Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase

The extracellular matrix (ECM) facilitates pancreatic cancer cells survival, which is of central importance for pancreatic adenocarcinoma that is highly fibrotic. Here, we show that reactive oxygen species (ROS) mediate the prosurvival effect of ECM in human pancreatic cancer cells. Fibronectin and laminin stimulated ROS production and NADPH oxidase activation in pancreatic cancer cells. Both pharmacological and molecular approaches show that fibronectin stimulated ROS production through activation of NADPH oxidase and NADPH oxidase-independent pathways and that 5-lipoxygenase (5-LO) mediates both these pathways. Analyses of the mechanisms of ROS production by ECM proteins and growth factors indicate that activation of NADPH oxidase (Nox4) is a common mechanism employed both by ECM proteins and growth factors to increase ROS in pancreatic cancer cells. We also found that Nox4 is present in human pancreatic adenocarcinoma tissues and that these tissues display membrane NADPH oxidase activity. ECM proteins and growth factors activate NADPH oxidase through different mechanisms; in contrast to ECM proteins, growth factors activate NADPH oxidase through 5-LO-independent mechanisms. Inhibition of 5-LO or NADPH oxidase with pharmacological inhibitors of these enzymes and with Nox4 or 5-LO antisense oligonucleotides markedly stimulated apoptosis in cancer cells cultured on fibronectin. Our results indicate that ROS generation via 5-LO and downstream NADPH oxidase mediates the prosurvival effect of ECM in pancreatic cancer cells. These mechanisms may play an important role in pancreatic cancer resistance to treatments and thus represent novel therapeutic targets.     

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.     

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.     

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.     

Integrins enhance platelet-derived growth factor (PDGF)-dependent responses by altering the signal relay enzymes that are recruited to the PDGF beta receptor.

Since the extracellular matrix (ECM) can promote platelet-derived growth factor (PDGF)-dependent responses, we hypothesized that the ECM mediates this effect by preventing the PDGF beta receptor (betaPDGFR) from associating with the negative regulator, RasGAP (the GTPase-activating protein of Ras). We found that binding of RasGAP to the wild-type betaPDGFR was decreased; the activation of Ras and Erk was enhanced, and [3H]thymidine uptake was better in cells cultured on fibronectin than in cells cultured on polylysine. To investigate the mechanism by which culturing cells on fibronectin diminished the recruitment of RasGAP to the betaPDGFR, we focused on SHP-2 since it dephosphorylates the betaPDGFR at the phosphotyrosine required for binding of RasGAP. Culturing cells on fibronectin increased the amount of SHP-2 that associated with the betaPDGFR. Furthermore, cells expressing receptor mutants that failed to associate with SHP-2 were insensitive to fibronectin. The ECM enhances PDGF-dependent responses by increasing the association of SHP-2 with the betaPDGFR, which in turn decreases the time that RasGAP interacts with the receptor. Thus, fibronectin changes PDGF-dependent signaling and biological responses by altering the signal relay enzymes that are recruited to the receptor.     

Tyrosine phosphorylation of the beta3-subunit of the alphaVbeta3 integrin is required for embrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor

Ligand occupancy of the alphaVbeta3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the alphaVbeta3 integrin on beta3 phosphorylation and studied the role of beta3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to alphaVbeta3 induced tyrosine phosphorylation of the beta3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with beta3. In confluent SMCs, the beta3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in beta3 phosphorylation and resulted in decreased SHP-2 association with beta3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant beta3 that had two tyrosines changed to phenylalanines showed a 89.9 +/- 1.2% decrease in beta3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated beta3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant beta3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of alphaVbeta3 regulates the SMC response to IGF-I.     

Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2.

Activation of insulin-like growth factor I receptor (IGF-IR) kinase is an important site of control of IGF-I-linked intracellular signaling pathways. One potentially important regulatory variable is IGF-IR dephosphorylation. It has been shown that SHP-2, a tyrosine phosphatase, can bind to the activated IGF-IR in vitro; however, its role in IGF-IR dephosphorylation in whole cells is unknown. These studies were undertaken to determine whether SHP-2 was a candidate for mediating IGF-IR dephosphorylation. The IGF-IR in smooth muscle cells was dephosphorylated rapidly beginning 10 min after ligand addition, and this was temporally associated with SHP-2 binding to the receptor. IGF-I stimulated SHPS-1 phosphorylation and the subsequent recruitment of SHP-2. In cells expressing a SHPS-1 mutant that did not bind SHP-2 there was no recruitment of SHP-2 to the IGF-IR. Cells expressing a catalytically inactive form of SHP-2 showed SHP-2 recruitment to SHPS-1, but this did not result in SHPS-1 dephosphorylation, and there was a prolonged IGF-IR phosphorylation response after IGF-I stimulation. These studies indicate that IGF-IR stimulates phosphorylation of SHPS-1 which is critical for SHP-2 recruitment to the plasma membrane and for its recruitment to the IGF-IR. Recruitment of SHP-2 to the receptor then results in receptor dephosphorylation. The regulation of this process may be an important determinant of IGF-IR-mediated signaling.     

Integrin alpha5/beta1 mediates fibronectin-dependent epithelial cell proliferation through epidermal growth factor receptor activation

Human integrin alpha5 was transfected into the integrin alpha5/beta1-negative intestinal epithelial cell line Caco-2 to study EGF receptor (EGFR) and integrin alpha5/beta1 signaling interactions involved in epithelial cell proliferation. On uncoated or fibronectin-coated plastic, the integrin alpha5 and control (vector only) transfectants grew at similar rates. In the presence of the EGFR antagonistic mAb 225, the integrin alpha5 transfectants and controls were significantly growth inhibited on plastic. However, when cultured on fibronectin, the integrin alpha5 transfectants were not growth inhibited by mAb 225. The reversal of mAb 225-mediated growth inhibition on fibronectin for the integrin alpha5 transfectants correlated with activation of the EGFR, activation of MAPK, and expression of proliferating cell nuclear antigen. EGFR kinase activity was necessary for both MAPK activation and integrin alpha5/beta1-mediated cell proliferation. Although EGFR activation occurred when either the integrin alpha5-transfected or control cells were cultured on fibronectin, coprecipitation of the EGFR with SHC could be demonstrated only in the integrin alpha5-transfected cells. These results suggest that integrin alpha5/beta1 mediates fibronectin-induced epithelial cell proliferation through activation of the EGFR.     

TNFalpha-induced apoptosis and integrin switching in human extravillous trophoblast cell line

Differentiation of extravillous trophoblast cells (EVT) to an invasive phenotype plays an essential role in establishing and maintaining feto-placental organization during human pregnancy. A switch in integrin expression occurs during this differentiation and is accompanied by changes in the extracellular matrix (ECM). Alteration of EVT behavior is also modulated by cytokines. To investigate the molecular interactions involved in the EVT differentiation, we examined the effects of cytokines and ECM on the human EVT cell line, TCL1 cells. We found that tumor necrosis factor alpha (TNFalpha) induced apoptosis in TCL1 cells but not in JEG3 cells derived from choriocarcinoma while the addition of interleukin-1beta, leukemia inhibitory factor, or transforming growth factor had no effect on TCL1 cells. This apoptosis was suppressed when TCL1 cells were seeded on fibronectin (Fn), collagen type I (C1), collagen type IV (C4), or laminin (Ln). Wortmannin, a specific PI3 kinase inhibitor, inhibited this suppression. Spreading assays and adhesion blocking assays indicated that TCL1 cells express integrin-alpha5 and -alpha6 and beta1 and beta4 subunits. Adhesion on Fn is mediated by alpha5beta1, and adhesion on C1, C4, or Ln is mediated by alpha6beta1 integrins. TNFalpha suppressed alpha6 integrin expression and enhanced alpha1 integrin expression in a dose-dependent manner. In addition, aggregation of beta1 subunits on C4 was detected after addition of TNFalpha. Taken together, these results suggest that TNFalpha and ECM, through activation of PI3 kinase mediated by beta1 integrin signaling, might collaboratively regulate differentiation of trophoblast cells through integrin signaling in establishing and maintaining successful pregnancy.     

Interaction between insulin-like growth factor-I receptor and alphaVbeta3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs

Integrins are heterodimeric transmembrane proteins that mediate cell attachment to extracellular matrix, migration, division, and inhibition of apoptosis. Because growth factors are also important for these processes, there has been interest in cooperative signaling between growth factor receptors and integrins. IGF-I is an important growth factor for vascular cells. One integrin, alphaVbeta3, that is expressed in smooth muscle cells modulates IGF-I actions. Ligand occupancy of alphaVbeta3 is required for IGF-I to stimulate cell migration and division. Src homology 2 containing tyrosine phosphatase (SHP-2) is a tyrosine phosphatase whose recruitment to signaling molecules is stimulated by growth factors including IGF-I. If alphaVbeta3 ligand occupancy is inhibited, there is no recruitment of SHP-2 to alphaVbeta3 and its transfer to downstream signaling molecules is blocked. Ligand occupancy of alphaVbeta3 stimulates tyrosine phosphorylation of the beta3-subunit, resulting in recruitment of SHP-2. This transfer is mediated by an insulin receptor substrate-1-related protein termed DOK-1. Subsequently, SHP-2 is transferred to another transmembrane protein, SHPS-1. This transfer requires IGF-I receptor-mediated tyrosine phosphorylation of SHPS-1, which contains two YXXL motifs that mediate SHP-2 binding. The transfer of SHP-2 to SHPS-1 is also required for recruitment of Shc to SHPS-1. Ligand occupancy of alphaVbeta3 results in sustained Shc phosphorylation and enhanced Shc recruitment. Shc activation results in induction of MAPK. Inhibition of the Shc/SHPS-1 complex formation results in failure to achieve sustained MAPK activation and an attenuated mitogenic response. Thus, within the vessel wall, a mechanism exists whereby ligand occupancy of the alphaVbeta3 integrin is required for assembly of a multicomponent membrane signaling complex that is necessary for cells to respond optimally to IGF-I.     

Mechanism of BMP-2 stimulated adhesion of osteoblastic cells to titanium alloy.

Cell adhesion is dependent on many factors, including the repertoire of extracellular matrix (ECM) proteins and their receptors, e.g. integrins, synthesized by the cell, the composition of the ECM adsorbed to the surface, and the intrinsic chemistry of the surface. Factors that govern bone cell, i.e. osteoblast, adhesion and ECM elaboration significantly influence its re-modeling into mature bone, and ultimately its ability to integrate with biomaterials used for orthopedic prostheses. In this study, we have investigated how treatment with bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta) superfamily that promotes ectopic bone formation, modulates the organization and expression of osteoblastic cell proteins. Specifically, we analyzed how BMP-2 treatment affects cytoskeletal components, ECM, and alpha 5 and beta 1 integrin receptor subunits in osteoblastic cells plated on Ti6A14V, a titanium alloy widely used for orthopedic implants that interacts with bone cells in vitro and in vivo. Osteoblastic cells were pre-treated with BMP-2 for 12 h prior to plating; BMP-2 treatment stimulated adhesion and proliferation of osteoblastic cells and this adhesive advantage was reflected in enhanced long-term matrix mineralization in the BMP-2 pretreated cultures. Confocal laser scanning microscopic analysis of BMP-2 treated cells showed that enhanced cytoskeletal organization and focal contact formation occurred. These changes were accompanied by a concomitant increase in the spatial organization of fibronectin, whereas vitronectin, collagen type I, osteopontin, and osteocalcin showed little change. The changes in ECM organization correlated with increased fibronectin, alpha 5 and beta 1 integrin subunit, and focal adhesion kinase (p125FAK) expression, as well as increased p125FAK phosphorylation. By confocal microscopy, the alpha 5 integrin subunit was more concentrated in lamellipodia after BMP-2 treatment. These results demonstrate that BMP-2 significantly altered osteoblastic cytoskeletal and ECM organization and enhanced expression of fibronectin and of specific integrin receptor subunits, with concomitant changes in the levels and phosphorylation of p125FAK. These effects may contribute to downstream cellular responses important for bone cell function, and growth.     

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by beta1 integrins

We show here that beta1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The beta1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, beta1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, beta1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the beta1 cytodomain plays an important role in mediating beta1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, beta1A associates with IRS-1 and beta1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.     

Dual function of the extracellular matrix: stimulatory for cell cycle progression of naive T cells and antiapoptotic for tissue-derived memory T cells

Tissue T cells encounter Ag in a distinct microenvironment, where they are embedded in the interstitial extracellular matrix (ECM). In contrast, while naive T cells are exposed to Ag in the lymph node, immediately after naive T cells are activated they must extravasate into the ECM to function effectively. Because integrin-mediated adhesion to the ECM modulates cell cycle progression and survival in adherent nonimmune cells, we hypothesize that blood and tissue-derived T cells have similarly adapted their behavior to their first or continued encounter with ECM. T cells from peripheral blood (PBT) and tissue (the intestinal lamina propria T cell (LPT)) were stimulated with anti-CD3-coated beads in the presence or absence of native ECM derived from intestinal fibroblasts, plate-immobilized fibronectin, or collagen type I. Native ECM and collagen, but not fibronectin, induced in anti-CD3 activated PBT a 4- to 5-fold increase in the entry, progression, and completion of the cell cycle over that triggered by anti-CD3 alone. Neutralizing beta1 integrin Abs abrogated this increase. None of these ECM proteins stimulated cell cycle progression in LPT. In contrast, anti-CD3 activation of LPT in the presence of native ECM and fibronectin reduced activation-induced cell death by 40%. These results demonstrate that naive and effector/memory T cells respond differently upon exposure to specific ECM components. When naive PBT encounter Ag in the context of ECM, their progression through the cell cycle is enhanced, favoring clonal expansion; while tissue T cell longevity may be mediated by interactions with the ECM.     

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.     

Regulation of expression of fibronectin and its receptor, alpha 5 beta 1, during development and regeneration of peripheral nerve.

The extracellular matrix glycoprotein, fibronectin, is a potent promoter of peripheral neurite outgrowth. Interactions of peripheral neurons with fibronectin have been shown to be primarily mediated by the beta 1 class of integrin heterodimers. In the present study, we have examined the expression and regulation of fibronectin and its integrin receptor, alpha 5 beta 1, in developing and regenerating chick peripheral nerve. We show that fibronectin and alpha 5 beta 1 are expressed at comparatively high levels in developing nerve with alpha 5 beta 1 expression on axons and non-neuronal cells. With nerve maturation, both proteins are less prominently expressed and the cellular pattern of alpha 5 beta 1 expression becomes more restricted. Following lesion of mature nerve, both fibronectin and alpha 5 beta 1 are strongly induced with prominent expression of alpha 5 beta 1 on regenerating neurites and Schwann cells. The elevation in fibronectin levels in the regenerating nerve is highest in the vicinity of the lesion, an area undergoing extensive cellular remodeling including Schwann cell migration and growth cone extension. Our results suggest that fibronectin and its receptor, alpha 5 beta 1, may mediate functionally important interactions in the development and regeneration of peripheral nerve.     

Conformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive activities of thrombospondin-1

The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by alpha4beta1 and alpha5beta1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by alpha3beta1 integrin and required that the alpha3beta1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified alpha3beta1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by alpha3beta1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.     

Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor

To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration.     

Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration

The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit beta1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and beta1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either beta1 integrin or PP2A in a complex at the IGF-IR.     

Extracellular matrix regulates endothelial functions through interaction of VEGFR-3 and integrin alpha5beta1

Endothelium extracellular matrix (ECM) interactions can provide distinct spatial and molecular signals which control cellular proliferation, migration, and differentiation. Here, we investigated the role of fibronectin (FN), a major ECM protein, on the functions of lymphatic endothelial cells (LEC). We observed that FN, the ligand for integrin alpha5beta1, selectively promoted the growth of LEC as compared with vitronectin (VN) in the presence of the ligand for vascular endothelial growth factor receptor 3 [VEGFR-3 (VEGF-C156S)]. Upon investigating the mechanisms whereby ECM components regulate VEGFR-3 signaling, we found that FN transactivated VEGFR-3 and significantly enhanced the phosphorylation of VEGFR-3 induced by VEGF-C156S as compared to VN. An enhanced association of the integrin subunit alpha5 or beta1 with VEGFR-3, after stimulation with VEGF-C156S, was observed by co-immunoprecipitation. While blockade of integrin alpha5beta1 inhibited the VEGF-C156S-induced phosphorylation of VEGFR-3, no similar effect was obtained by blocking integrin alphavbeta3. FN also protected the endothelial cells from serum deprivation-induced apoptosis. Moreover, while the specific PI3 kinase inhibitor, LY294002, abolished this FN-mediated cell survival, the MAPK kinase inhibitor, PD98059, had no significant effect. Furthermore, a dominant-negative mutant of VEGFR-3 (G857R) reduced VEGF-C156S or FN-mediated cell survival, as well as the activities of PI3 kinase/Akt. Our results indicate that integrin alpha5beta1 participates in the activation of both VEGFR-3 and its downstream PI3 kinase/Akt signaling pathway, which is essential for FN-mediated lymphatic endothelial cell survival and proliferation.     

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line

We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. alpha3beta1, alpha5beta1, alpha6beta1/beta4, alpha8beta1 and alpha(v)beta1/beta5/beta6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, alpha2beta1, alpha(v)beta6 and alpha6beta1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas alpha(v)beta5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.