L4 - Laminin and integrins

What is laminin?

a major component of BMs that self assembles into a network to present binding sites for cells

What is the structure of laminin?

• High MW glycoprotein (800kDa)

• 3 alpha helix chains (alpha, beta and gamma) assembled into a cruciform structure wrapped in coiled coil

• 3 shorts arms with globular domains at N termini

• 1 long coiled coil arm with globular domain C terminal - alpha chain

• Spacers of EGF repeats

  

What brings about the laminin coiled coil?

The formation of the laminin coiled-coil trimer is brought about by a repeated sequence of 7 amino acids in the long arms of each chain which maximises non-covalent bonds between the chains

What is the triple helix of laminin stabilised by?

disulphide cross links at each end of the coiled coil domain

What does laminin 1 form in vitro?

Spontaneous forms a network

o N-terminal globular domains promote polymerisation into a network

o Nidogen also interacts with domains and stabilises network

What does the alpha chain globular domain consist of?

5 LG domains which interact with cell surface receptors

LG1,2,3 interact with intergrins

LG4,5 interacts with dystroglycan and heparin

What is the laminin network linked to?

Collagen IV by accessory molecules

o Collagen IV 2D network forms - structural backbone

o Laminin forms integrated network - Cell binding sites stick out

How many laminin genes are there and how many heterotrimeric combinations do they make?

There are 11 laminin genes, but these only associate to form 15 different heterotrimeric combinations - there are more laminin isoforms than there are collagen IV isoforms

There are more laminin isoforms than collagen IV isoforms, what is the result of this?

• More variability - more tissue specific expression of laminin than collagen IV

  • Most BM will be the same type of collagen IV (a1a1a2)

  • Most BM will have different laminin in tissues

• A lot more diseases through mutations in laminin

• There are diseases associated with tissue restricted collagen isoforms

What does genetic deletion in mice show?

That some laminins are essential throughout the animal

Different laminin isoforms show tissue specific expression

Where is a1b1g1 found and what does its deficiency cause?

laminin found in the embryonic BM

• a1 is found in the BM of epithelial tissues during embryogenesis and some epithelial BM in adults

• Deficiency in a1 = embryonic lethality – only essential In extra embryonic tissue

Where is alpha 2 found and what does a deficiency cause?

 A2 is in the BM of skeletal and cardiac muscle, peripheral nervous system and central nervous system

 Deficiency in a2 = severe congenital muscular dystrophy, lethal 5 weeks after birth

Where is alpha 3 found and what does a deficiency cause?

 A3 is primarily in BMs of stratified epithelia

 Deficiency in a3 = lethal post natal skin blistering, dies 3 days after birth

Where is beta 2 found and what does a deficiency cause?

 B2 has wide expression pattern

 Deficiency in b2 = postnatally lethal because of defects in glomerular filtration and NMJ

What is pierson syndrome?

• Rare and lethal congenital

• Caused by a loss of laminin beta2 isoform

• Similar condition to loss of GBM collagen IV

• Frame shift mutations makes non-functional laminin

What are the symptoms of loss of laminin beta?

• Congenital nephrotic syndrome progressing to end stage renal disease

• Eye abnormalities

• Severe muscular hypotonia

What causes pierson disease phenotype?

Laminin 11 (a5b2g1) is expressed in the GBM, eye, and synaptic BM

What do mice phenocopying pierson syndrome show?

• Deficient for laminin beta 2 gene

• GBM shows beta 1 expression instead

• This does not form an effective filtration barrier

• Abnormal retinal and neuromuscular junctions

• High levels of protein in urine

• Collagen IV is not affected

What is the epidermis attached to?

The epidermis is attached to the underlying dermis via a basement membrane. This is mechanically strong

What is epidermolysis bullosa (EB)

• Related group of conditions where skin blisters following mechanical trauma

• Mutations affect the mechanical strength of the dermal/epidermal junction

• Position of the break depends upon the genetic defect

Where is laminin 5 found and what is its isoform?

a3b3g2

Found in skin BM

What does laminin 5 do?

• Links integrin a6b4 on epidermal layer to collagen IV, which is connected to anchoring fibre collagen VII, which links to collagen I in the dermis

• Integrins are in hemidesmosomes

• Collagen VII is also specific to the BM

What does Mutations in laminin 5 (a3b3g2) cause?

Junctional EB

genes = LAMA3, LAMB3, LAMC2

Autosomal recessive

What is herlitz type JEB?

Complete loss of any Laminin 5 chain leads to Herlitz type JEB - lethal within the first few months after birth

No laminin in epidermis so no mechanical strength so will lift off dermis

What is non-herlitz JEB?

Other mutations with perturbed laminin 5 function lead to milder forms of condition (non-Herlitz JEB) - misssense, splice site mutations leading to reduced Lm5 expression or variant with partial function

What have genetic models of JEB in mice prove?

• Prove JEB is caused by loss of laminin 5

• Laminin 5 deletion in mice phenocopies Herlitz JEB

• No difference at birth

• Develop blisters post natal

• Die by day 3

• Skin detaches and loses water barrier so dehydrates

Difference between herlitz and non-herlitz

Herlitz = complete loss of one of the chains eg major gene deletion or rearrangement or chain terminating mutation

Non-Herlitz = reduced expression or partial loss of function – can survive a few years

What is the gene therapy for Laminin β3 chain mutations?

• Seven-year-old with splice site mutation in exon 14 of LAMB3 (non-Herlitz JEB)

• Suffered severe blisters since birth. Presented at hospital following S. aureas infection, leading to loss of ~60% of epidermis

• Took some skin and grew as you would a skin graft – culture epidermal cells

• Used a RETROVIRUS to deliver a functional LAMB3 gene to patients own keratinocytes

• Holoclones are proliferative and contain stem cells. Paraclones, meroclones - more differentiated

• After 8 months, the skin was almost entirely derived from holoclones

What are the problems with using retroviruses for gene therapy?

• Safety

  • Retroviruses can cause replication-competent retroviruses (RCR) and oncogenesis

• Immune response

  • The body's immune system can fight off viruses, and an unwelcome response could cause serious illness

• Cell division

  • Retroviruses can only infect actively dividing cells

• Low transduction efficiency

• Inactivation by complement cascade

What are retroviruses?

RNA viruses so very small

Use reverse transcriptase to copy RNA into DNA which integrates into host cell genome permanently

How are retroviruses used in gene therapy?

• Take wild type retrovirus and modify it so it is unable to make more viruses

• Only able to infect a host cell

• Then hijack it so instead of having core viral gene, has a copy of gene of interest

• Integrates a copy into host cell DNA

SUMMARY

What are cell-ECM junctions in the skin?

• Structural links between cytoskeleton and the matrix

• Provides physical strength to tissues

• Continuous linkage between cytoskeleton and ECM fibres

o Whole cell is a mechanical system interacting with ECM

What are cell-cell junctions

Anchor cells strongly to each other to help the tissue withstand mechanical stress

What are tight junctions?

Seal gaps between epithelial cells

What are adherens junctions?

Connects actin filament bundle in one cell with that in the next cell

What are desmosomes?

Connect intermediate filaments in one cell to those in the next

What are gap junctions?

Allows passage of small water-soluble molecules from cell to cell

What are hemidesmosomes?

Anchors immediate filaments in cell to ECM

What are actin-linked cell-matrix junction?

Anchors actin filaments in cell to ECM

What are integrins?

Cell/ECM adhesion receptors on most cells

What is the structure of integrins?

• Heterodimers of alpha and beta subunits

• Lots of heterodimers with different specifities for different ECM proteins

• Large extracellular domain

• Single transmembrane spanning domain

• Each chain goes through the plasma membrane with a single alpha helix

• Short cytoplasmic domain that will connect to cytoskeleton (with one exception in b4)

• B4 has a long cytoplasmic domain

There are many different α and β subunits, with numerous known αβ heterodimers, what are there different specificities?

They have distinct but overlapping specificties for different ECM

eg a6 can be connected with b1 or b4

Wh

What heterodimer is important in EB?

a6b4

What does integrin do?

Connects ECM with cytoskeleton

Most integrins associate with actin myosin cytoskeleton

What does the integrin cytosplasmic domain do?

Interacts with the cytsoskeleton

What are focal adhesions?

Integrins linking to actiin

What are hemidesmosomes?

o B4 in hemidesmosomes they link to intermediate filaments made up of keratin (important for EB)

o Keratin is used to resist mechanical forces

What is the tissue specific distribution of beta1 integrin isoform?

o When deleted, peri-implantation lethality, ICM deteriorates, embryos fail to gastrulate

o Expressed in early embryos

What is the tissue specific distribution of beta2 integrin isoform?

Leukocytosis, impaired inflammatory responses, skin infections, cell proliferation defects

What is the tissue specific distribution of beta3 integrin isoform?

Hemorrhage, no platelet aggregation, osteosclerosis, hypervascularisation of tumours

What is the tissue specific distribution of beta4 integrin isoform?

• Severe skin blistering, other epithelial tissue defective

• Hemidesmosome specific b4 integrins are required for the integrity of the skin

What does deletion of b4 integrins lead to?

integrins leads to same phenotype as with laminin 5 deletions

• Loss of b4 integrin in mice results in the loss of hemidesomosmes, but not the cell/cell adhesions that link to IF’s

• Cells attach to each other but not basement membrane

• Similar to the col IV and laminin deletions, loss of b4 also results in the absence of the a6 integrin subunit

What is Junctional epidermolysis bullosa with pyloric atresia (PA-JEB)?

• Rare autosomal recessive condition associated with loss of a6b4 integrin

• Neonatal mucocutaneous blistering and gastric outlet obstruction through loss of function in gastrointesinal, genitourinary and respiratory epithelium

• Problem in digestive tract

• Fatal

• Seeing effects in other tissues which is not seen with laminin mutations

What is the tissue specific distribution of alpha 5 integrin isoform?

If deleted, defects in mesoderm and vascular development, neural crest apoptosis and muscular dystrophy

What is the tissue specific distribution of alpha 6 integrin isoform?

o Severe skin blistering, other epithelial tissues defective, lamination defects in cortex and retina

o Deletion of α6 integrin has the same effect as loss of β4 - in mice and humans

What is EB simplex?

• Milder phenotype than other forms of EB

• Usually restricted to blisters on regions subject to mechanical stress

• More localised blisters

• Skin is weaker but has some mechanical stability

• Can heal without significant scaring

• Compare with JEB which has 40% mortality in first year

What is EB simplex associated with?

mutations in keratins 5 and 14

o Affects cytoskeleton connecting to integrin

o Mechanical integrity inside the cell is weakened

What else can cause EB simplex?

deletion of b4 cytoplasmic tail in mice results in EB simplex

• Cells are weakened internally, but the BM is intact

• Weak intercellular linkage

• Cells rip apart leaving part still attached to BM via the integrin

What shows similar phenotypes as b4 integrin cytoplasmic domain deletions?

Patients can also have mutations in any of the cytoplasmic proteins that link the b4 integrin cytoplasmic domain with intermediate filaments. These show similar phenotype as b4 integrin cytoplasmic domain deletions

EB summary