THE EXTRACELLULAR MATRIX

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THE EXTRACELLULARMATRIX

• Four lectures to cover the following topics
• What is ECM and where can you find it?
• Building blocks of ECM Did you know that 25 of
  total protein in your body is collagen.
• More ECM components Laminin, Fibronectin etc.
  (and what the heck is a heparan-sulphate
  proteoglycan?)
• ECM functions it is not only to keep cells in
  place
• Cell-matrix interactions. Integrins.

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WHAT IS THE EXTRACELLULAR MATRIX

• Complex arrangements of molecules filling in
  spaces between the cells.
• Not an amorphous jelly or glue but highly
  organised structure.
• Mostly found in connective tissues, such as
  tendon, cartilage, bone or dermis of the skin.
• Diverse structures created by different amounts
  and organisation of ECM components
• ECM is a local product for local cells. Cells
  secrete ECM that is finally assembled outside the
  cell.

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SOME FUNCTIONS OF ECM
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MAJOR TYPES OF ECM MOLECULES

• Glycosaminoglycans polysaccharide chains usually
  found attached to proteins to form proteoglycans
• Fibrillar proteins such as collagens (mainly
  structural role) or fibronectin (adhesive
  glycoprotein)

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CONNECTIVE TISSUES
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EXAMPLES OF ORGANISATION OF ECM- DERMIS
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EXAMPLES OF ORGANISATION OF ECM- CARTILAGE
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EXAMPLES OF ORGANISATION OF ECM- BASEMENT
MEMBRANE
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THE COLLAGEN FAMILY

• Triple helical domain
• Repeated Gly - X - Y amino acid sequence, where X
  is often proline and Y hydroxyproline
• 19 different collagen types ( possibly 4 more)
  containing polypeptides encoded by at least 38
  genes

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COLLAGENS -examples out 19 different types
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Collagens assemble to diverse structures
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COMMON THEMES IN ECM SYNTHESIS

• Extensive post-translational modification
• Route ER - Golgi - Secretory vesicles
• During this journey protein are glycosylated or
  decorated with long GAG chains
• Amino acid recidues can be modified (in collagens
  proline -gt hydroxyproline

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Collagen Biosynthesis - intracellular steps
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COLLAGEN
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Collagen Biosynthesis - extracellular steps
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Collagen Biosynthesis - extracellular assembly
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Collagen Biosynthesis - extracellular assembly
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Non-collagenous domains

• Triple-helical collagen rods are not the only
  functional domains
• Example Type XVIII collagen that is found in
  many tissues associated with basal lamina.
• Endostatin is a 22kDa polypeptide that is
  proteolytically cleaved from the C-terminus of
  type XVIII collagen
• Endostatin found in blood vessel walls and
  basement membranes
• Endostatin is a potent inhibitor of angiogenesis
  and tumour growth !!!
• Endostatin is currently being tested in clinical
  trials
• Similar domains in other collagen family members

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Collagens in disease

• Inherited diseases with mutations in collagen
  genes
• Osteogenesis Imperfecta
• Fibrotic diseases with accumulation of ECM
• Liver Chirrosis
• Lung Fibrosis

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Collagens in disease

• Osteogenesis Imperfecta - Brittle bone disease
  (not to be confused with osteoporosis)
• Variable from mild to embryonic lethal
• Often a point mutation in one of type I collagen
  genes can cause disease
• Glycine substitutions to another amino acid more
  severe than mutations of X or Y in Gly - X - Y
  triplet. Why?
• Dominant negative effect of some mutations.
• Predisposing mutations (e.g. Type II collagen in
  osteoarthrosis)

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Collagens in disease

• Fibrotic diseases such as liver chirrosis are
  characterised by accumulation of ECM
• Collagen synthesis is mainly regulated by the
  level of gene activity.
• Some growth factors such as TGF-b signal to
  increase collagen synthesis.
• Enzymes in the collagen synthesis are
  investigated as drug targets to treat fibrotic
  diseases

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Collagens a summary

• All collagens contain a repeating Gly-X-Y
  sequence and fold into a characteristic
  triple-helical structure
• Collagens assemble to fibrils or networks
• Procollagen chains are modified in ER where they
  also assemble into a triple helix
• Type I collagen is the most abundant type it is
  a major structural protein of bone, tendon and
  dermis
• Mutations in collagen chaisn can render the
  fibrils unstable

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Fibronectin

• Large extracellular glycoprotein
• Name fibro nectere (to bind)
• Multiple domains with different binding sites for
  other ECM proteins or for receptors on cell
  surface
• Present in tissues and in blood plasma

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Fibronectin structure
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Fibronectin structure domains and interactions
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Plasma Fibronectin
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Fibronectin binds cell surfaces by an RGD sequence
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Fibronectin is essential for embryonic development

• Gene targeting gt complete lack of fibronectin
• Embryonic lethal.
• Gross malformations, notchord and somites
  missing, heart malformation
• Problems in cell adhesion, migration and
  differentiation

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Proteoglycans have long sugar chains attached to
a core protein
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Proteoglycan biosynthesis

• Signal peptide directs the nascent polypeptide to
  ER
• Protein modifications starts in late ER. GAG side
  chains elongation and modification takes place in
  Golgi.
• Several specific enzymes to add disaccharide
  units and to modify them (e.g. sulphation). For
  example over 30 enzymes are needed in synthesis
  of aggrecan, a cartilage matrix proteoglycan.

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Cell-surface proteoglycans
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Syndecans and glypicans

• Syndecans are transmembrane proteins. Four family
  members. Short cytoplasmic tail contains highly
  conserved sequences that bind to adaptor
  proteins. Variable part of syndecan-4 cytoplasmic
  domain binds protein kinase C and affect cell
  signalling
• Glypicans (6 known family members) are lipid
  anchored to plasma membrane. GPI
  glycosylphosphatidylinositol.
• Both families individual family members have
  distinct expression patterns e.g. syndecan-1 in
  epithelia and syndecan-3 in neural cells

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Sugar sequence in GAG chains is functionally
important
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Sugar sequence in GAG chains is functionally
important
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Proteoglycans modulate growth factor activity
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Proteoglycans modulate growth factor activity

• Matrix associated PG sequestration
• Membrane-bound PG presentation
• Certain sugar sequences promote FGF signalling
  and others inhibit
• Membrane-bound PGs can be cleaved from cell
  surface into matrix
• Sugar chains can be cleaved by heparanase enzymes
  to oligosaccharides.

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Heparin binding proteins

• Certain growth factors, especially Fibroblast
  Growth Factor family (FGF)
• Enzymes and their inhibitors, e.g. proteases
• Blood coagulation factors
• ECM proteins
• Note proteoglycans can bind several proteins at
  the same time

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Proteoglycans can modulate cell adhesion to ECM
proteins
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More functions for proteoglycans- syndecan-3
regulates appetite

• Serendipitous discovery in transgenic mice
  over-expressing syndecan-1 under a viral promoter
  gt maturity-onset obesity.
• Heparan-sulphate sugar cahins potentiate
  signalling in hypothalamus that induces
  over-eating.
• In normal mice syndecan-3 is present in
  hypothalamus (in addition to other neural
  tissues). Food deprivation induces syndecan-3
  expression several fold and triggers reflex
  hyperphagia.

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Aggrecan Example of Matrix Proteoglycans
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Proteoglycans in human diseases
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Hyaluronic acid
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Hyaluronic acid
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CD44

• Adhesive glycoprotein
• Numerous isoforms from alternative splicing
• Originally found as a homing receptor in
  T-lymphocytes
• Some splice isoforms suggested to play a role in
  tumour metastasis
• Cytoplasmic tail of CD44 binds to ERM proteins
  (ezrin-radixin-moiesin family) that can regulate
  dynamics of actin cytoskeleton

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Basement membrane

• Also known as basal lamina
• Thin sheetlike network
• Epithelial, endothelial, muscle and Schwann cells
• Physical support, developmental control,
  filtering functions
• Major constituents laminins, collagen type IV,
  perlecan (a proteoglycan)

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Basement membrane
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Laminins
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Laminins

• Molecular composition of basement membranes is
  tissue-specific
• Laminins at least 11 heterotrimers
• Five alternative alpha chains,
• Three alternative beta chains
• Two alternative gamma chains
• For example in skin in the BM between epidermis
  and dermis, Laminin-5 (a3b3g2) is the predominant
  laminin isoform.

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Interactions of laminins
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Type IV collagen
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More Basal Lamina Proteins

• Perlecan a large heparan sulfate proteoglycan.
  HS chains bind other BM components and contribute
  to filtering functions
• Entactin interacts with laminin and type IV
  collagen
• Nidogen, a laminin binding protein

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Hemidesmosome a cell - basement membrane
adhesion site

• In some epithelia epidermis, bladder, trachea,
  breast and amnion
• Shares some ultrastructural features with
  desmosomes both display dense,
  membrane-associated cytoplasmic plaques that are
  connected to intermediate fialments. But
  molecular composition is different.
• Transmembrane glycoproteins connect basement
  membrane to intracellular plaque

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Hemidesmosomes and basement membrane-
ultrastructural view
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Hemidesmosomes and basement membrane- molecular
composition
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Basal lamina functions-structural support
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Basal lamina functions-filter
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Basal lamina functions-developmental guidance

• Early embryo keeps 4 and 8 cell stages together
• Differentiation of epithelial organs epithelial
  - mesenchymal interactions
• Neurite outgrowth guidance of axon growth by ECM
  containing laminin sububits

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Basal lamina functions-developmental guidance
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ECM Turnover - MMPs

• Matrix metalloproteinases are enzymes that cleave
  components of ECM
• Over 20 different enzymes with differenrt
  specificities.
• Common theme expressed as an inactive proenzyme
• Also other substrates than ECM proteins
• TIMPs tissue inhibitors of MMPs

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MMPs - some examples

• Old names collagenases, gelatinases and
  stromelysisn replaced by numbers (e.g. MMP-1)
• MMP-1 (collagenase-1) cuts triple helical
  collagens
• MMP-9, (Gelatinase-B) chops e.g. type IV collagen
  and laminins
• MT-MMPs are membrane-bound enzymes

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MMPs - some functions

• Regulate amount of ECM - degradation and
  remodelling
• Cell migration, wound healing, angiogenesis
• Activate other MMPs
• Release or activate growth factors and other
  bioactive molecules

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MMPs in diseases

• Extensive matrix degradation in e.g. in
  periodontitis, rheumatoid arthritis
• Tumour cell invasion and metastasis
• Carcinoma breaks basement membrane and invades
  surrounding stroma.
• MMP inhibitors tested for therapeutic use

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Integrins
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Integrins

• At least 24 different heterodimers from 9 b
  subunits and 18 a subunits.
• Variable pairing b1 integrin can have 11
  different a partners.
• Overlapping ECM binding e.g. 8 different
  integrins can bind fibronectin
• An integrin can bind one or several ECM proteins

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Integrins and cell behaviour

• Clustering of integrins (velcro effect in
  adhesion)
• Responses to cell adhesion include spreading,
  cytoskeletal re-organisation, polarisation,
  migration, proliferation, activation of specific
  genes
• Cell survival epithelial cells that are detached
  commit suicide (this type of apoptosis is called
  anoikis).
• Also, inside out signalling integrins can have
  inactive conformation that does not bind matrix
  unless first activated.

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Integrins - variety in functions
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SUMMARY

• Collagens Triple helical rod and non-collagenous
  domains. Important structural role. Extensive
  post-translational modifications
• Fibronectin adhesive glycoprotein in matrix and
  plasma
• Proteoglycans GAG-chains attached to core
  protein.
• Laminins major components of basement membranes
• Matrix metalloproteinases degrade and re-model
  matrix
• Integrins heterodimeric proteins that mediate
  cell adhesion to extracellular matrix.
• Cell-matrix interactions important regulator of
  cell behaviour

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SUMMARYFUNCTIONS OF ECM