Functional imaging of pericellular proteolysis in cancer cell invasion

Proteolytic interactions between cells and extracellular matrix (ECM) are involved in many physiological and pathological processes, such as embryogenesis, wound healing, immune response, and cancer. The visualization of cell-mediated proteolysis towards ECM is thus required to understand basic mechanisms of tissue formation and repair, such as the breakdown and structural remodelling of ECM, inflammatory changes of tissue integrity, and the formation of proteolytic trails by moving cells. A panel of synergistic techniques for the visualization of pericellular proteolysis in live and fixed samples allow monitoring the of proteolytic tumor cell invasion in three-dimensional (3D) fibrillar collagen matrices in vitro. These include the quantification of collagenolysis by measuring the release of collagen fragments, the detection of protease expression and local activity by dequenching of fluorogenic substrate, and the staining of cleavage-associated neoepitopes together with changes in matrix structure. In combination, these approaches allow the high-resolution mapping of pericellular proteolysis towards ECM substrata including individual focal cleavage sites and the interplay between cell dynamics and alterations in the tissue architecture.     

Epilysin (MMP-28) is deposited to the basolateral extracellular matrix of epithelial cells

Epilysin (MMP-28) is a conserved member of the matrix metalloproteinase (MMP) family. It is expressed in various normal tissues, and induced in wounds and in developing and regenerating nerves. Epilysin induces TGF-β mediated epithelial to mesenchymal transition, but its other functions are largely unknown. We have characterized the localization of both catalytically active and mutated inactive, overexpressed epilysin in established epithelial cell lines. We found that epilysin was localized abundantly to the basolateral side of the cells and associated with the extracellular matrix (ECM) as verified by immunoblotting and confocal microscopy. Overexpression of epilysin in MDCK cells resulted in a drastic reduction of basolateral ECM, as observed by the disappearance of collagen type IV, laminin and fibronectin. Cultivation of epilysin expressing MDCK cells in defined serum free medium resulted in the restoration of these proteins to the ECM. The levels of fibronectin and collagen IV were, however, reduced in epilysin expressing cells under the serum free conditions, and degradation fragments of collagen IV were detected supporting the activation of proteolysis by epilysin. Epilysin was observed in its unprocessed 50 kDa active form in the ECM of MDCK cells under serum free conditions whereas in cells cultured in serum containing it was processed to the 48 kDa form. Current results indicate that epilysin associates with the basolateral ECM of cultured epithelial cells, where it plausibly plays a role in the regulation of matrix composition and turnover.     

Gelatinases and their inhibitors in tumor metastasis: from biological research to medical applications

The involvement of matrix metalloproteinase (MMPs)-2 and -9, also known as gelatinases, in cancer cell migration and invasion has been well documented, although it is not yet clear how they facilitate metastasis formation in the course of malignancies. The idea that gelatinases are responsible for degradation of extracellular matrix (ECM) components and breakdown of basement membrane (BM) tissue boundaries has turned out not to be entirely correct. An action by remodelling the ECM components of the BM exposing new cryptic sites, or releasing growth factors, cytokines, or active ECM proteolysed fragments seems to be nearer to the truth. On the other hand, tissue inhibitors of gelatinase activity (TIMP-2), are involved both in the MMP-2 activation process; in concert with membrane type 1-MMP (MT1-MMP), and in the inhibition of gelatinolytic activity. Therefore proteolysis, the central step for cancer metastasis, should occur as a result of an imbalance between MMP-2 and TIMP-2. Many studies have reported the importance of this balance in patients with different malignancies, and considerable effort is currently being spent on the study of molecules that can shift the balance in favour of inhibition of MMP proteolytic activity. In this review we focus on the role of gelatinase activity in cancer invasion, addressing the following issues: how and where proteolysis occurs in cancer tissues, how it can be regulated, what the clinical implications are of the studies reported in literature so far, and finally what the future developments in this field that could have an impact on the management of patients affected by malignancies may be.     

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.     

Oscillatory behavior of a simple kinetic model for proteolysis during cell invasion.

Extracellular proteolysis during cell invasion is thought to be tightly organized, both temporally and spatially. This work presents a simple kinetic model that describes the interactions between extracellular matrix (ECM) proteins, proteinases, proteolytic fragments, and integrins. Nonmonotonous behavior arises from enzyme de novo synthesis consecutive to integrin binding to fragments or entire proteins. The model has been simulated using realistic values for kinetic constants and protein concentrations, with fibronectin as the ECM protein. The simulations show damped oscillations of integrin-complex concentrations, indicating alternation of maximal adhesion periods with maximal mobility periods. Comparisons with experimental data from the literature confirm the similarity between this system behavior and cell invasion. The influences on the system of cryptic functions of ECM proteins, proteinase inhibitors, and soluble antiadhesive peptides were examined. The first critical parameter for oscillation is the discrepancy between integrin affinity for intact ECM proteins and the respective proteolytic fragments, thus emphasizing the importance of cryptic functions of ECM proteins in cell invasion. Another critical parameter is the ratio between proteinase and the initial ECM protein concentration. These results suggest new insights into the organization of the ECM degradation during cell invasion.     

A bit of give and take: the relationship between the extracellular matrix and the developing chondrocyte

The extracellular matrix (ECM), once thought to be a static structural component of tissues, is now known to play a complex and dynamic role in a variety of cellular functions in a number of diverse tissues. A significant body of literature attests to the ability of the ECM to communicate both spatial and temporal information to adherent cells, thereby directing cell behavior via interactions between the ECM and cell-surface receptors. Moreover, volumes of experimental data show that a great deal of communication travels in the opposite direction, from the cell to the ECM, allowing for regulation of the cues transmitted by the ECM. As such, the ECM, with respect to its components and their organization, is not a fixed reflection of the state the local microenvironment in which a cell finds itself at a particular time, but rather is able to respond to and effect changes in its local microenvironment. As an example of the developmental consequences of ECM interactions, this review gives an overview of the 'give and take' relationship between the ECM and the cells of the developing skeletal elements, in particular, the chondrocyte.     

Emerging Role of Syndecans in Extracellular Matrix Remodeling in Cancer

The extracellular matrix (ECM) offers a structural basis for regulating cell functions while also acting as a collection point for bioactive molecules and connective tissue cells. To perform pathological functions under a pathological condition, the involved cells need to regulate the ECM to support their altered functions. This is particularly common in the development of cancer. The ECM has been recognized as a key driver of cancer development and progression, and ECM remodeling occurs at all stages of cancer progression. Thus, cancer cells need to change the ECM to support relevant cell surface adhesion receptor–mediated cell functions. In this context, it is interesting to examine how cancer cells regulate ECM remodeling, which is critical to tumor malignancy and metastatic progression. Here, we review how the cell surface adhesion receptor, syndecan, regulates ECM remodeling as cancer progresses, and explore how this can help us better understand ECM remodeling under these pathological conditions     

Structure-function relationships in class CA1 cysteine peptidase propeptides

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at a wrong time and location may be lethal. Proteases are synthesized as inactive or less active precursor molecules in order to prevent such inappropriate proteolysis. They are activated by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize the structural background of three functions of clan CA1 cysteine peptidase (papain family) proparts, namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Today, we know more than 500 cysteine peptidases of this family from the plant and animal kingdoms, e.g. papain and the lysosomal cathepsins L and B. As it will be shown, the propeptide functions are determined by certain structural motifs conserved over millions of years of evolution.     

Role of the extracellular matrix in lymphocyte migration

The extracellular matrix (ECM) exists in various biochemical and structural forms that can act either as a barrier to migrating leukocytes, in the case of basement membranes, or provide a physical scaffold supporting or guiding migration (interstitial matrix). This review focuses on basement membranes and our current knowledge of the way that leukocytes transmigrate this protein barrier, with emphasis on T lymphocytes. Recent data suggest that the classical concept of cell-matrix adhesion requires revision with respect to leukocyte-ECM interactions. Whereas specific receptors may be required for leukocyte recognition of ECM molecules or three-dimensional structural domains, the role of adhesion in migration as perceived from the traditional studies of adherent cell-ECM interactions is less clear. Further, the indirect effects of ECM such as the binding and presentation of cytokines or chemotactic factors may more profoundly influence the directed migration of normally non-adherent leukocytes than the migration of adherent cells such as epithelial cells or fibroblasts. Proteases (in particular matrix metalloproteinases) released at sites of inflammation can selectively process ECM, cell surface molecules or soluble factors, which may result in the release of bioactive fragments that can function as chemoattractants for different leukocyte subsets or may modulate the activity/function of resident mesenchymal and immune cells. Current findings suggest that different leukocyte types employ different mechanisms to migrate across or through the ECM; this might be determined by the composition and organization of the ECM itself.     

Suprastructures of extracellular matrices: paradigms of functions controlled by aggregates rather than molecules

Extracellular matrices (ECM) not only serve as structural scaffolds in organs and tissues, but also determine critical cellular functions through cell-matrix interactions. These are mediated by cell surface receptors that recognise specific structural features of ECMs and, hence, overall physical properties of the acellular environment. ECM structures are subject to hierarchic organisations, which are tightly adapted to the functions of tissues and organs. Only a few specialised tasks are reserved for isolated ECM macromolecules. Instead, molecular ECM components attain their prominent functions only after polymerising into insoluble suprastructural elements, i.e. fibrils, microfibrils, or networks that, in turn, are assembled into regional tissue structures, such as fibres or basement membranes. As an outstanding feature, most, if not all, ECM suprastructures are co-polymers of more than one molecular species that differ in their identity and relative abundance. Thus, ECM suprastructures are composite biological amalgamates. The analogy to metal alloys refers to structural and functional characteristics of ECM composites, which differ from those of each homo-polymeric aggregate. At the tissue level, biological alloys can themselves be assembled into conglomerates that again assume properties distinct from those of each individual alloy. Nevertheless, most studies in matrix biology solely focus on molecular features and mechanisms. Progress has however been made in identifying principles of interactions within suprastructural elements and their functional consequences. We are now only beginning to understand the impact of suprastructural organisation on the assembly and the functions of whole tissues and many fundamental issues in this almost pristine field await discovery.     

The developmental roles of the extracellular matrix: beyond structure to regulation

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to serve a structural function providing support and strength to cells within tissues, is increasingly being recognized as having pleiotropic effects in development and growth. Elucidation of the role that the ECM plays in developmental processes has been significantly advanced by studying the phenotypic and developmental consequences of specific genetic alterations of ECM components in the mouse. These studies have revealed the enormous contribution of the ECM to the regulation of key processes in morphogenesis and organogenesis, such as cell adhesion, proliferation, specification, migration, survival, and differentiation. The ECM interacts with signaling molecules and morphogens thereby modulating their activities. This review considers these advances in our understanding of the function of ECM proteins during development, extending beyond their structural capacity, to embrace their new roles in intercellula signaling.     

The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis.

Metalloproteases are important in many aspects of biology, ranging from cell proliferation, differentiation and remodeling of the extracellular matrix (ECM) to vascularization and cell migration. These events occur several times during organogenesis in both normal development and during tumor progression. Mechanisms of metalloprotease action underlying these events include the proteolytic cleavage of growth factors so that they can become available to cells not in direct physical contact, degradation of the ECM so that founder cells can move across tissues into nearby stroma, and regulated receptor cleavage to terminate migratory signaling. Most of these processes require a delicate balance between the functions of matrix metalloproteases (MMPs) or metalloprotease-disintegrins (ADAMs) and natural tissue inhibitors of metalloproteases (TIMPs). In this review, we discuss recent progress in identifying an essential role for metalloproteases in axon outgrowth, as an example of a focal invasive event. We also discuss the evolving concept of how MMPs might regulate stem cell fate during tumor development.     

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.     

RGTA<Superscript>®</Superscript> or ReGeneraTing Agents mimic heparan sulfate in regenerative medicine: from concept to curing patients

The importance of extracellular matrix (ECM) integrity in maintaining normal tissue function is highlighted by numerous pathologies and situations of acute and chronic injury associated with dysregulation or destruction of ECM components. Heparan sulfate (HS) is a key component of the ECM, where it fulfils important functions associated with tissue homeostasis. Its degradation following tissue injury disrupts this delicate equilibrium and may impair the wound healing process. ReGeneraTing Agents (RGTA^®s) are polysaccharides specifically designed to replace degraded HS in injured tissues. The unique properties of RGTA^® (resistance to degradation, binding and protection of ECM structural and signaling proteins, like HS) permit the reconstruction of the ECM, restoring both structural and biochemical functions to this essential substrate, and facilitating the processes of tissue repair and regeneration. Here, we review 25 years of research surrounding this HS mimic, supporting the mode of action, pre-clinical studies and therapeutic efficacy of RGTA^® in the clinic, and discuss the potential of RGTA^® in new branches of regenerative medicine.     

Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis

Nothing in biology stimulates the imagination like the development of a single fertilized egg into a newborn child. Consequently, a major focus of biomedical research is aimed at understanding cell differentiation, proliferation, and specialization during child health and human development. However, the fact that the increase in size and shape of the growing embryo has as much to do with the extracellular matrix (ECM) as with the cells themselves, is largely overlooked. Cells in developing tissues are surrounded by a fiber-composite ECM that transmits mechanical stimuli, maintains the shape of developing tissues, and functions as a scaffold for cell migration and attachment. The major structural element of the ECM is the collagen fibril. The fibrils, which are indeterminate in length, are arranged in different tissues in exquisite supramolecular architectures, including parallel bundles, orthogonal lamellae, and concentric weaves. This article reviews our current understanding of the synthesis and assembly of collagen fibrils, and discusses challenging questions about how cells assemble an organized ECM during embryogenesis.     

Extracellular matrix, biotensegrity and tumor microenvironment. An update and overview

The extracellular matrix (ECM) constitutes a three-dimensional network that surrounds all cells, organs and tissues in the body. It forms a biophysical filter for protection, nutrition and cell innervation, as well as the medium for facilitating immune response, angiogenesis, fibrosis and tissue regeneration. It is the mechanism by which mechanical forces are transmitted to the basement membrane which, through the integrins, supports the tensegrity system and activates the epigenetic mechanisms of the cell. A review and update on current knowledge on this topic reveals how disturbance of the ECM leads to a loss of efficient filtering, nutrition, elimination, and cell denervation functions, in addition to loss of regeneration capacity and disorders in mechanotransduction. Furthermore, such disturbance results in a loss of substrate, and with it the ability to provide a proper immune response against tumor, toxic and infectious agents. Reciprocal communication between ECM stromal and parenchymatous cells directs gene expression. The oncogenic capacity of the stroma derives from the associated cells as well as from the tumor cells, the angiogenic microenvironment and from an alteration in tensegrity; all of which are dependent on the ECM. It has been shown that the malignant phenotype is reversible by correction of the altered cues of the ECM.     

Epithelial-mesenchymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors.

Morphogenesis and cell differentiation in the developing tooth are controlled by a series of reciprocal interactions between the epithelial and mesenchymal tissues. The exact molecular mechanisms operating in these interactions are unknown at present, but both structural components of the extracellular matrix (ECM) and diffusible growth factors have been suggested to be involved. In this review article we summarize our findings on the distribution patterns of three ECM molecules and two cell surface receptors during tooth morphogenesis through bud, cap, and bell stages of development. The examined molecules include fibronectin, type III collagen, and tenascin, which all represent components of the mesenchymal ECM, the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix, and the cell surface receptor for epidermal growth factor. Based on the observed changes in distribution patterns and on experimental evidence, roles are suggested for these molecules in epithelial-mesenchymal interactions during tooth development. Fibronectin is suggested to be involved in the cell-matrix interaction that controls odontoblast differentiation. Epidermal growth factor and its receptors are suggested to be involved in a paracrine fashion in the epithelial-mesenchymal interactions regulating morphogenesis of bud- and cap-stage teeth. Tenascin and syndecan are accumulated in the dental mesenchyme during the bud stage of development, and it is suggested that they represent a couple of a cell surface receptor and its matrix ligand and that they are involved in mesenchymal cell condensation during the earliest stages of tooth morphogenesis.     

Functional role of periostin in development and wound repair: implications for connective tissue disease

Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell-matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair.