Fibronectin–integrin mediated signaling in human cervical cancer cells (SiHa)

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin–integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin–integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-κB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.     

14-3-3beta-Rac1-p21 activated kinase signaling regulates Akt1-mediated cytoskeletal organization, lamellipodia formation and fibronectin matrix assembly

Akt1 belongs to the three-gene Akt family and functions as a serine-threonine kinase regulating phosphorylation of an array of substrates and mediating cellular processes such as cell migration, proliferation, survival, and cell cycle. Our previous studies have established the importance of Akt1 in angiogenesis and absence of Akt1 resulted in impaired integrin activation, adhesion, migration, and extracellular matrix assembly by endothelial cells and fibroblasts. In this study, we identify the downstream signaling pathways activated by Akt1 in the regulation of these cellular events. We demonstrate here that Akt1 is necessary for the growth factor stimulated activation of 14-3-3beta-Rac1-p21 activated kinase (Pak) pathway in endothelial cells and fibroblasts. While activation of Akt1 resulted in translocation of Rac1 to membrane ruffles, enhanced Rac1 activity, Pak1 phosphorylation, and lamellipodia formation, resulting in enhanced adhesion and assembly of fibronectin, inhibition of Akt1 resulted in inhibition of these processes due to impaired Rac1-Pak signaling. Formation of lamellipodia, adhesion, and fibronectin assembly by myristoylated Akt1 expression in NIH 3T3 fibroblasts was inhibited by co-expression with either dominant negative Rac1 or dominant negative Pak1. In contrast, impaired lamellipodia formation, adhesion, and fibronectin assembly by dominant negative-Akt1 expression was rescued by co-expression with either constitutively active-Rac1 or -Pak1. Moreover, previously reported defects in adhesion and extracellular matrix assembly by Akt1(-/-) fibroblasts could be rescued by expression with either active-Rac1 or -Pak1, implying the importance of Rac1-Pak signaling in growth factor stimulated cytoskeletal assembly, lamellipodia formation and cell migration in endothelial cells and fibroblasts downstream of Akt1 activation.     

Akt1 signaling regulates integrin activation, matrix recognition, and fibronectin assembly

Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1(-/-) fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin beta(1) blocking antibodies, treatment with beta(1)-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1(-/-) fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin alpha(5)beta(1).     

Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in extracellular matrix-induced cell migration and the activation of extracellular signal-regulated kinase (ERK). We showed here that transfection of the MT1-MMP gene into HeLa cells promoted fibronectin-induced cell migration, which was accompanied by fibronectin degradation and reduction of stable focal adhesions, which function as anchors for actin-stress fibers. MT1-MMP expression attenuated integrin clustering that was induced by adhesion of cells to fibronectin. The attenuation of integrin clustering was abrogated by MT1-MMP inhibition with a synthetic MMP inhibitor, BB94. When cultured on fibronectin, HT1080 cells, which endogenously express MT1-MMP, showed so-called motile morphology with well-organized focal adhesion formation, well-oriented actin-stress fiber formation, and the lysis of fibronectin through trails of cell migration. Inhibition of endogenous MT1-MMP by BB94 treatment or expression of the MT1-MMP carboxyl-terminal domain, which negatively regulates MT1-MMP activity, resulted in the suppression of fibronectin lysis and cell migration. BB94 treatment promoted stable focal adhesion formation concomitant with enhanced phosphorylation of tyrosine 397 of focal adhesion kinase (FAK) and reduced ERK activation. These results suggest that lysis of the extracellular matrix by MT1-MMP promotes focal adhesion turnover and subsequent ERK activation, which in turn stimulates cell migration.     

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.     

Thromboxane receptor signaling is required for fibronectin-induced matrix metalloproteinase 9 production by human and murine macrophages and is attenuated by the Arhgef1 molecule

During an inflammatory response, resident and newly recruited tissue macrophages adhere to extracellular matrix and cell-bound integrin ligands. This interaction induces the expression of pro-inflammatory mediators that include matrix metalloproteinases (MMPs). Arhgef1 is an intracellular signaling molecule expressed by myeloid cells that normally attenuates murine macrophage MMP production in vivo and in vitro after cell culture on the extracellular matrix protein, fibronectin. In this study, we have extended the characterization of this fibronectin-induced Arhgef1-regulated signaling pathway in both human and murine myeloid cells. Our results show that MMP9 production by fibronectin-stimulated monocytes and macrophages depends on autocrine thromboxane receptor signaling and that under normal conditions, this signaling pathway is attenuated by Arhgef1. Finally, we show that the expression of ARHGEF1 by human peripheral blood monocytes varies between individuals and inversely correlates with fibronectin-mediated MMP9 production.     

Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.     

MEKK2 regulates focal adhesion stability and motility in invasive breast cancer cells

MEK Kinase 2 (MEKK2) is a serine/threonine kinase that functions as a MAPK kinase kinase (MAP3K) to regulate activation of Mitogen-activated Protein Kinases (MAPKs). We recently have demonstrated that ablation of MEKK2 expression in invasive breast tumor cells dramatically inhibits xenograft metastasis, but the mechanism by which MEKK2 influences metastasis-related tumor cell function is unknown. In this study, we investigate MEKK2 function and demonstrate that silencing MEKK2 expression in breast tumor cell significantly enhances cell spread area and focal adhesion stability while reducing cell migration. We show that cell attachment to the matrix proteins fibronectin or Matrigel induces MEKK2 activation and localization to focal adhesions. Further, we reveal that MEKK2 ablation enhances focal adhesion size and frequency, thereby linking MEKK2 function to focal adhesion stability. Finally, we show that MEKK2 knockdown inhibits fibronectin-induced Extracellular Signal-Regulated Kinase 5 (ERK5) signaling and Focal Adhesion Kinase (FAK) autophosphorylation. Taken together, our results strongly support a role for MEKK2 as a regulator of signaling that modulates breast tumor cell spread area and migration through control of focal adhesion stability.     

Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways

Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.     

The copper binding domain of SPARC mediates cell survival in vitro via interaction with integrin beta1 and activation of integrin-linked kinase

Secreted protein acidic and rich in cysteine (SPARC) is important for the normal growth and maintenance of the murine lens. SPARC-null animals develop cataracts associated with a derangement of the lens capsule basement membrane and alterations in lens fiber morphology. Cellular stress and disregulation of apoptotic pathways within lens epithelial cells (LEC) are linked to cataract formation. To identify molecular targets of SPARC that are linked to this disorder, we stressed wild-type (WT) and SPARC-null LEC by serum deprivation or exposure to tunicamycin. SPARC enhanced signaling by integrin-linked kinase (ILK), a serine/threonine kinase known to enhance cell survival in vitro. In response to stress, an ILK-dependent decrease in apoptosis was observed in WT relative to SPARCg-null LEC. Co-immunoprecipitation and cross-linking of cell lysates revealed enhanced levels of a SPARC-integrin beta1 complex during stress. Competition with monoclonal antibodies and peptides indicated that the copper binding domain of SPARC is required for SPARC-mediated response to stress. Inhibiting the binding and/or activity of ILK, integrin beta1, or SPARC resulted in increased apoptosis of stressed LEC. We conclude that SPARC protects cells from stress-induced apoptosis in vitro via an interaction with integrin beta1 heterodimers that enhances ILK activation and pro-survival activity.     

Secreted protein acidic, rich in cysteine (SPARC), mediates cellular survival of gliomas through AKT activation

Secreted protein acidic, rich in cysteine (SPARC), is an extracellular matrix protein expressed in many advanced cancers, including malignant gliomas. We and others have previously shown that human glioma cell lines engineered to overexpress SPARC adopt an invasive phenotype. We now show that SPARC expression increases cell survival under stress initiated by serum withdrawal through a decrease in apoptosis. Phosphatidylinositol 3-OH kinase/AKT is a potent pro-survival pathway that contributes to the malignancy of gliomas. Cells expressing SPARC display increased AKT activation with decreased caspase 3/7 activity. Exogenous SPARC rapidly induces AKT phosphorylation, an effect that is blocked by a neutralizing SPARC antibody. Furthermore, AKT activation is essential for the anti-apoptotic effects of SPARC as the decreased apoptosis and caspase activity associated with SPARC expression can be blocked with dominant-negative AKT or a specific AKT inhibitor. As tumor cells face stressful microenvironments particularly during the process of invasion, these results suggest that SPARC functions, in part, to promote tumor progression by enabling tumor cells to survive under stressful conditions.     

Insulin-like growth factor-I receptor mediates the prosurvival effect of fibronectin

We recently showed that extracellular matrix (ECM) proteins, which are abundant in desmoplastic pancreatic tumor, are as potent as growth factors in inhibiting apoptosis in pancreatic cancer (PaCa) cells. Here we show that fibronectin, a major ECM component, engages insulin-like growth factor-I receptor (IGF-IR) to inhibit PaCa cell death. We found that fibronectin-induced protection from apoptosis is fully mediated by IGF-IR and is independent of IGF-I. Pharmacologic and molecular inhibitions of IGF-IR stimulated apoptosis and prevented the prosurvival effect of fibronectin in PaCa cells. Our data indicate that fibronectin protects from apoptosis through trans-activation of IGF-IR. We showed that fibronectin stimulated complex formation between its receptor beta3 integrin and protein-tyrosine phosphatase SHP-2. This process of complex formation, in turn, prevents SHP-2 from dephosphorylating IGF-IR resulting in sustained phosphorylation of IGF-IR and leading to the downstream activation of Akt kinase, up-regulation of antiapoptotic Bcl(xL), and inhibition of apoptosis. Among ECM proteins tested only fibronectin and laminin but not vitronectin and collagen I stimulated trans-activation of IGF-IR. Interaction of fibronectin with beta3 but not beta1 integrin receptors mediates the survival pathway. In contrast, fibronectin-induced adhesion is mediated through beta1 integrin receptor and is IGF-IR-independent. Thus, our results indicate that the prosurvival effect of fibronectin in PaCa cells is mediated by trans-activation of IGF-IR induced by the beta3 integrin receptor. The data suggest IGF-IR as a key target for prevention of the prosurvival effects of ECM proteins and growth factors in pancreatic cancer.     

Regulation of gene expression by SPARC during angiogenesis in vitro. Changes in fibronectin, thrombospondin-1, and plasminogen activator inhibitor-1.

Angiogenesis in vitro, the formation of capillary-like structures by cultured endothelial cells, is associated with changes in the expression of several extracellular matrix proteins. The expression of SPARC, a secreted collagen-binding glycoprotein, has been shown to increase significantly during this process. We now show that addition of purified SPARC protein, or an N-terminal synthetic peptide (SPARC4-23), to strains of bovine aortic endothelial cells undergoing angiogenesis in vitro resulted in a dose-dependent decrease in the synthesis of fibronectin and thrombospondin-1 and an increase in the synthesis of type 1-plasminogen activator inhibitor. SPARC decreased fibronectin mRNA by 75% over 48 h, an effect that was inhibited by anti-SPARC immunoglobulins. Levels of thrombospondin-1 mRNA were diminished by 80%. Over a similar time course, both mRNA and protein levels of type 1-plasminogen activator inhibitor (PAI-1) were enhanced by SPARC and the SPARC4-23 peptide. The effects were dose-dependent with concentrations of SPARC between 1 and 30 micrograms/ml. In contrast, no changes were observed in the levels of either type I collagen mRNA or secreted gelatinases. Half-maximal induction of PAI-1 mRNA or inhibition of fibronectin and thrombospondin mRNAs occurred with 2-5 micrograms/ml SPARC and approximately 0.05 mM SPARC4-23. Strains of endothelial cells that did not form cords and tubes in vitro had reduced or undetectable responses to SPARC under identical conditions. These results demonstrate that SPARC modulates the synthesis of a subset of secreted proteins and identify an N-terminal acidic sequence as a region of the protein that provides an active site. SPARC might therefore function, in part, to achieve an optimal ratio among different components of the extracellular matrix. This activity would be consistent with known effects of SPARC on cellular morphology and proliferation that might contribute to the regulation of angiogenesis in vivo.     

Features of fibronectin-dependent activation of ribosomal protein S6 kinase (S6K1 and S6K2)

Integrin family of adhesion receptors play an important role in organizing the actin cytoskeleton and in signal transduction from the extracellular matrix. The previous studies have shown that exposure of fibroblast cells to extracellular matrix proteins activates ribosomal S6 kinase 1 (S6K1) pathway in a ligand dependent manner. Recently, a new, highly homologous ribosomal S6 kinase, termed S6K2, was identified. It has 70% amino acid identity in the overall sequence with S6K1, and the potential phosphorylation sites of S6K1 are conserved in S6K2. However, the N- and C-terminal domains of S6K2 are quite different from those of S6K1. In this study we have examined dynamics of fibronectin-induced activation of these two kinases, transiently expressed in human HEK 293 cells. Differences between profiles of activation of S6K1 and S6K2 were observed in the early period of fibronectin stimulation. Fibronectin-induced changes in S6K2 activity were closely correlated with phosphorylation at Ser423, which is homologues to Ser 434 of S6K1. Although we didn't observe considerable changes in phosphorylation of S6K1 at Ser434, suggesting potential differences in the regulation of these homologous kinases upon fibronectin stimulation.     

Inhibition of integrin-linked kinase/protein kinase B/Akt signaling: mechanism for ganglioside-induced apoptosis

Ganglioside GT1b inhibits keratinocyte attachment to and migration on a fibronectin matrix by binding to alpha(5)beta(1) and preventing alpha(5)beta(1) interaction with fibronectin. The role of gangliosides in triggering keratinocyte apoptosis, however, is unknown. Addition of GT1b to keratinocyte-derived SCC12 cells, grown in serum-free medium but exposed to fibronectin, suppressed Bad phosphorylation, activated caspase-9, and inhibited cyclin D and E expression, resulting in cell cycle arrest at G(1) phase and initiation of apoptosis. The mechanism of GT1b activation of caspase-9 involved inhibition of beta(1) integrin serine/threonine phosphorylation and decreased phosphorylation of both integrin-linked kinase and protein kinase B/Akt at its Ser-473 site, leading to cytochrome c release from mitochondria. Consistently, blockade of GT1b function with anti-GT1b antibody specifically activated the Ser-473 site of Akt, markedly suppressing apoptosis. The ganglioside-induced inhibition of Akt phosphorylation was GT1b-specific and was not observed when cells were treated with other keratinocyte gangliosides, including GD3. These studies suggest that the modulation of keratinocyte cell cycle and survival by GT1b is mediated by its direct interaction with alpha(5)beta(1) and resultant inhibition of the integrin/integrin-linked kinase/protein kinase B/Akt signaling pathway.     

The mechanical hierarchies of fibronectin observed with single-molecule AFM

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.     

Coregulation of fibronectin signaling and matrix contraction by tenascin-C and syndecan-4

Syndecan-4 is a ubiquitously expressed heparan sulfate proteoglycan that modulates cell interactions with the extracellular matrix. It is transiently up-regulated during tissue repair by cells that mediate wound healing. Here, we report that syndecan-4 is essential for optimal fibroblast response to the three-dimensional fibrin-fibronectin provisional matrix that is deposited upon tissue injury. Interference with syndecan-4 function inhibits matrix contraction by preventing cell spreading, actin stress fiber formation, and activation of focal adhesion kinase and RhoA mediated-intracellular signaling pathways. Tenascin-C is an extracellular matrix protein that regulates cell response to fibronectin within the provisional matrix. Syndecan-4 is also required for tenascin-C action. Inhibition of syndecan-4 function suppresses tenascin-C activity and overexpression of syndecan-4 circumvents the effects of tenascin-C. In this way, tenascin-C and syndecan-4 work together to control fibroblast morphology and signaling and regulate events such as matrix contraction that are essential for efficient tissue repair.     

Integrins and cell signaling in chondrocytes

Integrins are adhesion receptor heterodimers that transmit information from the extracellular matrix (ECM) to the cell through activation of cell signaling pathways. Chondrocytes express several members of the integrin family including alpha5beta1 which is the primary chondrocyte receptor for fibronectin. Cell signaling mediated through integrins regulates several chondrocyte functions including differentiation, matrix remodeling, responses to mechanical stimulation and cell survival. Integrin-mediated activation of members of the mitogen-activated protein kinase family likely plays a key role in transmitting signals regulating chondrocyte gene expression. Upstream mediators of mitogen-activated protein kinase (MAP kinase) activation include focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (pyk2) which are both expressed by chondrocytes. A better understanding of chondrocyte integrin signaling is needed to define the mechanisms by which the ECM regulates chondrocyte function.