Epithelial cell adhesion to the surrounding extracellular matrix (ECM) is necessary for their proper behaviour and function. During pregnancy and lactation mammary epithelial cells (MECs) require signals imparted by specific β1 integrin-laminin interactions for their functional differentiation in response to Prolactin (Prl) and for the correct formation of polarised secretory acini. Downstream of β1 integrin (β1Itg), the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both Prl driven lactational differentiation and the establishment of apico-basal polarity in MECs. ILK is a multifunctional adaptor protein that links integrins to the actin cytoskeleton and Rho GTPases such as Rac1. ILK forms a ternary IPP (ILK-PINCH-Parvin) complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known which of ILKs interacting partners are important for controlling tissue-specific gene expression, or what acts downstream of the IPP complex. In this thesis I have now established that inducible ILK deletion in MECs from ILKfl/flCreER mice, prevents phosphorylation of Stat5 leading to a failure of Prl induced milk expression. In addition I have established a 3-dimensional culture model using the EpH4 mammary epithelial cell line, which respond to Prl treatment and form polarised acini similar to primary cells. In these cells knocking down β1Itg and ILK by lentiviral shRNA delivery was confirmed to have a profound effect on β-Casein production. Expression of ILK mutants that disrupt its protein-protein interactions, showed that mutation of K220 and E359 in the kinase domain also reduced milk production. This means that ILKs kinase domain is important for MEC differentiation, and suggests that Parvin binding (which is disrupted by these mutations) is key in mediating ILKs differentiation functions. Using a complimentary shRNA approach, knockdown of the βParvin binding Rac guanine nucleotide exchange factor αPix also prevented MEC differentiation. This identified for the first time that αPix is required for differentiation and suggests a route by which ILK, via it’s interaction with Parvin, can link integrins to αPix and Rac activity. Interestingly, αPix depletion did not disrupt the IPP complex or polarity, suggesting that αPix represents a differentiation specific bifurcation point in β1Itg-ILK adhesive signalling. Together, this work has helped to establish how ILK is involved in MEC differentiation and has identified a new role for the downstream Rac GEF αPix. In addition, this work contributes to our understanding of the molecular mechanisms by which cell adhesion regulates fundamental cell biological behaviours.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:603219 |
| Date | January 2014 |
| Creators | Rooney, Nicholas |
| Contributors | Streuli, Charles; Ballestrem, Christoph |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-role-of-integrin-linked-kinase-in-mammary-epithelial-cell-differentiation(7ea57786-1b8a-4391-bfbf-2cda9977877e).html |
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