Biology of the Extracellular Matrix




Abstract


Extracellular matrices (ECMs) consist of highly organized networks of collagens, elastin, glycoproteins, and proteoglycans. In addition to establishing tissue architecture and providing mechanical strength, ECMs are biologically active, regulating cellular behavior, fate, and communications. The functions of the ECM are adapted to meet the specific needs of each tissue, with tissue-specific molecular composition that is fine-tuned by intricate post-translational modifications. Genetic defects that lead to a disorganized and dysfunctional ECM underlie a broad spectrum of heritable diseases with cutaneous manifestations. This chapter describes the molecular composition of the dermal ECM and provides an overview of genetic ECM disorders affecting the skin as well as potential molecular therapies.




Keywords

Extracellular matrices, collagens, extrafibrillar matrix, glysosaminoglycans, proteoglycans, elastic fibers, elastin

 





Key features





  • Extracellular matrices (ECMs) represent specifically organized networks of collagens, elastin, glycoproteins, and proteoglycans that have distinct structural roles and specific functional properties in all tissues



  • ECMs are biologically active, interact with cells, and regulate their functions during development, regeneration, and normal tissue turnover



  • Mutations in ECM genes cause a broad spectrum of human diseases, from Ehlers–Danlos syndrome to epidermolysis bullosa, and components of the ECM are targeted in autoimmune diseases, e.g. bullous pemphigoid, bullous systemic lupus erythematosus, lichen sclerosus



  • The collagen family contains 28 different subtypes. All collagens consist of three polypeptide α-chains, which are folded into a triple helix. In each chain, every third amino acid is glycine (Gly), and thus the sequence can be expressed as (Gly-X-Y) n . A hallmark of collagens is the presence of hydroxyproline (Hyp) in the Y position of this repeat sequence. Collagens are expressed in all tissues of the human body, and distinct sets of collagens co-polymerize into highly organized suprastructures, e.g. fibrils and filaments, in a tissue-specific manner



  • Elastin provides tissues with elasticity. Elastin monomers contain repetitive hydrophobic sequences and are highly cross-linked. The cross-links between several individual molecules provide both elasticity and insolubility to elastic fibers, which can be stretched by 100% or more and still return to their original form. The elastic fibers in the dermis also contain a microfibrillar component which attaches the fibers to surrounding structures.





Introduction


The different types of extracellular matrix (ECM) represent specifically organized assemblies of the matrix macromolecules listed in Table 95.1 . These macromolecules have characteristic patterns of aggregation into insoluble suprastructures with a high degree of order at successive hierarchic levels . Each of these structures is tissue-specific and adapted to the particular needs of a given tissue. The major constituents are often similar in functionally diverse ECMs. However, different types of quantitatively minor molecular components associate with major elements in tissue-specific suprastructural arrays determined by their relative compositions. The matrix suprastructures may be likened to alloys, each having metallurgic properties that differ from each other and those of the pure metals ( Fig. 95.1 ).



Table 95.1

Components of the extracellular matrix (ECM).

They belong to several protein superfamilies, and the molecules assemble into mixed fibrils and networks in a tissue-specific manner. Integrins are the main cellular receptors for the ECM. LTBP, latent TGF-β binding protein.





















COMPONENTS OF THE EXTRACELLULAR MATRIX
Collagens (28 types) Laminins (15 types)
Elastin Proteoglycans ( Table 95.3 )
Fibrillins (3 types) Glycoproteins ( Table 95.4 )
LTBPs (4 types) Integrins
Fibulins * (7 types) Enzymes that modify ECM assemblies

* Fibulins are believed to function as intra-molecular bridges that stabilize ECM structural networks (e.g. elastic fibers, microfibrils, basement membrane structures).




Fig. 95.1


Dermal extracellular matrix networks.

Different molecules polymerize into distinct fibril networks and, within the mesh of the networks, cells are embedded in the amorphous extrafibrillar matrix. The fibril networks interact with each other, with the extrafibrillar matrix and with the cells. These networks have a dual function: support of the tissue and regulation of cellular functions.


Individual ECM macromolecules are usually oligomers composed of one or several polypeptides. Intimate contacts between the subunits are formed by coiled-coil structures, such as the collagen triple helix or supercoiled α-helices comprised of three or more polypeptides. In addition, large matrix macromolecules can be regarded as linear sequences of structural modules that are similar in a large variety of proteins . The modules can be recognized by several cellular receptors, but receptor clustering will be determined in a tissue-specific manner and the response may be different in different tissues.


Our knowledge of matrix macromolecules has expanded dramatically in recent years due to advances in molecular genetics and proteomics. A multitude of molecules have been characterized and their expression, regulation, tissue specificity, and functions discerned . The assembled ECM structures are generally adhesive, enabling the attachment of tissue-specific cells, leukocytes, tumor cells, and even microorganisms. Through integrin-mediated interactions with cells, matrix molecules control cell proliferation, differentiation, and migration, especially during development and regenerative processes. Without contact with the ECM, many cells undergo a form of apoptosis known as anoikis . Furthermore, the ECM can function as a reservoir of information; certain proteoglycans and proteins bind growth factors (e.g. transforming growth factor [TGF]-β), releasing and activating them as needed to control cellular functions . To date, mutations in >50 different genes encoding ECM molecules have been found to underlie heritable disorders in humans and mice.




Structure and Function of the Extracellular Matrix


Collagens


The collagen family of proteins plays an important role in maintaining the integrity of most tissues. The family currently includes 28 proteins formally defined as collagens ( Table 95.2 ). They contain at least 45 distinct polypeptide chains, each encoded by a different gene, and more than 15 other proteins have a collagen-like domain (e.g. macrophage scavenger receptor 1 and 2, ectodysplasin, pulmonary surfactant proteins).



Table 95.2

The collagen family of proteins.

The names of types found in the skin are in bold italics . Multiplexins are collagens with multiple triple helix domains and interruptions. ECM, extracellular matrix; FACITs, fibril-associated collagens with interrupted triple helices.









































































































































































THE COLLAGEN FAMILY OF PROTEINS
Type Chains Gene Tissue distribution
Fibril-forming collagens
Collagen I α 1 (I), α 2 (I) COL1A1, COL1A2 Skin, most ECM
Collagen II α 1 (II) COL2A1 Cartilage, vitreous humor
Collagen III α 1 (III) COL3A1 Skin (including fetal skin), lung, vasculature
Collagen V α 1 (V), α 2 (V), α 3 (V) COL5A1, COL5A2, COL5A3 Skin, with collagen I heterotypic fibrils
Collagen XI α 1 (XI), α 2 (XI), α 3 (XI) COL11A1, COL11A2, COL11A3 With collagen II heterotypic fibrils
Collagen XXIV α 1 (XXIV) COL24A1 Developing bone and cornea
Collagen XXVII α 1 (XXVII) COL27A1 Cartilage, eye, ear, lung
FACITs
Collagen IX α 1 (IX), α 2 (IX), α 3 (IX) COL9A1, COL9A2, COL9A3 With collagen II heterotypic fibrils
Collagen XII α 1 (XII) COL12A1 Skin, tissues containing collagen I
Collagen XIV α 1 (XIV) COL14A1 Skin, tissues containing collagen I
Collagen XVI α 1 (XVI) COL16A1 Skin, many tissues
Collagen XIX α 1 (XIX) COL19A1 Basement membranes, fetal muscle
Collagen XX α 1 (XX) COL20A1 Skin, cornea, cartilage, tendon
Collagen XXI α 1 (XXI) COL21A1 Many tissues, including skin
Collagen XXII α 1 (XXII) COL22A1 Tissue junctions (including between the anagen hair follicle and dermis)
Basement membrane collagen
Collagen IV α 1 (IV), α 2 (IV), α 3 (IV), α 4 (IV), α 5 (IV), α 6 (IV) COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6 All basement membranes, isoforms vary
Skin: α 1 (IV), α 2 (IV), α 5 (IV), and α 6 (IV)
Microfibrillar collagen
Collagen VI α 1 (VI), α 2 (VI), α 3 (VI) COL6A1, COL6A2, COL6A3, COL6A5,COL6A6 Skin, other microfibril-containing tissues
Network-forming collagens
Collagen VIII α 1 (VIII), α 2 (VIII) COL8A1, COL8A2 Skin, subendothelial matrices
Collagen X α 1 (X) COL10A1 Hypertrophic cartilage
Anchoring fibril collagen
Collagen VII α 1 (VII) COL7A1 Skin, mucous membranes, cornea
Transmembrane collagens
Collagen XIII α 1 (XIII) COL13A1 Skin, many tissues
Collagen XVII α 1 (XVII) COL17A1 Skin, mucous membranes, cornea
Collagen XXIII α 1 (XXIII) COL23A1 Lung, cornea, brain, skin, tendon, kidney
Collagen XXV α 1 (XXV) COL25A1 Brain, neurons
Multiplexins
Collagen XV α 1 (XV) COL15A1 Many tissues; parent molecule of restin
Collagen XVIII α 1 (XVIII) COL18A1 Many tissues, including skin, subendothelial matrices; parent molecule of endostatin
Other collagens
Collagen XXVI α 1 (XXVI) COL26A1 Testis, ovary
Collagen XXVIII α 1 (XXVIII) COL28A1 Schwann cells; fetal skin and calvaria

Inhibitors of angiogenesis.



Collagen triple helix


All collagens consist of three polypeptide chains, known as α-chains, which are folded into a triple helix. In some collagens, the α-chains are identical (homotrimers), while others contain two or three different α-chains (heterotrimers). In the collagenous repeat of each polypeptide chain, every third amino acid is glycine (Gly), and the sequence of an α-chain can be expressed as (Gly-X-Y) n , where X and Y represent other amino acids and n varies according to the length of the repeat. A high number of proline (Pro) and hydroxyproline (Hyp) residues are in the X and Y positions, respectively, and hydrogen bonds between the hydroxyl groups of Hyp contribute to the stability of the helix. The prototype collagen (type I) has an uninterrupted Gly-X-Y repeat sequence that is almost 1000 amino acid residues in length; this forms a rigid, rodlike structure with a diameter of 1.5 nm and length of 300 nm. In some collagens, the (Gly-X-Y) n repeats are interrupted by one or more amino acids. The interruptions may be numerous and longer than the (Gly-X-Y) n repeats, and they provide the molecule with flexibility, which is important for the specific functions of a given collagen type .


Biosynthesis of collagens


Collagen biosynthesis involves a number of post-translational modifications ( Fig. 95.2 ). Some collagens are first synthesized as procollagens that have propeptide extensions at their N-terminus, C-terminus, or both. The main intracellular steps in collagen biosynthesis include the following:




  • cleavage of signal peptides



  • hydroxylation of certain Pro and lysine (Lys) residues to 4-Hyp, 3-Hyp, and hydroxylysine (Hyl)



  • glycosylation of some of the Hyl residues to galactosyl-Hyl and glucosylgalactosyl-Hyl



  • glycosylation of certain asparagine residues



  • association of the α-chains in a specific manner



  • formation of intra- and interchain disulfide bonds



  • folding of the triple helix.

After the chains have become associated and sufficient Hyp residues have been formed in each chain, a nucleus of the triple helix forms (usually in the C-terminal region) and the triple helix propagates towards the other end of the molecule in a zipper-like fashion . The procollagen molecules are then transported from the endoplasmic reticulum across the Golgi apparatus without leaving the lumen of the Golgi cisternae. During this transport, the molecules begin to aggregate laterally and form early fibrils ready for secretion . Folding and transport of the large, rigid collagens require special machinery . Deficiencies in the proteins that interact with collagens during these events can lead to phenotypes similar to those of collagen deficiency disorders . The extracellular steps in biosynthesis include cleavage of the N- and/or C-terminal propeptides, assembly into suprastructures with other collagens and non-collagenous components, and formation of covalent cross-links.


Fig. 95.2


Biosynthesis of a “prototype” collagen.

The procollagen α-chains are synthesized by ribosomes in the rough endoplasmic reticulum (ER). Already during the synthesis of the nascent polypeptide, certain prolyl and lysyl residues are hydroxylated and modified by glycosylation. Three α-chains associate to form a trimer and fold into a triple helix. This process is facilitated by heat shock protein (HSP) 47, a collagen-specific molecular chaperone in the ER. The transport and Golgi organization (TANGO) protein 1 mediates secretion of the newly formed triple helical procollagen into the extracellular space, where the N- and C-terminal propeptides are cleaved by specific proteases. The mature collagen molecules assemble to form mixed fibrils with other collagens and non-collagenous molecules. The suprastructures are stabilized by covalent cross-links. Small leucine-rich proteoglycans (SLRPs) and other proteoglycans have roles in the regulation of collagen fibrillogenesis and cross-linking. EDS, Ehlers–Danlos syndrome.

Adapted from Myllyharju J, Kivirikko KI. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet. 2004;20:33–43.


The specific enzymes involved in the biosynthesis of collagens include prolyl-4-hydroxylase and prolyl-3-hydroxylase, which hydroxylate Pro residues to Hyp, and three lysyl hydroxylases, which hydroxylate Lys residues to Hyl. These enzymes require O 2 , Fe 2+ , α-ketoglutarate and ascorbate as cofactors for the reactions. In the rough endoplasmic reticulum, glycosyltransferases add glucosylgalactosyl disaccharides onto the α-chains. The same intracellular enzymes modify all collagen chains , whereas extracellular processing enzymes have a higher substrate specificity. Procollagen I N-proteinase cleaves the N-propeptide of procollagens I and II. This enzyme is a member of the disintegrin and metalloproteinase (ADAM) proteinase family and is also designated as ADAM with t hrombo s pondin type 1 motif (ADAMTS)-2. Procollagen C-proteinases that cleave the C-propeptide of collagens I, II, III, V, and VII belong to the astacin family and include bone morphogenetic protein-1 (BMP-1) and tolloid-like proteins; meprins in this family can cleave both N- and C-termini of collagens .


Cross-linking between collagen molecules involves the ε-amino groups of Lys and Hyl and is catalyzed by lysyl oxidase, a copper-requiring enzyme ; this process can be regulated by proteoglycans . Another enzyme that catalyzes cross-linking of some collagens is tissue transglutaminase. Collagen VII-containing anchoring fibrils in the skin appear to be transaminated, and collagen VII serves as a substrate for tissue transglutaminase in vitro .


The collagen family of proteins


The length and continuity of the triple helical domains vary among the collagen types. For practical purposes, the collagens have been divided into groups according to their ability to form supramolecular aggregates, which are depicted in Fig. 95.3 . The types and tissue distributions of the collagens in each group are listed in Table 95.2 , and more detailed information is available in reviews .




Fig. 95.3


Supramolecular assemblies of collagens.

The suprastructures formed by different collagens are shown. Non-collagenous components also interact with the fibrils and networks. The suprastructural organization of the transmembrane collagens XIII and XVII and the multiplexin collagens XV and XVIII is not known yet (panels 7 and 8). These collagens are in close vicinity to basement membranes and are likely to participate and/or interact with the different basement membrane networks. FACIT, fibril-associated collagens with interrupted triple helices; GAG, glycosaminoglycans.

Adapted from Myllyharju J, Kivirikko KI. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet. 2004;20:33–43.


Collagens of the skin


Collagens account for 75% of the dry weight and 20–30% of the volume of the dermis. Different collagens polymerize into distinct suprastructures and have specific functions in the dermis as well as in epidermal and vascular basement membranes. “Pure” collagen fibrils do not exist; these fibrils are always mixtures of several collagens and other molecules, e.g. proteoglycans . Classic, ultrastructurally recognizable, cross-banded fibrils in the dermis contain collagens I, III, V, XII, and XIV. The characteristic cross-banding ( Fig. 95.4 ) with a periodicity of 64 nm results from precise lateral packing of the different collagens within the fibrils (see Fig. 95.3 ). Collagen I is the major component of the fibrils, and the amount of other collagens varies. For example, during embryonic development and wound repair, the relative content of collagen III increases. Collagen VI, a highly glycosylated and disulfide-bonded collagen, is a component of almost all tissues, including the skin. In vitro , it polymerizes to form beaded filaments (see Fig. 95.3 ), but in vivo in the dermis, the ultrastructure of the collagen VI fibrils is reminiscent of microfibrils.




Fig. 95.4


Fibrillar and filamentous networks extracted from human skin.

The large cross-banded fibrils represent dermal mixed fibrils containing collagens I, III, V, other minor collagens, and decorin (a proteoglycan). The cross-banding has a characteristic periodicity of 64 nm. The filamentous network in the background contains microfibrillar and basement membrane components. In this immunoelectron photomicrograph, the black dots are colloidal gold particles coupled to anti-collagen IV antibodies, indicating that basement membrane networks are strongly associated with the dermal fibrillar networks.

Courtesy, Dr Uwe Hansen.


The collagen IV molecules in different basement membranes contain six genetically distinct but structurally homologous α-chains, which form three major networks – α 1 2 , α 3 4 5 , and α 1 2 5 6 . The chain composition is determined by the carboxy-terminal non-collagenous (NC1) domains; covalent interactions between these domains link α-chains to each other. In the skin, the α 1 2 -containing collagen IV network dominates within the dermal–epidermal junction (see Ch. 28 ), but the α 1 2 5 6 -containing network is also thought to be present .


Two collagens are essential for the cohesion of the epidermis and dermis (see Ch. 28 ). Collagen VII is the major, if not sole, component of the anchoring fibrils that attach the basement membrane to the dermal ECM. Collagen XVII (also known as bullous pemphigoid antigen 2) is a component of the anchoring filaments that bind the basal keratinocytes to the lamina densa of the basement membrane. It is a transmembrane collagen in type II orientation with a long extracellular C-terminal region containing a multiply-interrupted triple helix referred to as collagenous domains 1–15 (see Fig. 31.9 ). The ectodomains can be shed from the cell surface by transmembrane proteases , a process important for regulation of cell adhesion and migration. Basal keratinocytes also express other transmembrane collagens such as type XIII, which is a component of focal contacts, and type XXIII.


The vascular basement membranes in the skin contain yet other collagens – namely, collagens VIII and XVIII. Collagen VIII builds hexagonal networks below the endothelial basement membranes (see Fig. 95.3 ) and, thus, structurally strengthens the vascular wall. Collagen XVIII is localized at the dermal side of vascular basement membranes. It is present in both epidermal and vascular basement membranes. Its C-terminal fragment, endostatin, is proteolytically released from the collagen molecule and has independent antiangiogenic and anti-scarring activities .


Most collagens in the skin are products of dermal fibroblasts. Exceptions include: (1) collagen XVII, a surface component of epidermal keratinocytes; (2) collagen VII, which can be synthesized by both keratinocytes and fibroblasts; and (3) collagens VIII and XVIII, which can also be produced by endothelial cells. Several genetic and acquired diseases are associated with abnormalities of skin collagens or the enzymes that process them (see Tables 95.5 & 95.6 and below).


Elastic Fibers


The elasticity of many tissues, including the skin, depends upon the structure of elastic fibers, which have variable compositions. A characteristic property of these fibers is that they can be stretched by 100% or more and still return to their original form. The main components of elastic fibers are elastin and microfibrils.


Elastin


Elastin is a highly cross-linked protein . Its monomer, tropoelastin, exists in several tissue-specific splice variants. Ala- and Lys-rich repeats form critical cross-linking domains within a background of hydrophobic amino acid repeats. Lysyl oxidase, the same copper-dependent enzyme that catalyzes collagen cross-linking (see above), catalyzes the formation of desmosine cross-links between elastin molecules, which account for the elasticity and insolubility of the elastic fibers. Elastin makes up ~90% of mature elastic fibers. During fiber assembly, tropoelastin both self-assembles and interacts with fibulins 4 and 5, fibrillin 1, and microfibril-associated glycoprotein-1 (MAGP-1). A model for this spatially and temporally regulated process is shown in Fig. 95.5 .




Fig. 95.5


Microfibril and elastic fiber assembly.

A Following secretion of fibrillin 1, its N- and C-termini are processed by furin and then interact homotypically; this leads to axial and lateral fibrillin 1 assembly to form microfibrils. Beads may arise from folding of terminal regions, and transglutaminase cross-links provide stabilization. Fibronectin is thought to act as a microfibril assembly template and/or to facilitate assembly at fibrillar adhesions by stimulating cytoskeletal tension through α 5 β 1 integrin. Fibrillin 1 interacts with α 5 β 1 , α v β 3 , and α v β 6 integrins during microfibril assembly. Heparan sulfate proteoglycans (HSPGs) are thought to facilitate cell surface–fibrillin 1 interactions, and heparin inhibits microfibril assembly. Fibrillin 1 also binds microfibril-associated glycoproteins, tropoelastin, fibulins, and latent transforming growth factor (TGF)-β binding protein. B Elastic fiber formation includes pericellular elastin “microassembly” and subsequent “macroassembly” on a microfibrillar scaffold. Secreted tropoelastin forms globules at the cell surface, which become cross-linked by lysyl oxidase (LOX); this process may involve α v β 3 integrin interactions with tropoelastin, as well as interactions of HSPGs with integrins and tropoelastin. Fibulin 4 and fibulin 5 contribute to elastin cross-linking by lysyl oxidase and are also thought to direct the deposition of elastin globules onto preformed fibrillin-containing microfibrils, thereby forming elastic fibers. Microfibrils and elastic fibers are important matrix storage sites for bone morphogenic proteins (BMPs) and latent TGF-β1.

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Sep 15, 2019 | Posted by in Dermatology | Comments Off on Biology of the Extracellular Matrix
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