Coordinate regulation of fibronectin matrix assembly by the plasminogen activator system and vitronectin in human osteosarcoma cells

BACKGROUND: Plasminogen activators are known to play a key role in the remodeling of bone matrix which occurs during tumor progression, bone metastasis and bone growth. Dysfunctional remodeling of bone matrix gives rise to the osteoblastic and osteolytic lesions seen in association with metastatic cancers. The molecular mechanisms responsible for the development of these lesions are not well understood. Studies were undertaken to address the role of the plasminogen activator system in the regulation of fibronectin matrix assembly in the osteoblast-like cell line, MG-63. RESULTS: Treatment of MG-63 cells with P25, a peptide ligand for uPAR, resulted in an increase in assembly of fibronectin matrix which was associated with an increase in the number of activated β1 integrins on the cell surface. Overexpression of uPAR in MG-63 cells increased the effect of P25 on fibronectin matrix assembly and β(1 )integrin activation. P25 had no effect on uPAR null fibroblasts, confirming a role for uPAR in this process. The addition of plasminogen activator inhibitor Type I (PAI-1) to cells increased the P25-induced fibronectin polymerization, as well as the number of activated integrins. This positive regulation of PAI-1 on fibronectin assembly was independent of PAI-1's anti-proteinase activity, but acted through PAI-1 binding to the somatomedin B domain of vitronectin. CONCLUSION: These results indicate that vitronectin modulates fibronectin matrix assembly in osteosarcoma cells through a novel mechanism involving cross-talk through the plasminogen activator system

Fibronectin Matrix Assembly Suppresses Dispersal of Glioblastoma Cells

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12–15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal

The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9β1 Affects Cancer Cell Behaviour

Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9β1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9β1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9β1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9β1 to focal adhesions where active β1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions

Cell–fibronectin interactions and actomyosin contractility regulate the segmentation clock and spatio-temporal somite cleft formation during chick embryo somitogenesis

Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the unsegmented presomitic mesoderm (PSM). PSM cells undergo cyclic waves of segmentation clock gene expression, followed by Notch-dependent upregulation of meso1 in the rostral PSM which induces somite cleft formation. However, the relevance of the fibronectin matrix for these molecular processes remains unknown. Here, we assessed the role of the PSM fibronectin matrix in the spatio-temporal regulation of chick embryo somitogenesis by perturbing (1) extracellular fibronectin matrix assembly, (2) integrin–fibronectin binding, (3) Rho-associated protein kinase (ROCK) activity and (4) non-muscle myosin II (NM II) function. We found that integrin–fibronectin engagement and NM II activity are required for cell polarization in the nascent somite. All treatments resulted in defective somitic clefts and significantly perturbed meso1 and segmentation clock gene expression in the PSM. Importantly, inhibition of actomyosin-mediated contractility increased the period of hairy1/hes4 oscillations from 90 to 120 min. Together, our work strongly suggests that the fibronectin–integrin–ROCK–NM II axis regulates segmentation clock dynamics and dictates the spatio-temporal localization of somitic clefts.info:eu-repo/semantics/publishedVersio

Inhibition of fibronectin matrix assembly by the heparin-binding domain of vitronectin

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin\u27s heparin-binding domain inhibited both β1 and non-β1 integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin- associated proteins α-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton

Syndecan-2 is a novel target of insulin-like growth factor binding protein-3 and is over-expressed in fibrosis

Extracellular matrix deposition and tissue scarring characterize the process of fibrosis. Transforming growth factor beta (TGFβ) and Insulin-like growth factor binding protein-3 (IGFBP-3) have been implicated in the pathogenesis of fibrosis in various tissues by inducing mesenchymal cell proliferation and extracellular matrix deposition. We identified Syndecan-2 (SDC2) as a gene induced by TGFβ in an IGFBP-3-dependent manner. TGFβ induction of SDC2 mRNA and protein required IGFBP-3. IGFBP-3 independently induced production of SDC2 in primary fibroblasts. Using an ex-vivo model of human skin in organ culture expressing IGFBP-3, we demonstrate that IGFBP-3 induces SDC2 ex vivo in human tissue. We also identified Mitogen-activated protein kinase-interacting kinase (Mknk2) as a gene induced by IGFBP-3. IGFBP-3 triggered Mknk2 phosphorylation resulting in its activation. Mknk2 independently induced SDC2 in human skin. Since IGFBP-3 is over-expressed in fibrotic tissues, we examined SDC2 levels in skin and lung tissues of patients with systemic sclerosis (SSc) and lung tissues of patients with idiopathic pulmonary fibrosis (IPF). SDC2 levels were increased in fibrotic dermal and lung tissues of patients with SSc and in lung tissues of patients with IPF. This is the first report describing elevated levels of SDC2 in fibrosis. Increased SDC2 expression is due, at least in part, to the activity of two pro-fibrotic factors, TGFβ and IGFBP-3. © 2012 Ruiz et al

CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling

Integrin-dependent assembly of the fibronectin (Fn) matrix plays a central role in vertebrate development. We identify CD98hc, a membrane protein, as an important component of the matrix assembly machinery both in vitro and in vivo. CD98hc was not required for biosynthesis of cellular Fn or the maintenance of the repertoire or affinity of cellular Fn binding integrins, which are important contributors to Fn assembly. Instead, CD98hc was involved in the cell's ability to exert force on the matrix and did so by dint of its capacity to interact with integrins to support downstream signals that lead to activation of RhoA small GTPase. Thus, we identify CD98hc as a membrane protein that enables matrix assembly and establish that it functions by interacting with integrins to support RhoA-driven contractility. CD98hc expression can vary widely; our data show that these variations in CD98hc expression can control the capacity of cells to assemble an Fn matrix, a process important in development, wound healing, and tumorigenesis

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins.We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly.These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration