MAJOR EXTRACELLULAR MATRIX (ECM) ELEMENTS
The “core matrisome”1–3 comprises approximately 300 proteins. Major components include collagens, proteoglycans, elastin, and cell-binding glycoproteins, each with distinct physical and biochemical properties.
Collagen is composed of 3 polypeptide α chains that form a triple helical structure. In vertebrates, 46 distinct collagen chains assemble to form 28 collagen types2,4 that are categorized into fibril-forming collagens (eg, types I, II, III), network-forming collagens (eg, the basement membrane collagen type IV), fibril-associated collagens with interruptions in their triple helices (eg, types IX, XII), and others (eg, type VI). The fibril-forming collagens contain continuous triple helix–forming domains flanked by amino-terminal and carboxyl-terminal noncollagenous domains. These noncollagenous domains are proteolytically removed, and triple helices formed are associated laterally into fibrils. Nonfibril supramolecular structures, such as the networks of collagen IV in basement membranes and beaded filaments, are formed by nonfibrillar collagens. The fibril-associated collagens with interruptions in their triple helices do not assemble into fibrils by themselves, but are associated with collagen fibrils.
Specific proline residues in collagens are hydroxylated by prolyl 4-hydroxylase and prolyl 3-hydroxylase. Selected lysine residues are also hydroxylated by lysyl hydroxylase. The fibrillar procollagens, after processing, are secreted into the extracellular space where their propeptides are removed. The resulting collagens then assemble into fibrils by covalent cross-links formed between lysine residues of 2 collagen chains by a process catalyzed by extracellular enzyme lysyl oxidases (LOX). The collagenous backbone dictates the tissue architecture, shape, and organization.
Proteoglycans consist of a core protein to which glycosaminoglycan (GAG) side chains are attached. GAGs are linear, anionic polysaccharides made of repeating disaccharide units. There are 4 groups of GAGs: hyaluronic acid, keratan sulfate, chondroitin/dermatan sulfate, and heparan sulfate, including heparin. All except hyaluronic acid are sulfated. The highly negatively charged GAG chains allow the proteoglycans to sequester water and divalent cations, conferring space-filling and lubrication functions. Secreted proteoglycans include large proteoglycans, such as aggrecan and versican, small leucine-rich proteoglycans, such as decorin and lumican, and basement membrane proteoglycans, such as perlecan. Syndecans are cell surface associated, whereas serglycin is an intracellular proteoglycan. The molecular diversity of proteoglycans provides structural basis for a multitude of biological functions. For instance, aggrecan in cartilage generates elasticity and high biomechanical resistance to pressure. Decorin and lumican have a regulatory role in collagen fibril assembly. Proteoglycans also interact with growth factors and growth factor receptors, and are implicated in cell signaling5 and biological processes, including angiogenesis.
The laminin family comprises about 20 glycoproteins that are assembled into a cross-linked web, interwoven with the type IV collagen network in basement membranes. They are heterotrimers (400 to 800 kDa) consisting of 1 α, 1 β, and 1 γ chain. In vertebrates, 5 α, 3 β, and 3 γ chains have been identified. Many laminins self-assemble to form networks that remain in close association with cells through interactions with cell surface receptors. Laminins are essential for early embryonic development and organogenesis.6
Fibronectin is critical for the attachment and migration of cells, functioning as “biological glue.” The fibronectin monomer (∼250 kDa) is made of subunits that comprise 3 types of repeats: I, II, and III. Fibronectin is secreted as dimers linked by disulfide bonds and has binding sites to other fibronectin dimers, collagen, heparin, and cell surface receptors. In the FNIII10 repeat, there is an important Arg-Gly-Asp cell-binding site. The fibronectin dimers can form multimers. With continued deposition, the fibrils are lengthened and thickened and the fibronectin fibrils can be further processed into a deoxycholate-insoluble matrix.7
Elastin imparts elasticity to tissue subjected to repeated stretch, such as vascular vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60 to 70 kDa tropoelastin monomer. Tropoelastin, with the assistance of fibulins, associates with microfibrils to form elastic fibers. All tropoelastins share a characteristic domain arrangement of hydrophobic sequences alternating with lysine-containing cross-linking motifs. Microfibrillar proteins fibrillins and microfibril-associated glycoprotein-1 interact directly with elastin and are important for its nucleation and assembly.2 A crucial feature of the elastic fiber, critical to its proper function, is the extensive cross-linking of tropoelastin mediated by LOX, which oxidizes selective lysine residues in peptide linkage to allysine. There are 2 major bifunctional cross-links in elastin: dehydrolysinonorleucine, formed through the condensation of 1 residue of allysine and 1 of lysine, and allysine aldol, formed through the association of 2 allysine residues. These 2 cross-links can further condense with each other, or with other intermediates, to form desmosine or isodesmosine. Electron microscopy and imaging studies show that tropoelastin is assembled into small globular aggregates on the cell surface (microassembly). The cross-linking begins, which results in a loss of positive charges on the molecule, enabling the release of tropoelastin from the cell and assisting globular fusion in the presence of microfibrils (macroassembly). Fibulin-4 has a role in early stages of elastin assembly and fibulin-5 acts to bridge elastin between the matrix and cells.8
CELLULAR RECEPTORS FOR ECM MOLECULES
ECM molecules connect to the cells through integrins, syndecans, and other receptors. Integrins are heterodimeric receptors composed of α and β subunits.9 In vertebrates, the family encompasses 18 α and 8 β subunits that can assemble into 24 different integrins. Both α and β subunits are transmembrane proteins with large modular extracellular domains, single transmembrane helices, and short cytoplasmic regions that mediate cytoskeletal interactions. The integrins can be grouped into subgroups on the basis of ligand-binding properties or on the basis of their subunit composition. Major matrix-binding integrins are the β1 integrins with affinity to fibronectin, collagens, and laminins.
Integrins function as links between the ECM and the cytoskeleton. Activation of integrins by matrix ligands leads to conformational changes in the integrins, exposing their cytoplasmic domains to binding of focal complex proteins such as the focal adhesion kinase and integrin-linked kinase (ILK). Clustering of integrins in the membrane follows, and intracellular proteins including vinculin and talin along with actin stress fibers are assembled into focal adhesion complexes. The integrins subsequently trigger phosphorylation cascades and initiate signaling events including Rho and MAP kinase pathways to affect cell proliferation, differentiation, polarity, contractility, and gene expression in the so-called “outside-in” signaling process. Conversely, intracellular signals from proteins such as focal adhesion kinase, integrin-linked kinase, and talin can induce changes in integrin conformation and activation that alter its ligand binding activity in the “inside-out” signaling manner. Integrins thus act as a bidirectional conduit, transmitting signals and providing connections between intracellular and extracellular compartments.10,11
ECM REMODELING AND MODULATION
The ECM is constantly undergoing remodeling whereby its components are deposited, degraded, or modified. Intermolecular cross-linking by LOX is a key posttranslational modification for collagens and elastin. Expanded cross-linking due to excess LOX activity increases tissue tensile strength and matrix stiffness, affecting thereby cellular behaviors.
Collagens and other ECM elements are substrates for matrix metalloproteinases (MMPs), a disintegrin and metalloproteases (ADAMs), ADAM with thrombospondin motifs (ADAMTS), and proteases such as cathepsin G and elastase.1 MMPs are produced in precursor forms and remain inactive until activated. Most of the 23 members of the MMP family are secreted, but there are also membrane-type MMPs. Their activities are counteracted by tissue inhibitors of MMPs (TIMPs) and other inhibitors, and an imbalance may lead to tissue fibrosis and diseases.1 Besides ECM molecules, MMPs and others can also cleave precursor proteins, release ECM-bound growth factors, and release from ECM proteins bioactive fragments with new bioactivities, such as endostatin.4
In addition to the turnover process, the ECM is also modulated by exogenous stimuli such as cytokines, glucocorticoids, oxidative stress, pressure, and mechanical stretch. The most studied cytokine is transforming growth factor-β, which is known to enhance ECM production and upregulate ECM-related genes.
ECM AS RESERVOIR FOR BIOACTIVE MOLECULES
The ECM has the capacity to store and sequester growth factors and cytokines, establishing concentration gradients and regulating spatially and temporally their bioavailability. In particular, the fibroblast growth factor family strongly binds to heparan sulfate chains of proteoglycans such as perlecan. Heparan sulfate proteoglycans are also involved in binding, transporting, and activating developmental control factors including Wnt factors and hedgehog. The ECM is furthermore a reservoir of bioactive fragments released upon limited proteolysis. These fragments, with biological properties and activities of their own, regulate physiological and pathologic processes including angiogenesis. The ECM can additionally participate in ligand maturation. Transforming growth factor-β, secreted in the latent form, is stored in the ECM and remains inactive until activated by MMP-dependent proteolysis.1
ECM PROVIDES CHEMICAL AND PHYSICAL CUES
The biochemical properties of the ECM allow cells to sense and interact with their extracellular environment using various signal transduction pathways. The chemical cues are provided by ECM components, especially the adhesive proteins such as fibronectin, integrin, and nonintegrin receptors, as well as growth factors and associated signaling molecules. Interactions with different matrices by specific sets of receptors can trigger distinct cellular responses.10,11
The ECM functions as a physical barrier, an anchorage site, or a movement track for cell migration.1 The physical properties of the ECM, including its rigidity, density, porosity, insolubility, and topography (spatial arrangement and orientation), provide physical cues to the cells. The mechanical properties are essentially sensed by integrins that connect extracellular ECM to the actin cytoskeleton inside the cells. Stiff matrices induce integrin clustering, robust focal adhesions, and Rho and MAP kinase activation, leading to increased proliferation and contractility.1 Matrix rigidity also regulates differentiation. For example, on soft matrices, mesenchymal stem cells favor a neurogenic path, and on stiff ones they favor an osteogenic path.
The ECM provides a structural scaffold by means of a network of protein-protein and protein-proteoglycan interactions. These interactions are involved in the formation of supramolecular assemblies such as collagen fibrils and elastic fibers, in tissue architecture, and in cell-matrix interactions that regulate cell growth and behavior.
The cells and the ECM have a 2-way reciprocal relationship (Fig. 1).10,12 Cells produce, secrete, deposit, and remodel ECM to mediate ECM composition and topography. The ECM in turn transmits signals through ECM receptors to influence cell characteristics and activities (Fig. 2).13 Such a feedback mechanism is essential for rapid response of cells to surrounding environmental changes.
The multifunctional ECM is now recognized as a “niche” for stem cells (Fig. 3).13,14 ECM biology also has wide implications in areas ranging from embryonic development, wound healing, fibrosis, cancer invasion and metastasis, diabetes, and eye diseases including exfoliation syndrome, to biomedical engineering.
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