The Extracellular Matrix

Jeff Miner

7717 Wohl Clinic

362-8235

minerj@wustl.edu

Suggested Reading**Overview: Lodish, Molecular Cell Biology (2008), Chapter 19, pp 801-841.

Lecture 1: The Extracellular Matrix**Review: A Aszodi, KR Legate, I Nakchbandi, and R. Fassler: What mouse mutants teach us about extracellular matrix function. Annu. Rev. Cell Dev. Biol. 22:591-621, 2006.

**Review: JF Bateman, RP Boot-Handford, SR Lamandé: Genetic diseases of connective tissues: cellular and extracellular effets of ECM mutations. Nat. Rev. Genet. 10: 173-183, 2009.

KK McKee, D Harrison, S Capizzi, and PD Yurchenco: Role of laminin terminal globular domains in basement membrane assembly. J. Biol. Chem. 282:21437-21447, 2007.

G Ge and DS Greenspan: BMP1 controls TGFβ activation via cleavage of latent TGFβ-binding protein. J. Cell Biol. 175:111-120, 2006.

Lecture 2: Cell-Matrix Interactions**Review: B-H Luo, CV Carman, and TA Springer: Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25:619-647, 2007.

**Review: KR Legate, E Montanez, O Kudlacek, and Reinhard Fassler: ILK, PINCH, and parvin: the tIPP of integrin signalling. Nat. Rev. Mol. Cell Biol. 7:20-31, 2006.

Review: Y Mao and JE Schwarzbauer: Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biol. 24:389-399, 2005.

**Review: R Barresi and KP Campbell: Dystroglycan: from biosynthesis to pathogenesis of human disease. J. Cell Sci. 119:199-207, 2006.

“Half of the secrets of the cell are outside the cell.”

Dr. Mina BissellOct. 17, 2007Erlanger Auditorium

Basement Membrane Proteins Regulate Mammary Cell Gene Expression:

Streuli et al,J. Cell Biol. 1991

General Organization of Tissues

There are

Generic Tissue Structure

Stratified, pseudostratified, or monolayer

“Tube within a Tube” concept

(aka stroma)

“Tube Within a Tube”

PancreasA Compartmentalized Tissue

Shimizu, H. et al. Development 2002;129:1521-1532

“Mesoglea” (BM-like)separates ectoderm from endoderm

Even Hydra

Why do all multicellular animals have ECM?

• Act as structural support to maintain cell organization and integrity (epithelial tubes; mucosal lining of gut; skeletal muscle fiber integrity)

• Compartmentalize tissues (pancreas: islets vs. exocrine component; skin: epidermis vs. dermis)

• Provide hardness to bone and teeth (collagen fibrils become mineralized)

• Present information to adjacent cells:– Inherent signals (e.g., RGD motif in fibronectin)– Bound signals (BMP7, TGFβ, FGF, SHH)

• Serve as a highway for cell migration during development (neural crest migration), in normal tissue maintenance (intestinal mucosa), and in injury or disease (wound healing; cancer)

Types of ECMs

• Basement membrane (basal lamina)– Epithelia, endothelia, muscle, fat, nerves

• Elastic fibers– Skin, lung, large blood vessels

• Stromal or interstitial matrix

• Bone, tooth, and cartilage

• Tendon and ligament

Cells Need Receptors to Recognize and Respond to ECM

• Integrins

• Dystroglycan

• Syndecans

• Muscle-Specific kinase (MuSK)

• Others

Types of ECM Components

• Collagens• Proteoglycans

– Perlecan, aggrecan, agrin, collagen XVIII

• Hyaluronan (no protein core)• Large Glycoproteins

– Laminins, nidogens, fibronectin, vitronectin

• Fibrillins, elastin, LTBPs, MAGPs, fibulins• “Matricellular” Proteins

– SPARC, Thrombospondins, Osteopontin, tenascins

Generalizations• Most ECM proteins are large, modular,

multidomain glycosylated or glycanated proteins

• Some domains recur in different ECM proteins

– Fibronectin type III repeats– Immunoglobulin repeats– EGF-like repeats– Laminin Globular (G) domain– von Willebrand factor

Perlecan

Basement Membranes

• Specialized layers of extracellular matrix surrounding or adjacent to all epithelia, endothelia, peripheral nerves, muscle cells, and fat cells

• Originally defined by electron microscopy as ribbon-like extracellular structures beneath epithelial cells

J. Schwarzbauer, Curr. Biol. 1999M. Loots, Univ. of Pretoria, S.A.

Basement Membrane

Lamina Densa + Lamina Lucidae

Kidney Glomerular Basement Membrane

Fredrik Skarstedt and Carrie Phillips

Deep-Etch Electron Microscopy

• In general, basement membranes appear very similar to each other by EM.

• But all are not alike!• There is a wealth of molecular and

functional heterogeneity among basement membranes, due primarily to isoform variations of basement membrane components.

Basement Membranes

Kidney Basement Membranes

Laminin Laminin ββ11 Laminin Laminin ββ22

• Influence cell proliferation, differentiation, and migration

• Maintain cell polarization and organization, as well as tissue structure

• Act as a filtration barrier in the kidney between the vasculature and the urinary space

• Separate epithelia from the underlying stroma/mesenchyme/interstitium, which contains a non-basement membrane matrix

Basement Membranes are Involved in a Multitude of Biological Processes

Primary Components of All Basement Membranes

• Collagen IV 6 chains form α chain heterotrimers• Laminin 12 chains form several α-β-γ heterotrimers• Entactin/Nidogen 2 isoforms• Sulfated proteoglycans Perlecan and Agrin are the

major ones; Collagen XVIII is another

History: The Engelbreth-Holm-Swarm (EHS) tumor:A blessing with a caveat.

LamininHeterotrimers are composed of one α, one β, and one γ chain.

• 400 to 800 kDa cruciform, Y, or rod-shaped macromolecules.

• Major glycoprotein of basement membranes—it’s required!

• Chains are evolutionarily related.• 5 alpha, 4 beta, and 3 gamma chains are

known. They assemble with each other non-randomly.

• 15 heterotrimers described to date.LM-521

Laminin• All laminin chains share

structural homology• Contain globular, rod (EGF-like

repeats), and coiled-coil domains

• Alpha chains are unique, contain a C-terminal laminin globular “LG” domain, ~100 kDa

(New nomenclature)

The Laminin Trimers

Miner and Yurchenco, 2004

Laminin Trimers Polymerize

• Laminin chains assemble into trimers in the ER and are secreted as trimers into the extracellular space.

• Full-sized laminin trimers can self-polymerize into a macromolecular network through short arm-short arm interactions.

• The α chain LG domain is left free for interactions with cellular receptors.

Receptor-mediated AssemblyReceptor-mediated Assembly

Involves LG domains and receptors on the surface of cells.Results in laminin polymerization and signal transduction.

Laminin Mutations in Mice (M) and Humans (H) Have Consequences

Lama1, Lamb1, Lamc1: Peri-implantation lethality (M)

Lama2: Congenital muscular dystrophy (M, H)

Lama3, Lamb3, Lamc2: Junctional epidermolysis bullosa (skin blistering) (M, H)

Lama4: Mild bleeding disorder, moto-nerve terminal defects (M); cardiac and endothelial defects (H)

Lama5: Neural tube closure, placenta, digit septation, lung, kidney, tooth, salivary gland defects (M)

Lamb2: Neuromuscular junction and kidney filtration defects (M); Iris muscle, neuromuscular, kidney filtration defects (H; Pierson syndrome)

Lamc3: Brain malformations, autism spectrum disorder? (H)

Sulfated Proteoglycans

• Have protein cores with large glycosaminoglycan (GAG) side chains (from 1 to >100) attached to serines

• Some PGs contain heparan sulfate– Perlecan, Agrin, Collagen XVIII

(endostatin)• Others contain chondroitin, keratan or

dermatan sulfate• GAG chains are responsible for most of

the biological properties of proteoglycans and provide charge to basement membranes Heparan sulfate:

Composed of D-glucuronate-2-sulfate +N-sulfo-D-glucosamine-6-sulfate

Some Major Proteoglycan Family Members

From: Iozzo, R.V. (1998) Ann. Rev. Biochem. 67:609 From: Iozzo, R.V. (2001) J. Clinic. Invest. 108:165

Perlecan• Found widely in basement membranes and in

cartilage.• Contains domains similar to LDL receptor, laminin,

and N-CAM• Binds to Collagen IV and to Entactin/Nidogen

Endorepellin: Domain V of Perlecan

• Exhibits anti-angiogenic activity

• Targets tumor vasculature

Endostatin: Noncollagenous Tail of Collagen XVIII

• Exhibits anti-angiogenic activity

• Targets tumor vasculature

Type IV Collagen NC1 Domains

• Exhibit anti-angiogenic activity

• Target tumor vasculature

Proteases Release Anti-Cancer Peptides

MMP = Matrix MetalloproteinaseMT-MMP = Membrane Tethered MMP

From Zent and Pozzi, 2005

Laminin cleavages

Agrin• A HSPG found widely in basement membranes• A modular protein containing domains homologous to

follistatin, laminin, and perlecan• Isolated due to its ability to cluster pre-existing

acetylcholine receptors in skeletal muscle fibers• BM form binds to laminin

Agrin

• Several splice variants exist; critical for function in skeletal muscle.– The “Z” exon is present in nerve-derived but not muscle-

derived agrin and is necessary for its AChR clustering activity.• Agrin may be the most dramatic example of a basement

membrane component with a specific, well-defined signaling role.

The CollagensThe Collagens

• The most ubiquitous structural protein. Characterized as a triple helical protein containing peptide chains with repeating Gly-Xaa-Yaa (usually Pro) triplets.

• The triple helix forms through the association of three related polypeptides (α-chains) forming a coiled coil, with the side chain of every third residue directed towards the center of the superhelix. Steric constraints dictate that the center of the helix be occupied only by Glycine residues.

• Many Proline and Lysine residues are enzymatically converted to hydroxyproline and hydroxylysine.

• ~28 distinct collagen types; each is assigned a Roman numeral that generally delineates the chronological order in which the collagens were isolated/characterized.

Diversity of CollagensType I fibrils Skin, tendon, bone, ligaments, dentin,

interstitium

Type II Fibrils Cartilage, vitreous humor

Type III Fibrils Skin, muscle, bv

Type IV 2D sheets All basement membranes

Type V Fibrils with globular end

Cornea, teeth, bone, placenta, skin, smooth muscle

Type VI Fibril-assoc. (I) Most interstitial tissues

Type VII Long anchoring fibril

Skin--connects epidermal basement membrane/hemidesmosome to dermis

Type IX Fibril-assoc. (II) Cartilage, vitreous humor

Type XIII Transmembrane Hemidesmosomes in skin

Type XV HSPG Widespread; near basement membranes in muscle

Type XVII Transmembrane Hemidesmosomes in skin (aka BPAG2 or BP180)

Collagen IV: Network or Sheet Forming

• Six genetically distinct α chains: α1- α6, ~180 kDa each• Chains form three types of heterotrimers:

– (α1)2(α2), α3α4α5, (α5)2(α6)

• Like all Collagens, comprised mainly of Gly-x-y repeats, y is frequently proline

• Gly-x-y pattern has multiple interruptions– Provides flexibility to the collagen network and to the basement

membrane

Hudson et al., NEJM 2003

Collagen IV Trimer

• 7S domain at N-terminus• Interrupted Gly-x-y triple helical domain• C-terminal non-collagenous domain--NC1

Collagen IV Network

Trimers (aka protomers) associate with each other, four at the N-terminus and two at the C-terminus (hexamer), to form a chicken wire-like network that provides strength and flexibility to the basement membrane.

What Directs Chain-Chain-Chain Recognition and Hexamer Assembly?

Vanacore et al., Science 2009

Sulfilimine: The Bond that Crosslinks Type IV Collagen NC1 Domains

Type IV Collagen Mutations and Human Disease

• COL4A1 mutations– Small vessel disease/retinal vascular

tortuosity– Hemorrhagic stroke– Porencephaly– HANAC syndrome

• COL4A3/A4/A5 mutations– Alport syndrome/hereditary

glomerulonephritis

Kidney Glomerular BM

Fibrillar Collagens (I, II, III, V)

Fibrillar Collagens (I, II, III, V)

• Connective tissue proteins that provide tensile strength

• Triple helix, composed of three α chains

• Glycine at every third position (Gly-X-Y)

• High proline content– Hydroxylation required for proper

folding and secretion

• Found in bone, skin, tendons, cartilage, arteries

Biosynthesis of Fibril-forming Collagens

Adapted from: Keilty, Hopkinson, Grant. In: Connective Tissue and Its Inheritable Disorders, Wiley-Liss, 1993.

Prolyl hydroxylasesLysyl hydroxylaseGlycosyltransferases

Procollagen N- and C- proteinasesLysyl oxidase

Collagen CrosslinkingCollagen Crosslinking

• Once formed, collagen fibrils are greatly strengthened by covalent crosslinks that form between the constituent collagen molecules.

• The first step in crosslink formation is the deamination by the enzyme lysyl oxidase of specific lysine and hydroxylysine side chains to form reactive aldehyde groups.

• The aldehydes then form covalent bonds with each other or with other lysine or hydroxylysine residues.

• If crosslinking is inhibited (Lysyl hydroxylase mutations; vitamin C deficiency), collagenous tissues become fragile, and structures such as skin, tendons, and blood vessels tend to tear. There are also many bone manifestations of under-crosslinked collagen.

• Hydroxylation of specific lysines governs the nature of the cross-link formed, which affects the biomechanical properties of the tissue. Collagen is especially highly crosslinked in the Achilles tendon, where tensile strength is crucial.

Collagen CrosslinkingCollagen Crosslinking

Bone is Composed of Mineralized Type I Collagen Fibrils

Mineral is Dahllite,similar to hydroxyapatite(contains calcium, phosphate, carbonate)

Bone is 70% mineral and 30% protein, mostly collagen

Scurvy

• Liver spots on skin, spongy gums, bleeding from mucous membranes, depression, immobility

• Vitamin C deficiency• Ascorbate is required for prolyl

hydroxylase and lysyl hydroxylase activities

• Acquired disease of fibrillar collagen

Illustration from Man-of-War by Stephen Biesty (Dorling-Kindersley, NY, 1993)

Some Genetic Diseases of Collagen

• Collagen I– Osteogenesis imperfecta– Ehlers-Danlos syndrome type VII

• Collagen II– Multiple diseases of cartilage

• Collagen III– Ehlers-Danlos syndrome type IV

• Collagen IV– Alport syndrome, stroke, hemorrhage, porencephaly

• Collagen VII– Dystrophic epidermolysis bullosa (skin blistering)

Different Types of Mutations in Collagen I αChain Genes Cause Different Disease Severities

Gene location mutation SyndromeCOL1A1 17q22 Null alleles OI type I

Partial deletions; C-terminal substitutions

OI type II

N-terminal substitutions OI types I, III or IV

Deletion of exon 6 EDS type VII

COL1A2 7q22.1 Splice mutations; exon deletions OI type I

C-terminal mutations OI type II, IV

N-terminal substitutions OI type III

Deletion of exon 6 EDS type VII

Osteogenesis Imperfecta(brittle bone disease)

Clinical:

Ranges in severity from mild to perinatal lethal

bone fragility, short stature, bone deformities, teeth abnormalities, gray-blue sclerae, hearing loss

Biochemical:

reduced and/or abnormal type I collagen

Molecular:

mutations in either type I collagen gene, COL1A1 or COL1A2, resulting in haploinsufficiency or disruption of the triple helical domain (dominant negative: glycine substitutions most common)

COL1 Haploinsufficiency (Dominant)

Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.

(α1)2α2

Dominant Negative COL1 Mutations

Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.

Gly subst. in COL4A2*

*Gly subst. in COL4A1

½ of the trimers are abnormal

¾ of the trimers are abnormal

Elastin and Elastic Fibers Exhibit Elastin and Elastic Fibers Exhibit Rubber-Like PropertiesRubber-Like Properties

• Physiological importance lies in the unique elastomeric properties of elastin. Found in tissues in which reversible extensibility or deformability are crucial, such as the major arterial vessels (esp. aorta), the lung and the skin.

• Elastin is characterized by a high index of hydrophobicity (90% of all the amino acid residues are nonpolar). One-third of the amino acid residues are glycine with a preponderance of the nonpolar amino acids Ala, Val, Leu, and Ile. As in collagen, one-ninth of the residues are proline (but with very little hydroxylation).

• Early in development, the elastic fibers consists of microfibrils, which define fiber location and morphology. Over time, tropoelastin accumulates within the bed of microfibrils.

Elastic Fiber Biogenesis

• Elastic fibers are very complex, difficult to repair structures

• There are two morphologically distinguishable components

– Microfibrils– Elastin

• Assembly follows a well-defined sequence of events:

1. Assembly of microfibrils

2. Association of tropoelastin aggregates with microfibrils

3. Crosslinking of tropoelastins with each other by lysyl oxidase to form polymers Shifren and Mecham, 2006

Copyright ©2004 American Physiological Society

Ramirez, F. et al. Physiol. Genomics 19: 151-154 2004;doi:10.1152/physiolgenomics.00092.2004

Major steps underlying the assembly of microfibrils and elastic fibers

Microfibril Components: ~30

• Fibrillin--three forms• Microfibril-associated glycoproteins

(MAGPs)--two forms• Latent TGFβ Binding Proteins

(LTBPs)--four forms• Proteoglycans, MFAPs, Fibulins,

Emilins, Collagens, Decorin, et al.

Fibrillins

• Large glycoproteins (~350 kDa) whose primary structures are dominated by cbEGF domains that, in the presence of Ca2+, adopt a rodlike structure

• Limited intracellular assembly may occur, but microfibril assembly initiates at the cell surface after secretion, perhaps with the help of cellular receptors

Marfan Syndrome

• Caused by dominant Fibrillin-1 (FBN1) mutations– Haploinsufficiency is the culprit

• Skeletal, ocular, and cardiovascular defects

• Deficiency of elastin-associated microfibrils

• Syndrome may result from alterations in TGFβ signaling, rather than purely structural changes in microfibrils

• Members of the fibrillin superfamily

• Maintain TGFβ in the inactive state by forming the “large latent complex”

Latent TGFβ Binding Proteins

Annes, J. P. et al. J Cell Sci 2003;116:217-224

The TGFβ Large Latent Complex (LLC)

(e.g., fibrillin)

Potential Activators:ROSProteasesIntegrins(Assoc. with ECM Perturbations)

LAP: Latency-associated peptide

Evidence for FBN/BMP7 Interactions

Fbn2+/-; Bmp7+/- trans-heterozygous animals show limb patterning defects.

Artaga-Solis et al., J. Cell Biol. 2001

Specific fragments of Fibrillin 1, but not LTBP1, bind to BMP7

Gregory et al., JBC 2005

Elastic Fiber Biogenesis

• Elastic fibers are very complex, difficult to repair structures

• There are two morphologically distinguishable components

– Microfibrils– Elastin

• Assembly follows a well-defined sequence of events:

1. Assembly of microfibrils

2. Association of tropoelastin aggregates with microfibrils

3. Crosslinking of tropoelastins with each other by lysyl oxidase to form polymers Shifren and Mecham, 2006

Emphysema

• Damage to the lung air sacs (alveoli) that affects breathing

• Macrophages induced to “ingest” particles in smoke also secrete proteases that degrade elastic fibers

• Loss of lung elasticity makes exhalation difficult

• Increased alveolar size reduces the surface area for gas exchange