Transcript Extracellular matrix

THE EXTRACELLULAR
MATRIX
• 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.
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.
SOME FUNCTIONS OF ECM
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)
CONNECTIVE TISSUES
EXAMPLES OF ORGANISATION OF
ECM
- DERMIS
EXAMPLES OF ORGANISATION OF
ECM
- CARTILAGE
EXAMPLES OF ORGANISATION OF
ECM
- BASEMENT MEMBRANE
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
COLLAGENS -examples out 19 different
types
Collagens assemble to diverse structures
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 ->
hydroxyproline
Collagen Biosynthesis - intracellular steps
COLLAGEN
Collagen Biosynthesis - extracellular steps
Collagen Biosynthesis - extracellular
assembly
Collagen Biosynthesis - extracellular
assembly
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 Cterminus 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
Collagens in disease
• Inherited diseases with mutations in collagen genes
• Osteogenesis Imperfecta
• Fibrotic diseases with accumulation of ECM
• Liver Chirrosis
• Lung Fibrosis
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)
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
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
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
Fibronectin structure
Fibronectin structure: domains and
interactions
Plasma Fibronectin
Fibronectin binds cell surfaces by an RGD
sequence
Fibronectin is essential for embryonic
development
• Gene targeting => complete lack of fibronectin
• Embryonic lethal.
• Gross malformations, notchord and somites missing, heart
malformation
• Problems in cell adhesion, migration and differentiation
Proteoglycans have long sugar chains
attached to a core protein
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.
Cell-surface proteoglycans
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
Sugar sequence in GAG chains is
functionally important
Sugar sequence in GAG chains is
functionally important
Proteoglycans modulate growth factor
activity
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.
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
Proteoglycans can modulate cell adhesion to
ECM proteins
More functions for proteoglycans
- syndecan-3 regulates appetite
• Serendipitous discovery in transgenic mice over-expressing syndecan1 under a viral promoter => 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.
Aggrecan: Example of Matrix Proteoglycans
Proteoglycans in human diseases
Hyaluronic acid
Hyaluronic acid
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-radixinmoiesin family) that can regulate dynamics of actin cytoskeleton
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)
Basement membrane
Laminins
Laminins
• Molecular composition of basement membranes is tissue-specific
• Laminins: at least 11 heterotrimers
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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.
Interactions of laminins
Type IV collagen
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
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
Hemidesmosomes and basement membraneultrastructural view
Hemidesmosomes and basement membranemolecular composition
Basal lamina functions
-structural support
Basal lamina functions
-filter
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
Basal lamina functions
-developmental guidance
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
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
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
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
Integrins
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
Integrins and cell behaviour
• Clustering of integrins (“velcro effect” in adhesion)
• Responses to cell adhesion include spreading, cytoskeletal reorganisation, 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.
Integrins - variety in functions
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
SUMMARY:FUNCTIONS OF ECM