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The matrix reorganized: extracellular matrix remodeling and integrin signaling Melinda Larsen1, Vira V Artym1,2, J Angelo Green1 and Kenneth M Yamada1 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. Addresses 1 Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370, USA 2 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057-1469, USA (2D) surfaces and complex, malleable three-dimensional (3D) environments can therefore be sensed by integrins that respond to these surfaces with altered signaling (reviewed in [3–7]). In this review, we discuss recent findings regarding ECM remodeling, with emphasis on collagen, fibronectin and associated integrin signaling. We examine how ECM physical properties and higher-order organization influence cell behavior and integrin signaling pathways. ECM remodeling is required in vivo for proper development, but ECM alterations can also create an environment conducive to tumorigenesis. In this review, we will consider some examples of these processes. We apologize for the omissions imposed by the need for brevity. Fibroblast-mediated collagen matrix remodeling in vitro Introduction Increasing numbers of studies show that the morphology, cytoskeletal structure and signaling of cells grown on 2D surfaces differ from those of cells grown within 3D environments, where collagen fibers contact both ventral (lower) and dorsal (upper) surfaces of the cells. In fact, upon engagement of receptors on their dorsal surface, well-spread fibroblasts in 2D culture quickly convert to a bipolar or stellate morphology characteristic of fibroblasts in 3D environments [8]. A recent study [9] of cell interactions with collagen reveals that a2b1 integrinmediated transport of collagen fibers and subsequent contraction of in vitro 3D collagen matrices requires non-muscle myosin II-B. Although this myosin is important for fibroblast cell motility in 3D collagen matrices, it is not required for migration on 2D surfaces. Another difference is that myosin II-B only localizes to cellular extensions when cells are plated within 3D matrices rather than on 2D surfaces [9]. Extracellular matrix (ECM) remodeling is involved in development, fibrosis, tissue repair and tumor-associated desmoplasia (stromatogenesis). The ECM can be remodeled by many processes, including synthesis, contraction and proteolytic degradation. Integrins are the primary ECM receptors mediating ECM remodeling (reviewed in [1,2]). In response to changes in the ECM, integrin signaling also regulates many other interrelated cellular processes: proliferation, survival, cell migration and invasion (Figure 1). Integrins function as mechanotransducers and can transform mechanical forces created by the ECM or the cytoskeleton into chemical signals. Differences between simple, rigid and non-pliable two-dimensional Specific serum components can facilitate integrin-dependent 3D collagen gel contraction through different signaling pathways. PDGF-stimulated contraction of floating collagen matrices utilizes phosphatidylinositol 3-kinase (PI3K) and myosin II, whereas LPA-stimulated contraction depends on signaling by the monomeric Gprotein GaI, but does not require myosin II [10,11]. Using siRNA knockdown and inhibitors, it was demonstrated that signaling through PDGF and LPA converge on p21-activated kinase-1 (PAK1) to regulate collagen matrix contraction through cofilin-1 [11]. PAK1 therefore links these two distinct matrix remodeling pathways. Corresponding author: Yamada, Kenneth M ([email protected]) Current Opinion in Cell Biology 2006, 18:463–471 This review comes from a themed issue on Cell-to-cell contact and extracellular matrix Edited by Martin Schwartz and Alpha Yap Available online 17th August 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.08.009 www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:463–471 464 Cell-to-cell contact and ECM Figure 1 Generalized schematic diagram of integrin signaling, focusing on pathways specifically covered in this review. Integrins signal through recruitment of FAK, recruitment and activation of SFKs, and activation of PI3K. Src phosphorylates p130CAS and recruits Crk to activate Rac. Rac is also activated by FAK via stimulation from PIX/GIT/paxillin complexes. FAK activates ERK signaling that, together with Rac downstream signaling, exerts a regulatory effect on cell proliferation and survival. Signaling downstream of PI3K affects activation of Akt and the small GTPases Rac, Cdc42, and Rho to induce changes in the cytoskeleton, cell contractility, cell migration, invasion and gene expression. Crosstalk between integrin and GFR signaling pathways ensures proper integration of integrin- and GFR-mediated signaling required for optimal cell function. LPA, acting through a seven-transmembrane G-protein-coupled receptor, signals through PAK and cofilin, and cooperates with the ROCK/MLCP/myosin II pathway to promote collagen matrix contraction. Abbreviations: guanine nucleotide-exchange factors, GEFs; growth factor, GF; LIM kinase, LIMK; mammalian diaphanous, mDIA; myosin light chain phosphatase, MLCP; phosphatidylinositol-3,4,5-trisphosphate, PIP3; protein kinase C, PKC; Src-family kinases, SFKs; Wiskott-Aldrich syndrome protein, WASP. ROCK was specifically implicated in PDGF-induced and mDia1 in LPA-induced ECM contraction. These findings suggest that Rho effectors act parallel to and/or cooperatively with PAK1 to regulate contraction of floating collagen matrices. Fibronectin matrix remodeling by fibroblasts in vitro The major receptor for fibronectin, a5b1 integrin, can be found in different adhesion structures, such as focal complexes, focal adhesions, fibrillar adhesions and 3Dmatrix adhesions [3]. Fibronectin fibrillogenesis is mediated by actin-dependent, directed translocation of a5b1 out of focal adhesions into fibrillar adhesions [12], and a recent study indicates that this translocation depends upon a specific integrin conformation [13]. It Current Opinion in Cell Biology 2006, 18:463–471 is well known that conformation directly affects integrin activation state and ligand-binding activity; in 2D substrates, exogenous activating antibodies or manganese can activate integrins and induce matrix formation [1]. Recent studies implicate the urokinase-type plasminogen activator receptor (uPAR) in regulating integrin a5b1 activity. Addition of a uPAR ligand, the P-25 peptide, stimulated fibronectin fibril assembly [14] by activating integrin a5b1 through the EGF receptor and Src [15]. Interestingly, fibronectin fibril assembly by cells expressing activationdependent integrins can be stimulated specifically by a 3D fibronectin matrix without exogenous activators [16]. These data support the idea that a 3D matrix can activate integrins to induce fibronectin matrix assembly (Figure 2), although the mechanism remains to be elucidated. One www.sciencedirect.com Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 465 Figure 2 Extracellular factors promote ECM remodeling by stimulating integrin activation. On a 2D substrate in vitro, fibroblast cells are polarized such that only the ventral surface contacts the substrate. In this state, cells are typically well-spread and only a subset of integrins is activated: bent conformation, inactive integrin; extended conformation, activated integrin. Upon plating within a 3D matrix or following treatment with exogenous stimulators (e.g. Mn2+, activating antibodies or uPAR ligand), integrins undergo a conformational change and are activated, leading to enhanced integrin–ECM interactions and increased ECM production. As the matrix assumes a more 3D character, cell morphology becomes more bipolar or stellate, at least partly as a result of increased engagement of receptors on the dorsal cell surface. experimental approach to this question would be to use conformation-specific antibodies to detect activated integrins within different 3D environments. Cells in vivo exist in complex environments, where they constantly interact with multiple ECM molecules rather than a single component. In vitro studies show that specific cell–ECM interactions affect the dynamics of other ECM components and the physical state of the ECM. For example, cell-dependent fibronectin polymerization increases the tensile strength of a 3D collagen biogel without affecting rigidity [17]. Additionally, LPA induces both fibronectin and collagen assembly concurrently in smooth muscle cells. Inhibition of LPA-induced fibronectin assembly with an anti-integrin a5b1 antibody prevents collagen type I fibril assembly [18]. Other studies also show that fibrillar collagen deposition is dependent on fibronectin [19,20]. ECM remodeling in epithelial morphogenesis ex vivo Organ culture model systems provide powerful tools to study developmental events using intact epithelial embryonic tissues ex vivo. Branching organs, such as lung, kidney and salivary gland, can be studied as organ explants, since internally driven morphogenetic programs continue ex vivo and recapitulate in vivo processes. Branching morphogenesis involves a number of repetitive steps, starting with formation of a cleft or indentation in the basement membrane, which is a specialized ECM www.sciencedirect.com located at the basal surface of differentiated epithelial cells. ECM remodeling and integrin signaling are clearly important for branching, especially in the salivary gland, where fibronectin and its major receptor, integrin a5b1, are required (Figure 3a) [21]. Fibronectin accumulation in cleft sites is associated with a decrease in E-cadherin, the prototypic epithelial cadherin that mediates cell–cell adhesion. In this study [21], exogenous cellular fibronectin also locally decreased cadherin levels in a human salivary gland cell line, indicating that a function of fibronectin is negative regulation of E-cadherin, although the mechanism of this inhibition is not known. Many classical studies point to the importance of basement membrane remodeling during branching morphogenesis. Recently, cytoskeletal tension was found to be important for branching: inhibitors of ROCK, myosin light chain kinase and myosin ATPase inhibited branching of lung rudiments (Figure 3b) [22]. Interestingly, ROCK stimulated basement membrane thinning at the lung bud distal tips. This thinning may facilitate cell proliferation and promote bud elongation through decreased synthesis or increased protease-mediated degradation of basement membrane components. Fibronectin matrix remodeling in vivo Important new insights into the role of fibronectin matrix remodeling during development have emerged recently from work in model organisms. Somite formation is a major event during vertebrate development, whereby the unsegmented paraxial mesoderm undergoes a mesenchymal-toepithelial transition to form epithelial segments. Although fibronectin is known to be critical for somitogenesis because fibronectin-null mouse embryos lack somites, recent work has provided insight into the mechanism of this morphological segmentation (Figure 3b). Mutation of integrin a5 in zebrafish prevents fibronectin accumulation, resulting in a defect in somite boundaries and epithelialization [23]. Another recent study reports that fibronectin is required for somite maintenance but not initiation [24]. The mechanism by which fibronectin induces somite epithelialization involves focal adhesion kinase (FAK). In zebrafish integrin a5 mutants, no active FAK (phosphorylated at Y397) was detected [24]. Inhibition of FAK signaling by the dominant-negative form of FAK, FAKrelated nonkinase (FRNK), results in defective fibronectin matrix deposition and disrupted somite boundaries in Xenopus embryos [25]. It is interesting that FNRKinjected Xenopus embryos display a phenotype more severe than that of FAK-null mouse embryos. FRNKinjected Xenopus embryos also show defective localization of Xenopus Ena, an Ena/VASP family protein linking integrins with the actin cytoskeleton. Neutralization of Ena/VASP activity results in defective fibronectin accumulation around somites and impaired FAK activation [25]. Together, these data suggest the existence of a Current Opinion in Cell Biology 2006, 18:463–471 466 Cell-to-cell contact and ECM Figure 3 Current Opinion in Cell Biology 2006, 18:463–471 www.sciencedirect.com Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 467 bidirectional signaling pathway between FAK and Ena/ VASP protein. These studies imply that Ena/VASP proteins and FAK activate integrin a5 to stimulate fibronectin fibrillogenesis and induce downstream signaling leading to epithelialization. This provocative finding indicates that fibronectin can promote the transition of certain mesenchymal cells to an epithelial phenotype. It is not clear why fibronectin can decrease E-cadherin in some contexts [21], whereas in other contexts, such as somite formation, it induces epithelialization [24] and increased cadherin expression. However, integrin signaling often forms part of a signaling network. In the complex process of somitogenesis, fibronectin-integrin a5 signaling acts cooperatively with other pathways: Eph– Ephrin signaling cooperates with fibronectin–integrin signaling to maintain somite boundaries [24], and Notch/Delta and integrin a5 interdependently regulate somite epithelialization and fibronectin assembly [23]. The interaction between these molecules is complex, and it changes along the anterior–posterior axis. The cellular response to fibronectin can therefore depend on the local signaling context. ECM rigidity and cytoskeletal tension in tumor progression Stromatogenesis is a desmoplastic alteration in tumorassociated stroma, occurring in parallel with neoplasia, which is characterized by many changes, including increased expression of organized fibronectin and type I collagen by adjacent stromal fibroblasts [26,27]. A consequence of stromatogenesis is that the tumor-associated stroma becomes more rigid. Measurements of the rigidity of normal mammary tissue, malignant breast tissue and tumor-associated stroma revealed that malignancy is accompanied by substantial increases in ECM rigidity in both the tumor and stroma [28]. Cells sense elevated ECM rigidity through integrins and respond with modified signaling, according to many studies. Increased ECM rigidity stimulates integrin expression [29] and induces conformational changes to activate avb3 integrin [30]. Inappropriate increases in ECM rigidity perturb normal tissue architecture, activate Rho, induce Rhogenerated cytoskeletal tension and activate ERK-dependent growth [31,32], whereas subsequent increases in cytoskeletal tension promote growth and focal adhesion assembly [33,34]. Pharmacological inhibition of ROCK or myosin II can reverse the malignant phenotype, indicating a dependence of the transformed phenotype on Rhodependent tension [28]. ERK inhibition can also reverse the malignant phenotype, underscoring the cross-talk between mitogenic growth factor and mechanotransducing integrin pathways [28]. As a result of the functional link between ECM rigidity, Rho, cell contractility and cell behavior, a mechanosensitive positive feedback loop appears to amplify cell proliferation, transformation and ECM rigidity in tumors (Figure 4). Proteolytic remodeling of ECM in tumor progression Many proteases can cleave ECM molecules involved in tumor progression (reviewed in [35]), but recent studies have identified MT1-MMP as a major protease supporting the invasive phenotype. MT1-MMP confers an advantage on tumor cells in vitro and in vivo by enabling them to escape growth suppression by fibrillar type I collagen [36]. Membrane-bound MT1-MMP cleaves collagen and activates proMMP-2 in the immediate vicinity of cancer cells, creating a proliferation-promoting microenvironment of cleaved collagen. Interestingly, in MCF-7 cells expressing MT1-MMP, type I collagen increases cell-surface MT1-MMP activity by a novel mechanism: impaired clathrin-mediated internalization of MT1MMP [37]. MT1-MMP activity is essential for fibroblast and tumor cell invasion through 3D collagen gels, independent of plasminogen or secreted MMP activity [38]. However, it should be noted that because epithelial cells rarely express MT1-MMP [39], even though tumor cell lines and the surrounding stroma express it, there are conflicting views on the significance of MT1-MMP in human epithelial tumors. The heterogeneity of ECM organization (reviewed in [40]), from loose connective tissue to dense basement membranes, requires cancer cells to employ a range of migration modes. Two types of cancer cell migration through ECM have been identified: mesenchymal migration that utilizes integrins and proteases for adhesive and proteolytic interactions with ECM proteins, and amoeboid migration that is integrin-independent and non-proteolytic (reviewed in [41]). Experimental models predict that cells can switch from mesenchymal to amoeboid migration (Figure Legend 3) ECM remodeling during organogenesis. (a) ECM remodeling in branching morphogenesis. Branching morphogenesis in the salivary gland and other organs requires fibronectin. Acting through integrin a5b1, fibronectin locally decreases E-cadherin to stimulate cleft formation. In the lung, cytoskeletal contraction mediated through ROCK signaling is critical for branching morphogenesis. Cytoskeletal contraction leads to basement membrane remodeling, which may facilitate localized proliferation and subsequent bud elongation. (b) Integrin signaling during somite morphogenesis. Somite formation depends upon fibronectin assembly at the intersomitic boundary. Activation of integrin a5b1 induces fibronectin fibrillogenesis. Notch signaling stimulates integrin a5b1 signaling either directly, through Eph signaling, or through cytoskeletal alterations. This was concluded since expression of active notch induced high-affinity integrin b1. Eph signaling induces cytoskeletal changes and integrin a5b1 activation. FAK phosphorylation is required for somite formation, fibronectin fibrillogenesis, and colocalization of Ena/VASP with integrin a5b1. FAK localization depends upon integrin ligation, and Ena/VASP is required for FAK activation by phosphorylation (P). Activated integrin a5b1 signals downstream via FAK to induce epithelialization. www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:463–471 468 Cell-to-cell contact and ECM Figure 4 Effect of ECM rigidity on integrin signaling and tumorigenesis. Increased ECM rigidity activates integrins to promote focal adhesion formation, leading to stimulation of the Rho/ROCK pathway and increased cell contractility, cell migration and invasion. ERK is activated either directly by integrins or indirectly by growth factors to increase cell proliferation and possibly cell contractility. As transformation proceeds, increased cell contractility contributes to further ECM stiffening, leading to increased integrin expression, focal adhesion formation and amplified signaling, creating a self-sustained positive feedback loop (long red arrow) that may promote cell transformation. The phenotypic consequence of ECM stiffening associated with cellular transformation is disruption of tissue homeostasis and conversion from a differentiated to a malignant phenotype. Malignancy is associated with high Rho activity, lumen obstruction and loss of tissue polarity and adherens junctions in glandular tissues. if pericellular proteolysis is inhibited by protease inhibitors, if the Rho/ROCK signaling pathway is activated, or if integrin–ECM interactions are blocked [41]. Focal pericellular proteolysis of ECM molecules is a hallmark of mesenchymal migration by tumor cells [41]. Studies of malignant cells on 2D fluorescent matrices indicate that pericellular proteolysis and invasion are mediated by specialized cell membrane protrusions termed invadopodia (reviewed in [42]). MT1-MMP was identified as a key invadopodial protease responsible for local ECM degradation by carcinoma cells. The dynamics of the sequential accumulation of cortactin plus actin at Current Opinion in Cell Biology 2006, 18:463–471 invadopodia followed by MT1-MMP recruitment, and the subsequent onset of ECM degradation, have been revealed by live-cell imaging [43]. Although intravital imaging has detected invadopodium-like protrusions in extravasating carcinoma cells [44], further studies are required to detect invadopodial markers and test the ECM-degrading ability of these protrusions to verify the physiological importance of invadopodia in vivo. Remodeled ECM as a carcinogen and inducer of metastasis Although, in normal tissue, one function of fibroblasts is to maintain tissue homeostasis, the fibroblasts and ECM www.sciencedirect.com Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 469 associated with tumors (the tumor microenvironment) can function as a carcinogen. The tumor microenvironment can send erroneous signals to tumors that induce accumulation of MMPs, activate soluble growth factors and facilitate paracrine signaling. The epithelium often responds by undergoing an epithelial–mesenchymal transition (EMT), where epithelial cells lose epithelial characteristics, acquire mesenchymal characteristics and become invasive. Exposure of epithelial cells to MMP3 stimulates expression of Rac1b, an alternatively spliced and highly active form of Rac1, which induces cellular reactive oxygen species (ROS). ROS both stimulate EMT and cause oxidative damage to DNA, resulting in genomic instability [45]. Tumor microenvironments are often hypoxic. An interesting but puzzling recent study reports that lysyl oxidase, the well-known enzyme that crosslinks collagen in vivo, is induced under hypoxic conditions [46]. Although not required for primary tumor growth, lysyl oxidase is required for metastasis, and it induces FAK phosphorylation to stimulate cell motility. The role of this matrix-modifying enzyme in metastasis will be intriguing to resolve. Conclusions and future directions Great strides have been made in our understanding of ECM remodeling through the increasing use of 3D culture systems in recent years. Although it is now clear that integrin signaling and cytoskeletal organization are different in 2D and 3D environments, the factors responsible for these differences remain to be determined. Understanding the mechanistic differences between ECM remodeling on 2D surfaces and in 3D systems is critical for developing valid model systems characteristic of in vivo biology. Which factors are responsible for the differences: the complex linkages of ECM proteins, the physical forces associated with ECM presentation to the cell, or a combination of these and other factors? Clarification is needed concerning how ECM components structurally regulate each other to influence cell morphology and signaling. It may be possible to answer many questions regarding the impact of structure on cell behavior using techniques that can sense local ECM rigidity. Future improvements in atomic force microscopy (AFM) or alternative approaches are needed to measure, apply and evaluate the effects of mechanical stresses on morphology and signaling of live cells in 3D environments. Since ECM remodeling is now known to be a dynamic process involving cytoskeletal molecules and proteases, live imaging in vivo and in 3D culture systems should provide many new insights into mechanisms of ECM remodeling. One issue with studying matrix assembly is distinguishing new ECM from old. A recent report applied the Timer reporter, a DsRed derivative that changes from green to red over time, to overcome this problem in studies of elastic fiber formation [47]. Multicolor in vivo time-lapse imaging of fluorescently labeled www.sciencedirect.com ECM and cell adhesion proteins should illuminate the mechanisms of ECM assembly that control developmental processes, whereas time-lapse imaging of tumor cell proteases and specialized cell membrane structures (e.g. invadopodia) will help elucidate their role in ECM degradation. Studies employing electron microscopy will also be useful to validate the significance of invadopodia in vivo. Although the ECM and microenvironment are now known to be involved in tumor initiation and progression (e.g. through ECM rigidity), the mechanisms need elucidation. This gap could be addressed by development of in vitro 3D models in which matrix rigidity and biochemical content can be carefully controlled. A difficulty with studying tumor-associated fibroblasts has been maintaining their phenotype in culture, but a recent report showed that their phenotype can be maintained within a 3D matrix [48]. Developing a deeper understanding of how ECM remodeling occurs and how it modifies cell behavior should lead to better clinical interventions for pathologies that develop when ECM remodeling goes awry. Update A new study proposes that local translocation of a component of the 3D matrix guides branching morphogenesis. Live confocal imaging of glands labeled with twocolor fluorescently labeled fibronectin was used to establish that translocating wedges of fibronectin move steadily inward through a population of surprisingly highly motile epithelial cells. This 3D matrix translocation separates the cells to form clefts during embryonic mouse salivary gland development [49]. Acknowledgements The authors thank A. Doyle and S. Even-Ram for valuable comments and suggestions and H. Grant for excellent proofreading. Research support was provided by the Intramural Research Program of the NIH, NIDCR, to K.M.Y. 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