesis by protein kinase A

Receptors for the provisional ECM are important regulators of angiogenesis. One of these receptors, integrin alpha5beta1, plays a critical role in tumor- and growth factor-induced angiogenesis, because antagonists of this integrin potently inhibit angiogenesis and tumor growth. Here we show that the integrin alpha5beta1 promotes endothelial cell survival during angiogenesis in vivo by suppressing the activity of protein kinase A (PKA). Antagonists of integrin alpha5beta1 activate PKA, which then leads to the activation of caspase-8 and induction of apoptosis. Direct activation of PKA by cAMP or by expression of the PKA catalytic subunit also induces endothelial cell apoptosis, resulting in angiogenesis inhibition in vivo. Our studies indicate that ligation of integrin alpha5beta1 during angiogenesis suppresses an apoptotic program that is dependent on PKA. These studies also indicate that induction of endothelial cell apoptosis in vivo by genetic or pharmacological activation of PKA may be a useful strategy to inhibit angiogenesis.     

整合蛋白β1在藻蓝蛋白抑制白血病细胞系K562细胞增殖中的作用

本研究观察不同浓度藻蓝蛋白（phycocyanin）对K562细胞增殖的影响，并检测藻蓝蛋白处理后细胞表面整合蛋白β1表达和胞内黏着斑激酶（FAK）基因表达的变化，探讨整合蛋白β1在藻蓝蛋白抑制K562细胞增殖中的可能作用机制。用流式细胞术（FCM）检测藻蓝蛋白处理前后K562细胞表面整合蛋白β1表达变化；MTT法测定不同浓度藻蓝蛋白对K562细胞增殖的影响；RT—PCR检测不同浓度藻蓝蛋白对K562细胞内FAK基因表达的作用。结果表明：整合蛋白β1在K562细胞上高表达；藻蓝蛋白不能改变其表达量。不同浓度藻蓝蛋白对K562细胞的增殖均有抑制作用。藻蓝蛋白可上调胞内FAK的基因表达水平，恢复整合蛋白β1介导的细胞增殖抑制信号。结论：藻蓝蛋白可能通过增强细胞基质与K562细胞表面的整合蛋白β1结合，上调K562细胞内FAK基因表达水平，恢复整合蛋白β1介导的细胞增殖抑制信号而发挥抑制K562细胞增殖的作用。     

Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by absence or dysfunction of the platelet integrin alpha(IIb)beta3. Patient RM is a thrombasthenic variant whose platelets fail to aggregate in response to physiological agonists, despite the fact that they express abundant levels of alpha(IIb)beta3 on their surface. Binding of soluble fibrinogen or fibrinogen mimetic antibodies to RM platelets did not occur, except in the presence of ligand-induced binding site (LIBS) antibodies that transformed the RM integrin complex into an active conformation from outside the cell. Sequence analysis of PCR-amplified genomic DNA and platelet mRNA revealed a C2268T nucleotide substitution in the gene encoding the integrin beta3 subunit that resulted in an Arg724Ter mutation, producing a truncated protein containing only the first eight of the 47 amino acids normally present in the cytoplasmic domain. Functional analysis of both RM platelets and CHO cells stably expressing this truncated integrin revealed that the alpha(IIb)beta3Arg724Ter complex is able to mediate binding to immobilized fibrinogen, though downstream events, including cytoskeletally-mediated cell spreading and tyrosine phosphorylation of focal adhesion kinase, pp125FAK, fail to occur. These studies establish the importance of the membrane-distal portion of the integrin beta3 cytoplasmic domain in bidirectional transmembrane signaling in human platelets, and the role of integrin signaling in maintaining normal hemostasis in vivo.     

Antimigratory effect of TK1-2 is mediated in part by interfering with integrin alpha2beta1

The recombinant two kringle domain of human tissue-type plasminogen activator (TK1-2) has been shown to inhibit endothelial cell proliferation, angiogenesis, and tumor cell growth despite of sharing a low amino acid sequence homology with angiostatin. Here, we explored a possible inhibitory mechanism of action of TK1-2 by focusing on antimigratory effect. TK1-2 effectively inhibited endothelial cell migration induced by basic fibroblast growth factor or vascular endothelial growth factor in a dose-dependent manner and tube formation on Matrigel. It blocked basic fibroblast growth factor-induced or vascular endothelial growth factor-induced phosphorylation of extracellular signal-regulated kinase 1/2 and formation of actin stress fibers and focal adhesions. Interestingly, TK1-2 alone induced the weak phosphorylation of focal adhesion kinase, whereas it inhibited focal adhesion kinase phosphorylation induced by growth factors. When immobilized, TK1-2 promoted adhesion and spreading of endothelial cells compared with bovine serum albumin. However, treatment with anti-alpha(2)beta(1) blocking antibody markedly diminished endothelial cell adhesion to immobilized TK1-2 compared with anti-alpha(v)beta(3) or anti-alpha(5)beta(1) antibody. Pretreatment of soluble TK1-2 also altered the binding level of anti-alpha(2)beta(1) antibody to endothelial cells in fluorescence-activated cell sorting analysis. Indeed, a blocking antibody against integrin alpha(2)beta(1) or knocking down of integrin alpha(2) expression prevented the inhibitory effect of TK1-2 in cell migration. Therefore, these results suggest that TK1-2 inhibits endothelial cell migration through inhibition of signaling and cytoskeleton rearrangement in part by interfering with integrin alpha(2)beta(1).     

von Willebrand factor mediates platelet spreading through glycoprotein Ib and αIIbβ3 in the presence of botrocetin and ristocetin, respectively

BACKGROUND: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin alpha(IIb)beta3. OBJECTIVE: To evaluate the ability of GPIb and alpha(IIb)beta3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. RESULTS: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of alpha(IIb)beta3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon alpha(IIb)beta3 and PI3-kinase. Furthermore, alpha(IIb)beta3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an alpha(IIb)beta3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an alpha(IIb)beta3-dependent manner. CONCLUSIONS: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.     

Molecular basis for leukocyte integrin alpha(E)beta(7) adhesion to epithelial (E)-cadherin

Cadherins are expressed in tissue-restricted patterns and typically mediate homophilic adhesion. Cadherins also mediate lymphocyte adhesion, providing the opportunity for lymphocyte attachment to parenchymal cells. The best characterized example of lymphocyte adhesion to a tissue-specific cell adhesion molecule, as opposed to a vascular endothelial adhesion molecule, is the interaction between integrin alpha(E)beta(7) on intraepithelial lymphocytes and E-cadherin on epithelial cells. However, the molecular basis for an integrin-cadherin interaction is not well defined. Realization that the cadherin domain adopts a topology similar to the immunoglobulin (Ig) fold suggested that integrin recognition of E-cadherin might be similar to recognition of Ig superfamily ligands. Thus, we modeled domain 1 of human E-cadherin and studied the role of solvent-exposed loops that connect Ig-like core-forming beta strands. Mutational analyses localized the integrin alpha(E)beta(7) recognition site to the top of domain 1 at the face formed by the BC and FG loops, a site distinct from the region recognized in intercellular adhesion molecule (ICAM)-1, -2, and -3, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), vascular cell adhesion molecule 1 (VCAM-1), and fibronectin by their integrin ligands. Moreover, the integrin alpha(E)beta(7) binding site is distinct from the homophilic binding site on E-cadherin. These studies provide a conceptual basis for integrin-cadherin binding and extend the model that an Ig-like fold can serve as a scaffold for recognition.     

Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction

The integrin alpha L beta 2 (leukocyte function-associated molecule 1, CD11a/CD18) mediates activation-dependent adhesion of leukocytes. The cytoplasmic domains of alpha L beta 2 have been demonstrated to modulate adhesiveness of alpha L beta 2. Affinity changes of alpha L beta 2 for its ligand or postreceptor events can be responsible for this modulation of adhesiveness. To investigate the possible role of the alpha L beta 2 cytoplasmic domains in postreceptor events we constructed cDNA encoding chimeric proteins with intracellular alpha L beta 2 domains, which are responsible for alpha L beta 2 specific intracellular interactions, and extracellular alpha IIIb beta 3 (GP IIb/IIIa) domains, which allow the assessment of the receptor affinity state. The cDNA was stably transfected in Chinese hamster ovary cells and chimeric heterodimer formation proven by immunoprecipitations and flow cytometry. The chimeric receptors mediate adhesion to immobilized fibrinogen, and this adhesion is increased by phorbol myristate acetate and abolished by cytochalasin D. However, neither treatment affects the affinity state of the chimeric receptor, suggesting involvement of the cytoskeleton in the regulation of alpha L beta 2 mediated cell adhesion. To exclude the possibility of postoccupancy affinity changes of the chimeric receptors, we locked the receptors into a high affinity state by creating a deletion variant. The region deleted (VGFFK) is highly conserved in integrin alpha subunit cytoplasmic domains. Cotransfection of this deletion variant with a beta subunit truncation (beta 3 delta 724) and a triple mutation at 758-760 (TTT to AAA) of beta 2 abolishes adhesion without changing the affinity state. A single mutation (TTT to TAT) reduces adhesion by half without affinity change. Scanning electron microscopy reveals impaired spreading of these truncated/mutated chimeras. Immunofluorescence microscopy demonstrates a correlation between impaired adhesion and a decrease in the ability to form focal adhesions and to organize the cytoskeleton into stress fibers. These results describe the integrin/cytoskeleton interaction, the organization of the cytoskeleton, and cell spreading as postreceptor events modulating alpha L beta 2 cytoplasmic domain mediated cell adhesion. Furthermore, we demonstrate that the cytoplasmic domain of the beta 2 subunit, and within it the TTT region, are required for these postreceptor events. Additionally, we present a new approach, using deletion variants to lock integrins in a high affinity state without interfering with the investigated integrin/cytoskeleton interaction. This approach may be generally useful to investigate the role of postreceptor events in integrin- mediated cell adhesion and migration.     

Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte function-associated antigen-1 expression and function

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.     

The Pl(A2) polymorphism of integrin beta(3) enhances outside-in signaling and adhesive functions

Genetic factors are believed to influence the development of arterial thromboses. Because integrin alpha(IIb)beta(3) plays a crucial role in thrombus formation, we analyzed receptor adhesive properties using Chinese hamster ovary and human kidney embryonal 293 cells overexpressing the Pl(A1) or Pl(A2) polymorphic forms of alpha(IIb)beta(3). Soluble fibrinogen binding was no different between Pl(A1) and Pl(A2) cells, either in a resting state or when alpha(IIb)beta(3) was activated with anti-LIBS6. Pl(A1) and Pl(A2) cells bound equivalently to immobilized fibronectin. In contrast, significantly more Pl(A2) cells bound to immobilized fibrinogen in an alpha(IIb)beta(3)-dependent manner than did Pl(A1) cells. Disruption of the actin cytoskeleton by cytochalasin D abolished the increased binding of Pl(A2) cells. Compared with Pl(A1) cells, Pl(A2) cells exhibited a greater extent of polymerized actin and cell spreading, enhanced tyrosine phosphorylation of pp125(FAK), and greater fibrin clot retraction. These adhesion differences appear to depend on a signaling mechanism sensitive to receptor occupancy. Thus, the Pl(A2) polymorphism altered integrin-mediated functions of adhesion, spreading, actin cytoskeleton rearrangement, and clot retraction.     

Insulin-like growth factor receptor cooperates with integrin alpha v beta 5 to promote tumor cell dissemination in vivo.

Tumor cell interactions with adhesion proteins and growth factors likely contribute to the metastatic cascade. Evidence is provided that insulin or insulin-like growth factor-mediated signals cooperate with the commonly expressed integrin alpha v beta 5 to promote spontaneous pulmonary metastasis of multiple tumor cell types in both the chick embryo and severe combined immune deficiency mouse/human chimeric models. Expression of alpha v beta 5 in tumor cells promoted their adhesion to vitronectin in vitro. However, cell motility required cytokine stimulation, which caused redistribution of alpha-actinin to membrane-adhesive sites containing alpha v beta 5. Significantly, ligation of alpha v beta 5 and cytokine receptors were both required for spontaneous pulmonary metastasis of multiple tumor types even though it was not necessary for primary tumor growth. Thus, tumor cell metastasis can be regulated by a functional cooperation between cytokine signaling events and the adhesion receptor alpha v beta 5 in a manner independent of tumor cell growth. These findings provide evidence that integrin ligation, in conjunction with cytokine activation, plays an important role in the dissemination of malignant tumor cells.     

Direct association of pp125FAK with paxillin, the focal adhesion- targeting mechanism of pp125FAK

Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin- homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin- binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.     

Expression and functional significance of alternatively spliced CS1 fibronectin in rheumatoid arthritis microvasculature.

Expression of fibronectin (FN) isoforms containing CS1, a 25-amino acid sequence present within the alternatively spliced IIICS region of FN, has been analyzed in rheumatoid arthritis (RA) synovium. Unexpectedly, CS1-containing FN variants were exclusively found on endothelium but not extracellular matrix (ECM) of RA synovium. Lumenal expression of CS1 on RA endothelial cells, as observed by electron microscopy, correlated with inflammation in RA, since normal synovium expressed little CS1 without appreciable decrease in ECM FN. CS1 expression on human endothelial cells was further shown by FN mRNA analyses. In adhesion assays on frozen RA synovial sections, T lymphoblastoid cells expressing functionally activated alpha 4 beta 1 integrin specifically attached to the intravascular surface of RA endothelium. Binding was abrogated by both anti-alpha 4 integrin and CS1 peptides. Our observations suggest direct involvement of CS1-containing FN in recruitment of alpha 4 beta 1-expressing mononuclear leukocytes in synovitis, and provide basis for therapeutic intervention in RA.     

Evaluation of the role of platelet integrins in fibronectin-dependent spreading and adhesion.

BACKGROUND: Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation. OBJECTIVE AND METHODS: To evaluate the platelet integrins and subsequent activation pathways associated with fibronectin-dependent platelet adhesion utilizing both human and murine platelets. RESULTS: Platelets can adhere to fibronectin via the integrin alpha(IIb)beta(3), leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes alpha(IIb)beta(3)-mediated Ca(2+) mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C gamma2. In contrast, studies with blocking antibodies and mice demonstrate that alpha(5)beta(1) and alpha(v)beta(3) support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither alpha(5)beta(1) nor alpha(v)beta(3) is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen-related-peptide (CRP). CONCLUSIONS: These observations demonstrate that integrins alpha(5)beta(1) and alpha(v)beta(3) support platelet adhesion and the generation of filopodia but that, in contrast to the integrin alpha(IIb)beta(3), are unable to promote formation of lamellipodia.