The matrix reorganized: remodeling and signaling Melinda Larsen1, Vira V Artym1,2, J Angelo Green1 and Kenneth M Yamada1

Via , cells can sense dimensionality and other physical (2D) surfaces and complex, malleable three-dimensional and biochemical properties of the extracellular matrix (ECM). (3D) environments can therefore be sensed by integrins Cells respond differently to two-dimensional substrates and that respond to these surfaces with altered signaling three-dimensional environments, activating distinct signaling (reviewed in [3–7]). pathways for each. Direct integrin signaling and indirect integrin modulation of and other intracellular signaling In this review, we discuss recent findings regarding ECM pathways regulate ECM remodeling and control subsequent remodeling, with emphasis on , fibronectin and behavior and tissue organization. ECM remodeling is associated integrin signaling. We examine how ECM critical for many developmental processes, and remodeled physical properties and higher-order organization influ- ECM contributes to tumorigenesis. These recent advances in ence cell behavior and integrin signaling pathways. ECM the field provide new insights and raise new questions about remodeling is required in vivo for proper development, the mechanisms of ECM synthesis and proteolytic degradation, but ECM alterations can also create an environment as well as the roles of integrins and tension in ECM remodeling. conducive to tumorigenesis. In this review, we will con- Addresses sider some examples of these processes. We apologize for 1 Craniofacial Developmental and Regeneration Branch, the omissions imposed by the need for brevity. National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370, USA Fibroblast-mediated collagen matrix 2 Department of Oncology, Lombardi Comprehensive Center, remodeling in vitro Georgetown University Medical School, Washington, DC 20057-1469, Increasing numbers of studies show that the morphology, USA cytoskeletal structure and signaling of cells grown on 2D surfaces differ from those of cells grown within 3D Corresponding author: Yamada, Kenneth M ([email protected]) environments, where collagen fibers contact both ventral (lower) and dorsal (upper) surfaces of the cells. In fact, Current Opinion in 2006, 18:463–471 upon engagement of receptors on their dorsal surface, well-spread fibroblasts in 2D culture quickly convert to a This review comes from a themed issue on Cell-to-cell contact and extracellular matrix bipolar or stellate morphology characteristic of fibroblasts  Edited by Martin Schwartz and Alpha Yap in 3D environments [8 ]. A recent study [9] of cell interactions with collagen reveals that a2b1 integrin- Available online 17th August 2006 mediated transport of collagen fibers and subsequent 0955-0674/$ – see front matter contraction of in vitro 3D collagen matrices requires # 2006 Elsevier Ltd. All rights reserved. non-muscle myosin II-B. Although this myosin is impor- tant for fibroblast cell motility in 3D collagen matrices, it DOI 10.1016/j.ceb.2006.08.009 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 Introduction rather than on 2D surfaces [9]. Extracellular matrix (ECM) remodeling is involved in development, fibrosis, tissue repair and tumor-associated Specific serum components can facilitate integrin-depen- (stromatogenesis). The ECM can be remo- dent 3D collagen gel contraction through different sig- deled by many processes, including synthesis, contraction naling pathways. PDGF-stimulated contraction of and proteolytic degradation. Integrins are the primary floating collagen matrices utilizes phosphatidylinositol ECM receptors mediating ECM remodeling (reviewed 3- (PI3K) and myosin II, whereas LPA-stimulated in [1,2]). In response to changes in the ECM, integrin contraction depends on signaling by the monomeric G-  signaling also regulates many other interrelated cellular protein GaI, but does not require myosin II [10,11 ]. processes: proliferation, survival, and inva- Using siRNA knockdown and inhibitors, it was demon- sion (Figure 1). Integrins function as mechanotransducers strated that signaling through PDGF and LPA converge and can transform mechanical forces created by the ECM on p21-activated kinase-1 (PAK1) to regulate collagen or the into chemical signals. Differences matrix contraction through cofilin-1 [11]. PAK1 there- between simple, rigid and non-pliable two-dimensional fore links these two distinct matrix remodeling pathways. 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 and survival. Signaling downstream of PI3K affects activation of Akt and the small Rac, Cdc42, and Rho to induce changes in the cytoskeleton, cell contractility, cell migration, invasion and gene expression. 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 , 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 , SFKs; Wiskott-Aldrich syndrome protein, WASP.

ROCK was specifically implicated in PDGF-induced and is well known that conformation directly affects integrin mDia1 in LPA-induced ECM contraction. These findings activation state and -binding activity; in 2D sub- suggest that Rho effectors act parallel to and/or coopera- strates, exogenous activating antibodies or manganese can tively with PAK1 to regulate contraction of floating col- activate integrins and induce matrix formation [1]. lagen matrices. Recent studies implicate the urokinase-type plasminogen matrix remodeling by fibroblasts receptor (uPAR) in regulating integrin a5b1 activ- in vitro ity. Addition of a uPAR ligand, the P-25 peptide, stimu- The major receptor for fibronectin, a5b1 integrin, can be lated fibronectin fibril assembly [14] by activating integrin found in different adhesion structures, such as focal a5b1 through the EGF receptor and Src [15]. Interestingly, complexes, focal adhesions, fibrillar adhesions and 3D- fibronectin fibril assembly by cells expressing activation- matrix adhesions [3]. Fibronectin fibrillogenesis is dependent integrins can be stimulated specifically by a 3D mediated by -dependent, directed translocation of fibronectin matrix without exogenous activators [16]. a5b1 out of focal adhesions into fibrillar adhesions [12], These data support the idea that a 3D matrix can activate and a recent study indicates that this translocation integrins to induce fibronectin matrix assembly (Figure 2), depends upon a specific integrin conformation [13]. It although the mechanism remains to be elucidated. One

Current Opinion in Cell Biology 2006, 18:463–471 www.sciencedirect.com Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 465

Figure 2 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 fibronec- tin 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 base- ment membrane remodeling during branching morpho- Extracellular factors promote ECM remodeling by stimulating integrin genesis. Recently, cytoskeletal tension was found to be activation. On a 2D substrate in vitro, fibroblast cells are polarized such important for branching: inhibitors of ROCK, myosin that only the ventral surface contacts the substrate. In this state, cells light chain kinase and myosin ATPase inhibited branch- are typically well-spread and only a subset of integrins is activated: bent ing of rudiments (Figure 3b) [22]. Interestingly, conformation, inactive integrin; extended conformation, activated ROCK stimulated basement membrane thinning at the integrin. Upon plating within a 3D matrix or following treatment with exogenous stimulators (e.g. Mn2+, activating antibodies or uPAR ligand), lung bud distal tips. This thinning may facilitate cell integrins undergo a and are activated, leading to proliferation and promote bud elongation through enhanced integrin–ECM interactions and increased ECM production. As decreased synthesis or increased protease-mediated the matrix assumes a more 3D character, cell morphology becomes degradation of basement membrane components. more bipolar or stellate, at least partly as a result of increased engagement of receptors on the dorsal cell surface. Fibronectin matrix remodeling in vivo Important new insights into the role of fibronectin matrix experimental approach to this question would be to remodeling during development have emerged recently use conformation-specific antibodies to detect activated from work in model organisms. formation is a major integrins within different 3D environments. event during vertebrate development, whereby the unseg- mented paraxial undergoes a mesenchymal-to- Cells in vivo exist in complex environments, where they epithelial transition to form epithelial segments. Although constantly interact with multiple ECM molecules rather fibronectin is known to be critical for somitogenesis than a single component. In vitro studies show that because fibronectin-null mouse embryos lack , specific cell–ECM interactions affect the dynamics of recent work has provided insight into the mechanism of other ECM components and the physical state of the this morphological segmentation (Figure 3b). Mutation of ECM. For example, cell-dependent fibronectin polymer- integrin a5 in zebrafish prevents fibronectin accumulation, ization increases the tensile strength of a 3D collagen resulting in a defect in somite boundaries and epitheliali- biogel without affecting rigidity [17]. Additionally, LPA zation [23]. Another recent study reports that fibronectin is induces both fibronectin and collagen assembly concur- required for somite maintenance but not initiation [24]. rently in smooth muscle cells. Inhibition of LPA-induced fibronectin assembly with an anti-integrin a5b1 antibody The mechanism by which fibronectin induces somite prevents collagen type I fibril assembly [18]. Other epithelialization involves focal adhesion kinase (FAK). studies also show that fibrillar collagen deposition is In zebrafish integrin a5 mutants, no active FAK (phos- dependent on fibronectin [19,20]. phorylated at Y397) was detected [24]. Inhibition of FAK signaling by the dominant-negative form of FAK, FAK- ECM remodeling in epithelial related nonkinase (FRNK), results in defective fibronec- ex vivo tin matrix deposition and disrupted somite boundaries in culture model systems provide powerful tools to Xenopus embryos [25]. It is interesting that FNRK- study developmental events using intact epithelial injected Xenopus embryos display a more embryonic tissues ex vivo. Branching organs, such as lung, severe than that of FAK-null mouse embryos. FRNK- kidney and salivary gland, can be studied as organ injected Xenopus embryos also show defective localization explants, since internally driven morphogenetic programs of Xenopus Ena, an Ena/VASP family protein linking continue ex vivo and recapitulate in vivo processes. integrins with the actin cytoskeleton. Neutralization of Branching morphogenesis involves a number of repetitive Ena/VASP activity results in defective fibronectin accu- steps, starting with formation of a cleft or indentation in mulation around somites and impaired FAK activation the basement membrane, which is a specialized ECM [25]. Together, these data suggest the existence of a www.sciencedirect.com 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/ assembly [33,34]. Pharmacological inhibition of ROCK or VASP protein. These studies imply that Ena/VASP pro- myosin II can reverse the malignant phenotype, indicat- teins and FAK activate integrin a5 to stimulate fibronec- ing a dependence of the transformed phenotype on Rho- tin fibrillogenesis and induce downstream signaling dependent tension [28]. ERK inhibition can also leading to epithelialization. This provocative finding reverse the malignant phenotype, underscoring the indicates that fibronectin can promote the transition of cross-talk between mitogenic growth factor and mechan- certain mesenchymal cells to an epithelial phenotype. otransducing integrin pathways [28]. As a result of the functional link between ECM rigidity, Rho, cell contrac- It is not clear why fibronectin can decrease E-cadherin in tility and cell behavior, a mechanosensitive positive feed- some contexts [21], whereas in other contexts, such as back loop appears to amplify cell proliferation, somite formation, it induces epithelialization [24] and transformation and ECM rigidity in tumors (Figure 4). increased cadherin expression. However, integrin signal- ing often forms part of a signaling network. In the com- Proteolytic remodeling of ECM in tumor plex process of somitogenesis, fibronectin-integrin a5 progression signaling acts cooperatively with other pathways: Eph– Many proteases can cleave ECM molecules involved in Ephrin signaling cooperates with fibronectin–integrin tumor progression (reviewed in [35]), but recent studies  signaling to maintain somite boundaries [24 ], and have identified MT1-MMP as a major protease support- Notch/Delta and integrin a5 interdependently regulate ing the invasive phenotype. MT1-MMP confers an  somite epithelialization and fibronectin assembly [23 ]. advantage on tumor cells in vitro and in vivo by enabling The interaction between these molecules is complex, and them to escape growth suppression by fibrillar type I it changes along the anterior–posterior axis. The cellular collagen [36]. Membrane-bound MT1-MMP cleaves col- response to fibronectin can therefore depend on the local lagen and activates proMMP-2 in the immediate vicinity signaling context. of cancer cells, creating a proliferation-promoting micro- environment of cleaved collagen. Interestingly, in MCF-7 ECM rigidity and cytoskeletal tension in cells expressing MT1-MMP, type I collagen increases tumor progression cell-surface MT1-MMP activity by a novel mechanism: Stromatogenesis is a desmoplastic alteration in tumor- impaired clathrin-mediated internalization of MT1- associated stroma, occurring in parallel with neoplasia, MMP [37]. MT1-MMP activity is essential for fibro- which is characterized by many changes, including blast and tumor cell invasion through 3D collagen gels, increased expression of organized fibronectin and type independent of plasminogen or secreted MMP activity I collagen by adjacent stromal fibroblasts [26,27]. A con- [38]. However, it should be noted that because epithe- sequence of stromatogenesis is that the tumor-associated lial cells rarely express MT1-MMP [39], even though stroma becomes more rigid. Measurements of the rigidity tumor cell lines and the surrounding stroma express of normal mammary tissue, malignant breast tissue and it, there are conflicting views on the significance of tumor-associated stroma revealed that malignancy is MT1-MMP in human epithelial tumors. accompanied by substantial increases in ECM rigidity in both the tumor and stroma [28]. Cells sense elevated The heterogeneity of ECM organization (reviewed in ECM rigidity through integrins and respond with mod- [40]), from loose to dense basement ified signaling, according to many studies. Increased membranes, requires cancer cells to employ a range of ECM rigidity stimulates integrin expression [29] and migration modes. Two types of cancer cell migration induces conformational changes to activate avb3 integrin through ECM have been identified: mesenchymal migra- [30]. Inappropriate increases in ECM rigidity perturb tion that utilizes integrins and proteases for adhesive and normal tissue architecture, activate Rho, induce Rho- proteolytic interactions with ECM proteins, and amoeboid generated cytoskeletal tension and activate ERK-depen- migration that is integrin-independent and non-proteoly- dent growth [31,32], whereas subsequent increases in tic (reviewed in [41]). Experimental models predict that cytoskeletal tension promote growth and focal adhesion cells can switch from mesenchymal to amoeboid migration

(Figure Legend 3) ECM remodeling during . (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 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 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 inhibi- invadopodia followed by MT1-MMP recruitment, and the tors, if the Rho/ROCK signaling pathway is activated, or if subsequent onset of ECM degradation, have been integrin–ECM interactions are blocked [41]. revealed by live-cell imaging [43]. Although intravital imaging has detected invadopodium-like protrusions in Focal pericellular proteolysis of ECM molecules is a hall- extravasating carcinoma cells [44], further studies are mark of mesenchymal migration by tumor cells [41]. required to detect invadopodial markers and test the Studies of malignant cells on 2D fluorescent matrices ECM-degrading ability of these protrusions to verify the indicate that pericellular proteolysis and invasion are physiological importance of invadopodia in vivo. mediated by specialized cell membrane protrusions termed invadopodia (reviewed in [42]). MT1-MMP was Remodeled ECM as a carcinogen and identified as a key invadopodial protease responsible for inducer of local ECM degradation by carcinoma cells. The dynamics Although, in normal tissue, one function of fibroblasts is of the sequential accumulation of cortactin plus actin at to maintain tissue homeostasis, the fibroblasts and ECM

Current Opinion in Cell Biology 2006, 18:463–471 www.sciencedirect.com Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 469

associated with tumors (the ) ECM and cell adhesion proteins should illuminate the can function as a carcinogen. The tumor microenviron- mechanisms of ECM assembly that control developmen- ment can send erroneous signals to tumors that induce tal processes, whereas time-lapse imaging of tumor cell accumulation of MMPs, activate soluble growth factors proteases and specialized cell membrane structures (e.g. and facilitate . The often invadopodia) will help elucidate their role in ECM degra- responds by undergoing an epithelial–mesenchymal tran- dation. Studies employing electron microscopy will also sition (EMT), where epithelial cells lose epithelial char- be useful to validate the significance of invadopodia in acteristics, acquire mesenchymal characteristics and vivo. Although the ECM and microenvironment are now become invasive. Exposure of epithelial cells to MMP- known to be involved in tumor initiation and progression 3 stimulates expression of Rac1b, an alternatively spliced (e.g. through ECM rigidity), the mechanisms need elu- and highly active form of Rac1, which induces cellular cidation. This gap could be addressed by development of reactive oxygen species (ROS). ROS both stimulate in vitro 3D models in which matrix rigidity and biochem- EMT and cause oxidative damage to DNA, resulting ical content can be carefully controlled. A difficulty with in genomic instability [45]. Tumor microenvironments studying tumor-associated fibroblasts has been maintain- are often hypoxic. An interesting but puzzling recent ing their phenotype in culture, but a recent report showed study reports that lysyl oxidase, the well-known that their phenotype can be maintained within a 3D that crosslinks collagen in vivo, is induced under hypoxic matrix [48]. Developing a deeper understanding of how conditions [46]. Although not required for primary tumor ECM remodeling occurs and how it modifies cell beha- growth, lysyl oxidase is required for metastasis, and it vior should lead to better clinical interventions for pathol- induces FAK phosphorylation to stimulate cell motility. ogies that develop when ECM remodeling goes awry. The role of this matrix-modifying enzyme in metastasis will be intriguing to resolve. Update A new study proposes that local translocation of a com- Conclusions and future directions ponent of the 3D matrix guides branching morphogen- Great strides have been made in our understanding of esis. Live confocal imaging of glands labeled with two- ECM remodeling through the increasing use of 3D cul- color fluorescently labeled fibronectin was used to estab- ture systems in recent years. Although it is now clear that lish that translocating wedges of fibronectin move steadily integrin signaling and cytoskeletal organization are dif- inward through a population of surprisingly highly motile ferent in 2D and 3D environments, the factors respon- epithelial cells. This 3D matrix translocation separates sible for these differences remain to be determined. the cells to form clefts during embryonic mouse salivary Understanding the mechanistic differences between gland development [49]. ECM remodeling on 2D surfaces and in 3D systems is critical for developing valid model systems characteristic Acknowledgements of in vivo biology. Which factors are responsible for the The authors thank A. Doyle and S. Even-Ram for valuable comments and suggestions and H. Grant for excellent proofreading. Research support differences: the complex linkages of ECM proteins, the was provided by the Intramural Research Program of the NIH, NIDCR, physical forces associated with ECM presentation to the to K.M.Y. cell, or a combination of these and other factors? Clar- ification is needed concerning how ECM components References and recommended reading structurally regulate each other to influence cell morphol- Papers of particular interest, published within the last two years, have been highlighted as: ogy and signaling. It may be possible to answer many questions regarding the impact of structure on cell beha-  of special interest vior using techniques that can sense local ECM rigidity.  of outstanding interest Future improvements in atomic force microscopy (AFM) or alternative approaches are needed to measure, apply 1. Humphries MJ, Travis MA, Clark K, Mould AP: Mechanisms of and evaluate the effects of mechanical stresses on mor- integration of cells and extracellular matrices by integrins. Biochem Soc Trans 2004, 32:822-825. phology and signaling of live cells in 3D environments. 2. DeMali KA, Wennerberg K, Burridge K: Integrin signaling to the actin cytoskeleton. Curr Opin Cell Biol 2003, 15:572-582. Since ECM remodeling is now known to be a dynamic 3. Cukierman E, Pankov R, Yamada KM: Cell interactions with process involving cytoskeletal molecules and proteases, three-dimensional matrices. Curr Opin Cell Biol 2002, live imaging in vivo and in 3D culture systems should 14:633-639. provide many new insights into mechanisms of ECM 4. O’Brien LE, Zegers MM, Mostov KE: Building epithelial remodeling. One issue with studying matrix assembly is architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol 2002, 3:531-537. distinguishing new ECM from old. A recent report applied the Timer reporter, a DsRed derivative that 5. Grinnell F: Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol 2003, 13:264-269. changes from green to red over time, to overcome this 6. Debnath J, Brugge JS: Modelling glandular epithelial problem in studies of elastic fiber formation [47]. Multi- in three-dimensional cultures. Nat Rev Cancer 2005, color in vivo time-lapse imaging of fluorescently labeled 5:675-688. www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:463–471 470 Cell-to-cell contact and ECM

7. Griffith LG, Swartz MA: Capturing complex 3D tissue physiology 22. Moore KA, Polte T, Huang S, Shi B, Alsberg E, Sunday ME, in vitro. Nat Rev Mol Cell Biol 2006, 7:211-224. Ingber DE: Control of basement membrane remodeling and epithelial branching morphogenesis in embryonic lung by Rho 8. Beningo KA, Dembo M, Wang YL: Responses of fibroblasts to and cytoskeletal tension. Dev Dyn 2005, 232:268-281.  anchorage of dorsal extracellular matrix receptors. Proc Natl Acad Sci U S A 2004, 101:18024-18029. 23. Julich D, Geisler R, Holley SA: Integrin a5 and delta/notch The differences between cell morphologies on 2D and 3D substrates can  signaling have complementary spatiotemporal requirements be partially resolved when dorsal receptors on cells in 2D are engaged by during zebrafish somitogenesis. Dev Cell 2005, 8:575-586. anchored fibronectin or collagen, thereby mimicking a 3D environment. This paper identifies a role for integrin a5 in somite formation and These responses are dependent on substrate rigidity and . suggests that integrin a5 and Notch signaling are interdependent during the cell polarization and fibronectin matrix assembly that occur in somite 9. Meshel AS, Wei Q, Adelstein RS, Sheetz MP: Basic mechanism of formation. three-dimensional collagen fibre transport by fibroblasts. Nat Cell Biol 2005, 7:157-164. 24. Koshida S, Kishimoto Y, Ustumi H, Shimizu T, Furutani-Seiki M,  Kondoh H, Takada S: Integrin a5-dependent fibronectin 10. Abe M, Ho CH, Kamm KE, Grinnell F: Different molecular motors accumulation for maintenance of somite boundaries in mediate -derived growth factor and lysophosphatidic zebrafish embryos. Dev Cell 2005, 8:587-598. acid-stimulated floating collagen matrix contraction. In this manuscript, the authors describe somite formation as a two-step J Biol Chem 2003, 278:47707-47712. process: a fibronectin-independent initiation step and a maintenance step requiring integrin a5 and fibronectin. The maintenance step involves 11. Rhee S, Grinnell F: P21-activated kinase 1: convergence point somite epithelialization, which also involves Eph–Ephrin signaling that  in PDGF- and LPA-stimulated collagen matrix contraction by may function redundantly with the fibronectin pathway. human fibroblasts. J Cell Biol 2006, 172:423-432. Two important signaling pathways that are initiated by the serum com- 25. Kragtorp KA, Miller JR: Regulation of somitogenesis by ponents LPA and PDGF converge on PAK1 to regulate collagen matrix  Ena/VASP proteins and FAK during Xenopus development. contraction through cofilin 1. PDGF and LPA can also stimulate matrix Development 2006, 133:685-695. contraction in cooperation with PAK1, via Rho kinase and mDia1, respec- In this paper, details of the molecular mechanisms for morphogenesis of tively. somite formation are identified. FAK and Ena/VASP proteins are shown to be required for somite formation and fibronectin matrix formation, per- 12. Pankov R, Cukierman E, Katz BZ, Matsumoto K, Lin DC, haps by regulating integrin activity. Lin S, Hahn C, Yamada KM: Integrin dynamics and matrix assembly: tensin-dependent translocation of a5b1 integrins 26. Bissell MJ, Radisky D: Putting tumours in context. Nat Rev promotes early fibronectin fibrillogenesis. J Cell Biol 2000, Cancer 2001, 1:46-54. 148:1075-1090. 27. Sivridis E, Giatromanolaki A, Koukourakis MI: ‘Stromatogenesis’ 13. Clark K, Pankov R, Travis MA, Askari JA, Mould AP, Craig SE, and tumor progression. Int J Surg Pathol 2004, 12:1-9.  Newham P, Yamada KM, Humphries MJ: A specific a5b1 integrin conformation promotes directional integrin translocation and 28. Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, fibronectin matrix formation. J Cell Sci 2005, 118:291-300.  Gefen A, Reinhart-King CA, Margulies SS, Dembo M, Boettiger D et al.: Tensional homeostasis and the malignant phenotype. A novel conformation-dependent anti-a5 integrin antibody (SNAKA51), capable of promoting fibronectin matrix formation, is used as a tool to Cancer Cell 2005, 8:241-254. identify a subset of adhesion contacts associated with integrin transloca- This paper examines the relationship between tissue rigidity and tumor tion from focal contacts into fibrillar adhesions, a mechanism of fibro- behavior. It demonstrates that ECM stiffness perturbs epithelial morpho- nectin fibrillogenesis. genesis by clustering integrins and inducing focal adhesion assembly to enhance ERK activation and increase ROCK-mediated contractility. 14. Monaghan E, Gueorguiev V, Wilkins-Port C, McKeown-Longo PJ: The receptor for urokinase-type plasminogen activator 29. Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, regulates fibronectin matrix assembly in human Zahir N, Ming W, Weaver V, Janmey PA: Effects of substrate fibroblasts. J Biol Chem 2004, 279:1400-1407. stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton 2005, 60:24-34. 15. Monaghan-Benson E, McKeown-Longo PJ: Urokinase-type plasminogen activator receptor regulates a novel pathway 30. Katsumi A, Orr AW, Tzima E, Schwartz MA: Integrins in of fibronectin matrix assembly requiring Src-dependent . J Biol Chem 2004, 279:12001-12004. transactivation of epidermal growth factor receptor. 31. Wang F, Weaver VM, Petersen OW, Larabell CA, Dedhar S, J Biol Chem 2006, 281:9450-9459. Briand P, Lupu R, Bissell MJ: Reciprocal interactions between b1-integrin and epidermal growth factor receptor in three- 16. Mao Y, Schwarzbauer JE: Stimulatory effects of a three- dimensional basement membrane breast cultures: a different dimensional microenvironment on cell-mediated fibronectin  perspective in epithelial biology. Proc Natl Acad Sci U S A 1998, fibrillogenesis. J Cell Sci 2005, 118:4427-4436. 95:14821-14826. This paper demonstrates that a 3D fibronectin matrix can stimulate cellular fibronectin fibril assembly to a greater extent than a 2D fibronectin 32. Wozniak MA, Desai R, Solski PA, Der CJ, Keely PJ: ROCK- matrix. In addition, cells normally requiring activation of integrins for generated contractility regulates breast epithelial cell matrix formation on 2D substrates can assemble fibronectin matrix on differentiation in response to the physical properties of a 3D substrates without exogenous activation. three-dimensional collagen matrix. J Cell Biol 2003, 163:583-595. 17. Gildner CD, Lerner AL, Hocking DC: Fibronectin matrix polymerization increases tensile strength of model tissue. 33. Roovers K, Assoian RK: Effects of Rho kinase and actin stress Am J Physiol Heart Circ Physiol 2004, 287:H46-H53. fibers on sustained extracellular signal-regulated kinase activity and activation of G1- phase cyclin-dependent kinases. 18. Li S, Van Den Diepstraten C, D’Souza SJ, Chan BM, Pickering JG: Mol Cell Biol 2003, 23:4283-4294. Vascular smooth muscle cells orchestrate the assembly of type I collagen via a2b1 integrin, RhoA, and fibronectin 34. Burridge K, Wennerberg K: Rho and Rac take center stage. polymerization. Am J Pathol 2003, 163:1045-1056. Cell 2004, 116:167-179. 19. Velling T, Risteli J, Wennerberg K, Mosher DF, Johansson S: 35. Decock J, Paridaens R, Cufer T: Proteases and metastasis: Polymerization of type I and III is dependent on clinical relevance nowadays? Curr Opin Oncol 2005, fibronectin and enhanced by integrins a11b1 and a2b1. 17:545-550. J Biol Chem 2002, 277:37377-37381. 36. Hotary KB, Allen ED, Brooks PC, Datta NS, Long MW, Weiss SJ: 20. Sottile J, Hocking DC: Fibronectin polymerization regulates the Membrane type I usurps tumor composition and stability of extracellular matrix fibrils and growth control imposed by the three-dimensional cell-matrix adhesions. Mol Biol Cell 2002, 13:3546-3559. extracellular matrix. Cell 2003, 114:33-45. 21. Sakai T, Larsen M, Yamada KM: Fibronectin requirement 37. Lafleur MA, Mercuri FA, Ruangpanit N, Seiki M, Sato H, in branching morphogenesis. Nature 2003, 423:876-881.  Thompson EW: Type I collagen abrogates the clathrin-

Current Opinion in Cell Biology 2006, 18:463–471 www.sciencedirect.com Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 471

mediated internalization of membrane type 1 matrix This paper uses three-color live-cell imaging and multispectral confocal metalloproteinase (MT1-MMP) via the MT1-MMP hemopexin microscopy to reveal the dynamics of the stepwise formation of invado- domain. J Biol Chem 2006, 281:6826-6840. podia. Cortactin accumulates first and is followed by MT1-MMP, leading A new regulatory mechanism for cell surface MT1–MMP presentation is to matrix degradation. described in this report. Upon type I collagen binding to its , MT1-MMP internalization via clathrin-coated-pit-mediated 44. Yamaguchi H, Wyckoff J, Condeelis J: Cell migration in tumors. endocytosis is inhibited through interactions with the hemopexin domain. Curr Opin Cell Biol 2005, 17:559-564. 38. Sabeh F, Ota I, Holmbeck K, Birkedal-Hansen H, Soloway P, 45. Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE,  Balbin M, Lopez-Otin C, Shapiro S, Inada M, Krane S et al.: Tumor  Leake D, Godden EL, Albertson DG, Nieto MA et al.: Rac1b and cell traffic through the extracellular matrix is controlled by the reactive oxygen species mediate MMP-3-induced EMT and membrane-anchored collagenase MT1-MMP. J Cell Biol 2004, genomic instability. Nature 2005, 436:123-127. 167:769-781. This work identifies a novel molecular pathway by which MMP-3 induces This paper reports that fibroblasts and tumor cells are able to traverse the expression of Rac1b, leading to increased ROS and subsequent through dense cross-linked type I collagen barriers in vitro and in vivo via a oxidative damage to DNA, genomic instability and malignant transforma- proteolytic process that is uniquely mediated by MT1-MMP. Data pre- tion. sented in the paper identify MT1-MMP as a required pericellular collage- nolysin that confers tissue-invasive capability on normal and neoplastic 46. Erler JT, Bennewith KL, Nicolau M, Dornhofer N, Kong C, Le QT, cells. Chi JT, Jeffrey SS, Giaccia AJ: Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 2006, 440:1222-1226. 39. Holmbeck K, Bianco P, Yamada S, Birkedal-Hansen H: MT1-MMP: a tethered collagenase. J Cell Physiol 2004, 47. Kozel BA, Rongish BJ, Czirok A, Zach J, Little CD, Davis EC, 200:11-19. Knutsen RH, Wagenseil JE, Levy MA, Mecham RP: Elastic fiber formation: a dynamic view of extracellular matrix assembly 40. Even-Ram S, Yamada KM: Cell migration in 3D matrix. using timer reporters. J Cell Physiol 2006, 207:87-96. Curr Opin Cell Biol 2005, 17:524-532. 48. Amatangelo MD, Bassi DE, Klein-Szanto AJ, Cukierman E: 41. Friedl P: Prespecification and plasticity: shifting mechanisms Stroma-derived three-dimensional matrices are necessary of cell migration. Curr Opin Cell Biol 2004, 16:14-23. and sufficient to promote desmoplastic differentiation of 42. Ayala I, Baldassarre M, Caldieri G, Buccione R: Invadopodia: a normal fibroblasts. Am J Pathol 2005, 167:475-488. guided tour. Eur J Cell Biol 2006, 85:159-164. 49. Larsen M, Wei C, Yamada KM: Cell and fibroenctin dynamics 43. Artym VV, Zhang Y, Seillier-Moiseiwitsch F, Yamada KM,  during branching morphogenesis. J Cell Sci 2006, in press.  Mueller SC: Dynamic interactions of cortactin and membrane Two-color confocal time-lapse imaging is used to examine both cell and type 1 matrix metalloproteinase at invadopodia: defining the matrix dynamics in salivary glands in organ culture. These studies reveal stages of invadopodia formation and function. Cancer Res that both embryonic epithelial cells and local sites of 3D fibronectin matrix 2006, 66:3034-3043. are unexpectedly highly dynamic during development.

www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:463–471