Tales from the crypt[ic] sites of the extracellular matrix

Proteolytic cleavage of extracellular matrix (ECM) proteins by matrix metalloproteinases and/or conformational changes unmask "cryptic" sites and liberate fragments with biological activities that are not observed in the intact molecule. Cryptic sites and fragments of ECM macromolecules have been implicated in many events governed by cell-ECM interactions, such as migration, invasion, adhesion and differentiation. The unmasking of cryptic sites is a tightly controlled process, reflecting the importance of cryptic ECM functions. This review summarizes and evaluates the current developments regarding cryptic regulatory ECM signals found as ECM-tethered protein epitopes or fragments.     

Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution

Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.     

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-mediated chemotaxis can impede cell migration

Cells use a combination of changes in adhesion, proteolysis and motility (directed and random) during the process of migration. Proteolysis of the extracellular matrix (ECM) results in thecreation of haptotactic gradients which cells use to move in a directed fashion. The proteolytic creation of these gradients also results in the production of digested fragments of ECM. In this study we show that in the human fibrosarcoma cell line HT1080, matrix metalloproteinase-2(MMP-2)-digested fragments of fibronectin exert a chemotactic pull stronger than that of undigested fibronectin. During invasion, this gradient of ECM fragments is established in the wake of an invading cell, running counter to the direction of invasion. The resultant chemotactic pull is anti-invasive, contrary to the traditional view of the role of chemotaxis in invasion. Uncontrolled ECM degradation by high concentrations of MMP can thus result in steep gradients of ECM fragments, which run against the direction of invasion. Consequently, the invasive potential of a cell depends on MMP production in a biphasic mannerimplying that MMP inhibitors will upregulate invasion in high-MMPexpressing cells. Hence the therapeutic use of protease inhibitors against tumours expressing high levels of MMP could produce an augmentation of invasion.     

Nanoscale imaging and quantification of local proteolytic activity

Proteolytic cleavage of extracellular matrix (ECM) is a critical feature of tumor cell invasion, and affects cancer cell growth, differentiation, apoptosis, and migration. Malignant cells secrete most proteases as inactive proenzymes that undergo proteolytic cleavage for activation, and proteolytic activity is elevated in close proximity to these cells. Therefore, local activity rather than protease concentration determines ECM proteolysis. Precise quantification of local proteolytic activity, functional investigation, and high resolution imaging of morphological ECM alterations have proven difficult. In this study, we present a novel approach for measuring proteolytic activity in the microenvironment of cells by using atomic force microscopy (AFM). Amelanotic melanoma cells (A7-clone) were seeded on fluorescent gelatin or collagen-IV coatings. Proteolysis reduced fluorescence of these coatings. Fluorescence microscopy (FM) in combination with AFM was used to maneuver the AFM-tip to tumor cell induced proteolytic spots. AFM enabled nanoscale volume measurement, three-dimensional reconstruction of single proteins and demonstrated that ECM cleavage is restricted to the proteolytic microenvironment of cancer cells. This method detected significant decreases in molecular weight of protein clusters (-76.6%), matrix volume (-46.6%), and height (-38.1%) between intact and proteolyzed gelatin. Similar parameter changes were demonstrated without FM, by AFM-scanning gelatin in close proximity to invasive cells. Furthermore, AFM depicted significantly stronger local degradation of gelatin than collagen-IV by A7-cells. Taken together, AFM allows specific quantification and imaging of local proteolytic processes at a nanometer level, thus providing a unique method for the functional evaluation of invasiveness and metastatic potential of tumor cells in small scale samples.     

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.     

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

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.     

Syndecans in tumor cell adhesion and signaling

Anchorage of cells to "heparin" – binding domains that are prevalent in extracellular matrix (ECM) components is thought to occur primarily through the syndecans, a four-member family of transmembrane heparan sulfate proteoglycans that communicate environmental cues from the ECM to the cytoskeleton and the signaling apparatus of the cell. Known activities of the syndecans trace to their highly conserved cytoplasmic domains and to their heparan sulfate chains, which can serve to regulate the signaling of growth factors and morphogens. However, several emerging studies point to critical roles for the syndecans' extracellular protein domains in tumor cell behavior to include cell adhesion and invasion. Although the mechanisms of these activities remain largely unknown, one possibility involves "co-receptor" interactions with integrins that may regulate integrin function and the cell adhesion-signaling phenotype. Thus, alterations in syndecan expression, leading to either overexpression or loss of expression, both of which take place in tumor cells, may have dramatic effects on tumor cell invasion.     

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination

The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5–dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and β3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.     

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.     

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.     

Localization of a cryptic binding site for tenascin on fibronectin

Fibronectin and tenascin are large extracellular matrix proteins that interact with each other and with integrin receptors to regulate cell growth and movement. They are both modular proteins composed of independently folded domains (modules) that are arranged in linear fashion. Fibronectin is a covalent dimer and tenascin is a hexamer. The site on tenascin to which fibronectin binds has been localized to type III modules 3-5. In this study we use surface plasmon resonance to examine the interaction between various fragments of fibronectin and tenascin to further characterize and localize the binding sites. We found that tenascin fragments that contain type III modules 3-5 bind primarily to the N-terminal 29-kDa hep-1/fib-1 domain, which contains the first five type I modules of fibronectin. The dissociation constant, K(d), is approximately 1 microm. The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation.     

Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion

Invasive cell migration through tissue barriers requires pericellular remodelling of extracellular matrix (ECM) executed by cell-surface proteases, particularly membrane-type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Using time-resolved multimodal microscopy, we show how invasive HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells coordinate mechanotransduction and fibrillar collagen remodelling by segregating the anterior force-generating leading edge containing beta1 integrin, MT1-MMP and F-actin from a posterior proteolytic zone executing fibre breakdown. During forward movement, sterically impeding fibres are selectively realigned into microtracks of single-cell calibre. Microtracks become expanded by multiple following cells by means of the large-scale degradation of lateral ECM interfaces, ultimately prompting transition towards collective invasion similar to that in vivo. Both ECM track widening and transition to multicellular invasion are dependent on MT1-MMP-mediated collagenolysis, shown by broad-spectrum protease inhibition and RNA interference. Thus, invasive migration and proteolytic ECM remodelling are interdependent processes that control tissue micropatterning and macropatterning and, consequently, individual and collective cell migration.     

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.     

Local proteolytic activity in tumor cell invasion and metastasis

Proteolytic cleavage of extracellular matrix (ECM) is a critical regulator of many physiological and pathological events. It affects fundamental processes such as cell growth, differentiation, apoptosis and migration. Most proteases are produced as inactive proenzymes that undergo proteolytic cleavage for activation. Proteolytic activity is additionally modified by endogenous inhibitors. Mechanisms that localize and concentrate protease activity in the pericellular microenvironment of cells are prerequisites for processes like angiogenesis, bone development, inflammation and tumor cell invasion. Methods that enable real-time, high-resolution imaging and precise quantification of local proteolytic activity in vitro and in vivo remain major challenges. These methods will play an important role in the understanding of basic principles e.g. in cancer cell invasion, the identification of new therapeutical targets and hence drug design. This review highlights mechanisms and functions of local proteolytic activity with special emphasis on tumor cell invasion and metastasis, and focuses on techniques for the investigation of this process.