Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3

Abstract

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells–fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α₅ integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α_5β1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

Fibronectin increases matrix metalloproteinase 9 expression through activation of c-Fos via extracellular-regulated kinase and phosphatidylinositol 3-kinase pathways in human lung carcinoma cells

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.     

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.     

The tetraspanin CD151 regulates cell morphology and intracellular signaling on laminin-511

The tetraspanin CD151 forms a stable complex with integrin alpha3beta1, a widely expressed laminin receptor, and is implicated in the regulation of integrin alpha3beta1-mediated cellular responses, including cell attachment, spreading and migration. However, the molecular mechanism by which CD151 regulates integrin alpha3beta1 functions remains unclear. To address this issue, we knocked down CD151 expression in A549 human lung adenocarcinoma cells by RNA interference. When plated on laminin-511 (laminin-10), the CD151-knocked-down cells showed aberrant membrane protrusions and exhibited reductions in the tyrosine phosphorylation of focal adhesion kinase, Src, p130Cas and paxillin. The formation of membrane protrusions was attenuated when the cells were either plated on surfaces coated with higher concentrations of laminin-511 or treated with the integrin beta1-activating mAb TS2/16; however, neither treatment could rescue the reduced tyrosine phosphorylation. These results indicate that CD151 knockdown weakens the integrin alpha3beta1-mediated adhesion to laminin-511 and thereby provokes an aberrant morphology, but this reduced adhesive activity is not involved in the decline of signaling events in CD151-knocked-down cells. Thus, our results suggest that CD151 regulates integrin alpha3beta1 functions in two independent aspects: potentiation of integrin alpha3beta1-mediated cell adhesion and promotion of integrin alpha3beta1-stimulated signaling events involving tyrosine phosphorylation.     

Tyrosine phosphorylation of Crk-associated substrate lymphocyte-type is a critical element in TCR- and beta 1 integrin-induced T lymphocyte migration.

Crk-associated substrate (Cas) lymphocyte-type (Cas-L) is a 105-kDa cytoplasmic protein consisting of Src homology-3 domain and multiple YXXP motifs (substrate domain). Our previous studies showed that Cas-L is tyrosine-phosphorylated following the ligation of TCR and beta 1 integrins in T lymphocytes. Here we show that Cas-L is involved in T cell motility following the ligation of TCR and beta 1 integrin. Peripheral T lymphocytes showed a marked increase of migration on fibronectin (FN) after the ligation of TCR. In contrast, the migrating Jurkat cells, in which Cas-L was marginally expressed, were less than one-tenth in number on the same condition. Transfection of wild-type Cas-L into Jurkat cells resulted in restoring CD3 plus FN-induced cell migration. Furthermore, following the ligation of beta 1 integrin alone, the Cas-L transfectants significantly migrated better than the vector control. Mutational analysis of Cas-L revealed that the substrate domain is required for both FN- and CD3-induced tyrosine phosphorylation of Cas-L and cell migration caused by FN alone and CD3 plus FN. In contrast, the Src homology-3 domain is required only for the FN-induced tyrosine phosphorylation of Cas-L and cell migration, but not for CD3-induced tyrosine phosphorylation or CD3 plus FN-induced cell migration. These data strongly suggest that Cas-L is a key molecule in T cell migration induced by the ligation of CD3 and beta 1 integrins and that tyrosine phosphorylation of Cas-L is essential for T cell migration.     

JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.     

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.     

Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by alpha 3beta 1 integrin in oral keratinocytes

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.     

Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor

The functional interaction ("cross-talk") of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through alpha3beta1 and alpha5beta1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, alpha3beta1 or alpha5beta1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (alpha3beta1 versus alpha5beta1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with alpha3/alpha5/beta1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain.     

Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling.

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of alpha3beta1-TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of alpha3beta1-TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B alpha3beta1-TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.     

Cutting edge: a novel function for the SLAP-130/FYB adapter protein in beta 1 integrin signaling and T lymphocyte migration

The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that alpha4beta1 integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain-containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the alpha4beta1 integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59fyn. Activation of normal T cells, but not H9 T cells, via alpha4beta1 leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1alpha. These results identify SLAP-130/FYB as a new tyrosine phosphorylated substrate in beta1 integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.     

Integrin-associated protein stimulates alpha2beta1-dependent chemotaxis via Gi-mediated inhibition of adenylate cyclase and extracellular-regulated kinases.

Integrin-associated protein (IAP/CD47) augments the function of alpha2beta1 integrin in smooth muscle cells (SMC), resulting in enhanced chemotaxis toward soluble collagen (Wang, X-Q., and W.A. Frazier. 1998. Mol. Biol. Cell. 9:865). IAP-deficient SMC derived from IAP(-/-) animals did not migrate in response to 4N1K (KRFYVVMWKK), a peptide agonist of IAP derived from the COOH-terminal domain of thrombospondin-1 (TSP1). When normal SMC were preincubated with 4N1K or an anti-alpha2beta1 function-stimulating antibody, cell migration to soluble collagen was significantly enhanced. 4N1K-induced chemotaxis was blocked by treatment of SMC with pertussis toxin indicating that IAP acts through Gi. In agreement with this, 4N1K evoked a rapid decrease in cAMP levels which was intensified in the presence of collagen, and forskolin and 8-Br-cAMP both inhibited SMC migration stimulated via IAP. 4N1K strongly inhibited extracellular regulated kinase (ERK) activation in SMC attaching to collagen and reduced basal ERK activity in suspended SMC. Pertussis toxin treatment of SMC significantly activated ERK, suggesting that an inhibitory input was alleviated. Inhibition of ERK activity by (a) the MAP kinase kinase (MEK) inhibitor, PD98059, (b) antisense oligonucleotide depletion of ERK, and (c) expression of mitogen-activated protein (MAP) kinase phosphatase-1 in SMC all led to increased migration to collagen, 4N1K, or 4N1K plus collagen. Thus, IAP stimulates alpha2beta1 integrin-mediated SMC migration via Gi-mediated inhibition of ERK activity and suppression of cyclic AMP levels. Both of these signaling pathways could directly modulate the state of the integrin as well as impact downstream components of the cell motility apparatus.     

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.     

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.     

Integrin alphavbeta3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin alpha2beta1

The interplay between the collagen-binding integrin, alpha2beta1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type alpha2beta1 (alpha2beta1A) or alpha2beta1 with a Y783/795F mutation in the cytoplasmic tail of the beta1 subunit (alpha2beta1Amut) in the beta1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the alpha1 and alpha2 integrin subunits and do not adhere to collagen even after transfection with beta1A. Cells expressing alpha2beta1Amut contracted three-dimensional collagen lattices less efficiently than those expressing alpha2beta1A. PDGF-BB significantly stimulated lattice contraction by GD25-alpha2beta1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130(Cas) were phosphorylated when GD25-alpha2beta1A cells, but not GD25-alpha2beta1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130(Cas) only in GD25-alpha2beta1A cells. However, when cultured within collagen lattices, FAK and p130(Cas) phosphorylation were stimulated in both alpha2beta1A- and alpha2beta1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-alpha2beta1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-alpha2beta1Amut cells were mediated by the alphavbeta3 integrin. Phosphorylation of p130(Cas), but not FAK, in GD25-alpha2beta1Amut cells seeded in collagen lattices also depended on alphavbeta3. Our results show that PDGF-BB stimulation of fibroblast-collagen interactions is mediated by the alphavbeta3 integrin when beta1 integrin function is impaired.     

Insulin-like growth factor I controls adhesion strength mediated by alpha5beta1 integrins in motile carcinoma cells

One of the intriguing questions regarding cell motility concerns the mechanism that makes stationary cells move. Here, we provide the first physical evidence that the onset of breast cancer cell motility in response to insulin-like growth factor I (IGF-I) correlates with lowering of adhesion strength from 2.52 +/- 0.20 to 1.52 +/- 0.13 microdynes/microm2 in cells attached to fibronectin via alpha5beta1 integrin. The adhesion strength depends on the dose of IGF-I and time of IGF-I treatment. Weakening of cell-matrix adhesion is blocked significantly (p < 0.01) by the catalytically inactive IGF-I receptor (IGF-IR) and the phosphoinositide 3-kinase (PI-3 kinase) inhibitor LY-294002, but it is unaffected by mitogen-activated protein kinase kinase inhibitor UO-126 and Src kinase inhibitor PP2. Sustained blockade of Rho-associated kinase (ROCK) with Y-27632 down-regulates adhesion strength in stationary, but not in IGF-I-treated, cells. Jasplakinolide, a drug that prevents actin filament disassembly, counteracts the effect of IGF-I on integrin-mediated cell adhesion. In the absence of growth factor signaling, ROCK supports a strong adhesion via alpha5beta1 integrin, whereas activation of the IGF-IR kinase reduces cell-matrix adhesion through a PI-3K-dependent, but ROCK-independent, mechanism. We propose that disassembly of the actin filaments via PI-3 kinase pathway contributes to weakening of adhesion strength and induction of cell movement. Understanding how cell adhesion and migration are coordinated has an important application in cancer research, developmental biology, and tissue bioengineering.     

Ganglioside GM3 inhibits matrix metalloproteinase-9 activation and disrupts its association with integrin

Gangliosides GM3 and GT1b both inhibit epithelial cell adhesion and migration on fibronectin. GT1b binds to integrin alpha5beta1 and blocks the integrin-fibronectin interaction; GM3 does not interact with integrin, and its effect is poorly understood. We evaluated the effects of endogenous modulation of GM3 expression on epithelial cell motility on several matrices and the mechanism of these effects. Endogenous accumulation of GM3 decreased cell migration on fibronectin, types I, IV, and VII collagen matrices; depletion of GM3 dramatically increased cell migration, regardless of matrix. GM3 overexpression and depletion in vitro correlated inversely with the expression and activity of matrix metalloproteinase-9; consistently, the cell migration stimulated by GM3 depletion is reversed by inhibition of matrix metalloproteinase-9 activity. Accumulation and depletion of GM3 in epithelial cells grown on fibronectin also correlated inversely with epidermal growth factor receptor and mitogen activated protein kinase phosphorylation and with Jun expression. Ganglioside depletion facilitated the co-immunoprecipitation of matrix metal-loproteinase-9 and integrin alpha5beta1, while endogenous accumulation of GM3, but not GT1b, blocked the co-immunoprecipitation. These data suggest modulation of epidermal growth factor receptor signaling and dissociation of integrin/matrix metalloproteinase-9 as mechanisms for the GM3-induced effects on matrix metalloproteinase-9 function.