Breast remodelling in the reproductive cycle Flashcards
Describe the mammary gland
Ductal tree
Ducts connect to nipple on one end, form lobular alveloi on other
Growth during puberty
What regulates growth of the mammary gland
Hormones, GFs, ECM
Describe the microenvironment of the mammary gland
Ducts consist of luminal epithelium, myoepithelium + laminin-BM
Luminal epithelium = lactocytes = produce milk
Myoepithelial cells squeeze milk from lactocytes
Stromal collagen + stromal fibroblasts are responsible for producing collagen matrix
Whole structure embedded on fat pad of adipoctyes
How does the mammary gland convert into a milk producing organ
Development of alveoli
Lactation differentiation
Cell proliferation + differentiation
Cell polarity
What is the role of the basement membrane
Holds cells together and provides signals
Specialised ECM surrounds epithelial tissues + separates them from interstitial stromal collagen compartment
What is the composition of the ECM
Laminins 111 + 332
Collagen IV
Nidogen/entactin
Proteoglycans
How does cell adhesion to ECM occur
Through transmembrane receptors
Different subunits can cooperate to detect different ECM proteins
B1 integrin important
Integrins bound to ECM on outside, linked to cytoskeleton inside cell
Focal adhesion complex
What genetic approaches can be used to study gene function
CRISPR/Cas9 TALEN short hairpin RNA Cre-Lox In vivo tissue specific targeting
What does genetic deletion of B1 integrin in mammary gland show
Deleted only from epithelial cells in mammary glands
Reduced alveolar development, reduced proliferation
GFs and hormones can cause phosphorylation of ERK but need signals from integrins which bind to FAK
B1 integrins control cell cycle via Talin + ERK nuclear translocation
How does laminin ECM mediate milk protein expression in mammary epithelia
Prolactin binds receptor
Triggers signalling cascade involving Jak2 bound to TF Stat5
Translocates into nucleus to initiate milk production
If the BM is removed them no development so ECM factors required- Laminin
How do we know B1 integrin is mediated for lactational differentiation
Pups feeding off transgenic mothers are smaller, mothers have defect- reduced milk fat + milk fat protein
Laminin signals to B1 integrin
Is FAK required for lactational differentiation
No
Fak deletion doesnt affect pup weight, milk fat or protein
Not involved in differentiation downstream of integrin
What does genetic deletion of ILK show
Pup weights reduced but not as much as B1 integrin
Reduced lactation
What does ILK deletion in culture show
Ad-Cre virus removes ILK
Phenotype can be rescued with IL-GFP
ILK is downstream of B1 integrin
Describe the lactational pathway
Laminin > B1 integrin > ILK > Rac1 > b-casein
Also: Parvin, a-pix, vinculin
What does genetic deletion of Rac1 show
Lobular alveoli much smaller than WT
Not producing as much milk protein
What does consitutively active Rac1 do
Rescues lactation defect in integrin/ILK null organoids + MECs on non-permissive collagen I matrix
Why is cell polarity important in lumen formation
Hollow lumen essential for proper tissue function
Apicobasal polarisation ensures secretion is directed towards the lumen
What does lumen formation look like without B1 integrin
Failed to form lumen
Filled lumens frequent in early breat + epithelial cancers
What does deletion of adhesome components show in relation to lumen formation
ILK but not FAK controls lumen formation downstream of b1 integrin
What are the hypothetical mechanisms of lumen formation
Apoptotic clearance of cells in the core- not possible, cell death not affected by b1 integrin KO
Controlled cell proliferation- no, KO causes underproliferation
Cell division axis orientation
Cell adhesion- No, cells filling lumen space still have adhesion
Cell rearrangements
How is the lumen formed
Apical markers form all over, basal markers move outwards, apical move inwards.
Multiple lumens form then fuse together
B1 integrin required to reorient apical polarity
What is the role of B1 integrin in maintenance of polarity
If deleted after polarised lumens form then apicobasal polarity flips
Lumen collapse
Membranes remodelled
How is the membrane remodelled at the cell-BM interface
- Unpolarised cells with fragmented golgi + apical markers form at the membrane
- Engagement of b1 integrins with BM recruits ILK to instruct orientation of polarity by interacting with plus tips
Integrin/ILK polarise microtubules along apicobasal axis
Interactions include endocytosis of apical components - Integrins/ILK regulate internal cell polarity by positioning golgi apparatus subapically, aids polarised trafficking to membrane. New apical face on other membrane
