The matrix reorganized: extracellular matrix remodeling and integrin signaling

Via integrins, cells can sense dimensionality and other physical and biochemical properties of the extracellular matrix (ECM). Cells respond differently to two-dimensional substrates and three-dimensional environments, activating distinct signaling pathways for each. Direct integrin signaling and indirect integrin modulation of growth factor and other intracellular signaling pathways regulate ECM remodeling and control subsequent cell behavior and tissue organization. ECM remodeling is critical for many developmental processes, and remodeled ECM contributes to tumorigenesis. These recent advances in the field provide new insights and raise new questions about the mechanisms of ECM synthesis and proteolytic degradation, as well as the roles of integrins and tension in ECM remodeling.     

Extracellular matrix conditions T cells for adhesion to tissue interstitium

The activation and differentiation of peripheral blood T cells (PBT) are known to correlate with increased surface expression and adhesive capacity of beta(1) integrins, which mediate adhesion to the extracellular matrix (ECM). However, little is known about the regulation of integrin expression, affinity, and avidity on tissue T cells after they are embedded in the interstitial ECM. In this study we show that tissue T cells, freshly isolated from their residence in the interstitial ECM of the intestinal lamina propria, express a distinct subset of functionally active integrins that contribute to enhanced adhesion to purified collagen, fibronectin, and cell-derived ECM when compared with freshly isolated, short term activated, and long term cultured PBT. Furthermore, integrin usage is distinct between circulating and tissue-derived T cells, in that lamina propria T cells prefer to bind to collagen, while PBT lymphoblasts choose fibronectin when presented with a complex, three-dimensional, cell-derived matrix. To identify the extrinsic factors that regulate the conversion from a nonadhesive PBT to highly adhesive tissue T cell, we demonstrate that activation of PBT in the presence of fibronectin or collagen rapidly generates a surface integrin expression profile, an integrin usage pattern, and adhesive capacity mirroring that of a tissue T cell. These results indicate that the tissue ECM microenvironment instructs newly arrived T cells for further interactions with the underlying matrix and thereby imprints them with a signature tissue adhesive phenotype.     

Induction of migration of periodontal ligament cells by selective regulation of integrin subunits

The recruitment of tissue‐resident stem cells is important for wound regeneration. Periodontal ligament cells (PDL cells) are heterogeneous cell populations with stemness features that migrate into wound sites to regenerate periodontal fibres and neighbouring hard tissues. Cell migration is regulated by the local microenvironment, coordinated by growth factors and the extracellular matrix (ECM). Integrin‐mediated cell adhesion to the ECM provides essential signals for migration. We hypothesized that PDL cell migration could be enhanced by selective expression of integrins. The migration of primary cultured PDL cells was induced by platelet‐derived growth factor‐BB (PDGF‐BB). The effects of blocking specific integrins on migration and ECM adhesion were investigated based on the integrin expression profiles observed during migration. Up‐regulation of integrins α3, α5, and fibronectin was identified at distinct localizations in migrating PDL cells. Treatment with anti‐integrin α5 antibodies inhibited PDL cell migration. Treatment with anti‐integrin α3, α3‐blocking peptide, and α3 siRNA significantly enhanced cell migration, comparable to treatment with PDGF‐BB. Furthermore, integrin α3 inhibition preferentially enhanced adhesion to fibronectin via integrin α5. These findings indicate that PDL cell migration is reciprocally regulated by integrin α3‐mediated inhibition and α5‐mediated promotion. Thus, targeting integrin expression is a possible therapeutic strategy for periodontal regeneration.     

A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300-1000 micrograms/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300-1000 micrograms/ml), which mimics a binding domain of LM, and alpha-lactalbumin (1-10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and alpha-lactalbumin inhibited cell migration 50%. Antibodies against integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.     

Effect of hypoxia on cellular adhesion to vitronectin and fibronectin

Cellular invasion of extracellular matrix (ECM) occurs during normal and pathological settings. For cells to invade, they must adhere to the underlying substratum, break down barrier molecules, and detach from the substratum prior to migrating through the ECM. We previously demonstrated that incubation under reduced oxygen levels increases the in vitro invasiveness of trophoblast and breast carcinoma cells, an effect linked to elevated expression of the cell surface receptor for urokinase-type plasminogen activator (uPAR). This study examined the role of oxygen, integrins and the urokinase-type plasminogen activator (uPA) system on the adhesion of trophoblast and breast carcinoma cells to the ECM molecules vitronectin and fibronectin. Compared to exposure to 20 and 5% oxygen, exposure to 1% oxygen decreased adhesion of these cells to vitronectin and fibronectin, an effect that was reversible by re-exposure to 20% oxygen. Incubation in 1% oxygen also resulted in reduced expression of surface alpha(5) integrin. Furthermore, adhesion to vitronectin and fibronectin was reduced by compounds that interfere with integrin function, such as EDTA, anti-integrin antibodies, or by antibodies that interfere with the binding of pro-uPA to uPAR, soluble uPAR, soluble vitronectin, phosphatidylinositol-specific phospholipase C, as well as plasminogen activator inhibitor-1. These findings suggest an important role for oxygen in the regulation of cellular invasion, possibly in part through its effects on integrin and uPAR-mediated mechanisms of adhesion.     

Mechanical force regulates integrin turnover in Drosophila in vivo

Regulated assembly and disassembly,?or?turnover,?of integrin-mediated cell-extracellular matrix?(ECM)?adhesions is essential for dynamic cell movements?and?long-term tissue maintenance. For example, in Drosophila,?misregulation of integrin turnover disrupts muscle-tendon?attachment at myotendinous junctions (MTJs). We demonstrate that?mechanical force, which modulates integrin activity, also regulates integrin and intracellular adhesion complex (IAC) turnover in vivo. Using conditional mutants to alter the tensile force on MTJs, we found that the proportion of IAC components undergoing turnover inversely correlated with the force applied on MTJs. This effect was disrupted by point mutations in β-integrin that interfere with ECM-induced conformational changes and activation of β-integrin or integrin-mediated cytoplasmic signalling. These mutants also disrupted integrin dynamics at MTJs during larval development. Together, these data suggest that specific β-integrin-mediated signals regulate adhesion turnover in response to tension during tissue?formation. We propose that integrin-ECM adhesive stability is continuously controlled by force in vivo through integrin-dependent auto-regulatory feedback mechanisms so that tissues can quickly adapt to and withstand mechanical stresses.     

Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.     

A Novel Mutation in β Integrin Reveals an Integrin-Mediated Interaction between the Extracellular Matrix and cki-1/p27(KIP1)

The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1). These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo.     

Fibronectin-dependent collagen I deposition modulates the cell response to fibronectin

Communication between cells and the extracellular matrix (ECM) is critical for regulation of cell growth, survival, migration, and differentiation. Remodeling of the ECM can occur under normal physiological conditions, as a result of tissue injury, and in certain pathological conditions. ECM remodeling leads to alterations in ECM composition and organization that can alter many aspects of cell behavior, including cell migration. The cell migratory response varies depending on the type, amount, and organization of ECM molecules present, as well as the integrin and proteoglycan repertoire of the cells. We and others have shown that the deposition of several ECM molecules, including collagen types I and III, depends on the presence and stability of ECM fibronectin. Hence, the effect of fibronectin and fibronectin matrix on cell function may partially depend on its ability to direct the deposition of collagen in the ECM. In this study, we used collagen-binding fibronectin mutants and recombinant peptides that interfere with fibronectin-collagen binding to show that fibronectin-dependent collagen I deposition regulates the cell migratory response to fibronectin. These data show that the ability of fibronectin to organize other proteins in the ECM is an important aspect of fibronectin function and highlight the importance of understanding how interactions between ECM proteins influence cell behavior.     

A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300–1000 μg/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300–1000 μg/ml), which mimics a binding domain of LM, and α-lactalbumin (1–10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and α-lactalbumin inhibited cell migration 50%. Antibodies agasinst integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.     

The interaction between urokinase receptor and vitronectin in cell adhesion and signalling

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells present in all tissue and organs. Cell-matrix interactions play fundamental roles during embryonic development, morphogenesis, tissue homoeostasis, wound healing, and tumourigenesis. Cell-matrix communication is kept in balance by physical contact and by transmembrane integrin receptors providing the dynamic link between the extracellular and intracellular environments through bi-directional signalling. The urokinase-type plasminogen activator receptor (uPAR) is a plasma membrane receptor overexpressed during inflammation and in almost all human cancers. One of its functions is to endorse ECM remodelling through the activation of plasminogen and downstream proteases, including matrix-metalloproteases (MMPs). Beside its role in ECM degradation, uPAR modulates cell-matrix contact through a direct engagement with the ECM component, vitronectin (Vn), and by regulating the activity state of integrins thus promoting or inhibiting integrin signalling and integrin-mediated cell adhesion to other ECM components, like fibronectin and collagen. In this review we have centred our attention on the non-proteolytic function of uPAR as a mediator of cell adhesion and downstream signalling.     

MUC5AC production is downregulated in NCI-H292 lung cancer cells cultured on type-IV collagen

Mucus overproduction is an important feature of bronchial asthma. MUC5AC mucin is a major component of mucus and is overproduced in patients with asthma. Although regulation of MUC5AC production has been well investigated, its regulation through the signals from extracellular matrix (ECM) is less clear. In this study, we investigated whether the signals from ECM regulate MUC5AC production in the human lung epithelial cell line NCI-H292. We found that MUC5AC production is downregulated in NCI-H292 cells cultured on type-IV collagen, a major component of ECM, but shows no obvious changes when cultured on type-I collagen or fibronectin. In contrast, MUC5AC production was upregulated on laminin and on reconstituted basement membrane (Matrigel), a complex of ECM components. Antibody-mediated inhibition of integrin β1-subunit, a major receptor involved in the adherence of cells to type-IV collagen, upregulated the MUC5AC production in NCI-H292 cells, and also in the cells cultured on type-IV collagen. Although the major signaling pathway from integrins is via Src kinase activation, treatment of cells with PP2, a Src kinase inhibitor, did not recover the downregulation of MUC5AC on type-IV collagen. In contrast, on Matrigel, the inhibition of integrin β1-subunit did not abolish the upregulation of MUC5AC production, but PP2 reduced the upregulation. These results suggest that ECM and an integrin/Src pathway play an important role in the regulation of MUC5AC production in the cell line NCI-H292. The production of MUC5AC is downregulated on type-IV collagen through a Src-independent pathway. In contrast, MUC5AC is upregulated on Matrigel through a Src-dependent pathway in NCI-H292 cells.     

Regulation of integrin function through conformational complexity: not simply a knee-jerk reaction?

Such diverse biological processes as the maintenance of tissue architecture and the regulation of cell migration are controlled through dynamic changes in integrin receptor conformation. Early analyses of the mechanisms of shape change by integrins led to the definition of three inter-convertible conformational states: inactive, primed and ligand-occupied. Recent advances reviewed in this article have now shown that the integrin molecule contains a number of flexible joints and connections, leading to a broad spectrum of possible conformational states. This conformational complexity is likely to permit fine-tuning of integrin function through regulation of ligand-binding affinity and intracellular signalling.     

A Spatial Model for Integrin Clustering as a Result of Feedback between Integrin Activation and Integrin Binding

Integrins are transmembrane adhesion receptors that bind extracellular matrix (ECM) proteins and signal bidirectionally to regulate cell adhesion and migration. In many cell types, integrins cluster at cell-ECM contacts to create the foundation for adhesion complexes that transfer force between the cell and the ECM. Even though the temporal and spatial regulation of these integrin clusters is essential for cell migration, how cells regulate their formation is currently unknown. It has been shown that integrin cluster formation is independent of actin stress fiber formation, but requires active (high-affinity) integrins, phosphoinositol-4,5-bisphosphate (PIP2), talin, and immobile ECM ligand. Based on these observations, we propose a minimal model for initial formation of integrin clusters, facilitated by localized activation and binding of integrins to ECM ligands as a result of biochemical feedback between integrin binding and integrin activation. By employing a diffusion-reaction framework for modeling these reactions, we show how spatial organization of bound integrins into clusters may be achieved by a local source of active integrins, namely protein complexes formed on the cytoplasmic tails of bound integrins. Further, we show how such a mechanism can turn small local increases in the concentration of active talin or active integrin into integrin clusters via positive feedback. Our results suggest that the formation of integrin clusters by the proposed mechanism depends on the relationships between production and diffusion of integrin-activating species, and that changes to the relative rates of these processes may affect the resulting properties of integrin clusters.     

PTH-related protein enhances LoVo colon cancer cell proliferation, adhesion, and integrin expression

Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. Tumor cell adhesion to extracellular matrix (ECM) proteins plays a major role in the invasion and metastasis of tumor cells, and is mediated via integrin subunits. The LoVo human colon cancer cell line was used as a model system to study the effects of PTHrP on cell proliferation and adhesion to ECM proteins found in normal liver. Clones of LoVo cells engineered to overexpress PTHrP by stable transfection with a PTHrP cDNA showed enhanced cell proliferation vs. control (empty vector-transfected) cells. PTHrP-overexpressing cells also showed significantly higher adhesion to collagen type I, fibronectin, and laminin, and enhanced expression of the [symbol: see text] integrin subunits. These results indicate that PTHrP may play a role in colon cancer invasion and metastasis by increasing cell proliferation and adhesion to the ECM via upregulation of proinvasive integrin expression.     

Neurite outgrowth,RGD-dependent,and RGD-independent adhesion of identified molluscan motoneurons on selected substrates

The extracellular matrix (ECM) provides structural support to cells and tissues and is involved in the regulation of various essential physiological processes, including neurite outgrowth. Most of the adhesive interactions between cells and ECM proteins are mediated by integrins. Integrins typically recognize short linear amino acid sequences in ECM proteins, one of the most common being Arginine-Glycine-Aspartate (RGD). The present study investigated neurite outgrowth and adhesion of identified molluscan neurons on a selection of substrates in vitro. Involvement of RGD binding sites in adhesion to the different substrates was investigated using soluble synthetic RGD peptides. The cells adhered to native (i.e., nondenatured) laminin and type IV collagen, but not to native plasma fibronectin. Denaturation of fibronectin dramatically enhanced cell adhesion. Only the adhesion to denatured fibronectin was inhibited by RGD peptides, indicating that denaturation uncovers a RGD binding site in the protein. Laminin as well as denatured fibronectin, but not type IV collagen, induced neurite outgrowth from a percentage of the RPA neurons. These results demonstrate that molluscan neurons can attach to various substrates using both RGD-dependent and RGD-independent adhesion mechanisms. This suggests that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in these neurons. Moreover, the results show that vertebrate ECM proteins can induce outgrowth from these neurons, suggesting that the mechanisms involved in adhesion as well as outgrowth promoting are evolutionarily well conserved. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 37–52, 1998     

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.