Cells are connected to one another and also the ECM by a large number of various cellular junctions. Adherens junctions are particularly important for physical adhesions of cells to their surroundings, hence the name adherence or adherens junctions (Yap
et al., 1997). Adherens junctions are special forms of adhesive-based contacts that
1 INTRODUCTION
are cadherin based and these junctions are essential for tissue organisation during de- velopment, as well as playing a critical role in the determination and maintenance of tissue organisation in adult organisms (Yap et al., 1997).
Cadherins are one of the major family members of adhesion molecules and support calcium dependent cell-cell adhesions in all solid tissues in the body (Yapet al., 1997). Cadherins in association with the actin cytoskeleton can regulate cell-cell recognition events that then bring about morphological transitions that maintain the structure of tissues in adult organisms (Aberleet al., 1996). All members of the adhesion molecule superfamily are transmembrane proteins and have a variable number of extracellular cadherin domains (Overduinet al., 1995).
The classical cadherins are single-span transmembrane proteins located primarily within adherens junctions and mediate calcium-dependent cell-cell adhesion (Angst
et al., 2001). They can interact with a complex network of signalling and cytoskeletal molecules by transferring information intracellularly. E-Cadherin and N-Cadherin are the two classical cadherins that have been extensively studied and it has long been known that expression of either of these cadherins on the cell surface is involved in cell sorting (see Figure 1.7, page 39) (Yapet al., 1997).
1 INTRODUCTION
Figure 1.7: Cadherins involved in cell migrationCell migration and polarisation are under- pinned by a complex network of cellular trafficking pathways required. Signalling receptors need to be recycled in order for the cell to become polarised and move in the correct direction ie towards the wound. Integrins need to be recycled creating and remodelling the adhesions and allowing the cell to move. Cell to cell junctions ie cadherins need to be remodelled in order for the cell to be able to separate from its neighbours.
Cadherins are homophilic molecules and cells adhere to their neighbouring cells only if these are expressing the identical cadherin member to themselves. Therefore when cells are in culture the cells will sort themselves from each other by only adhering if they are expressing the same cadherin (Nose et al., 1988). During development, changes in the cadherins expressed in the cells allow segregation of cells to distinct tissues therefore influencing the organisation of a number of cell types into tissues (Takeichi, 1991). Adhesive specificity arises from both affinity differences between cadherin sub- types and their expression levels (Foty and Steinberg, 2005). Furthermore, cadherins will segregate at high shear force but can cross adhere at low shear force and be- cause cells remain in contact with each other longer at lower shear forces the kinetics of cadherin-cadherin interactions may play a role in the determination of cell specificity (Duguayet al., 2003).
In humans there are over 80 members of the cadherin superfamily (Derycke and Bracke, 2004). There are four cadherin subfamilies conserved between C. elegans,
1 INTRODUCTION
Drosophila and humans: classic cadherin, fat-like cadherins, seven-pass transmem- brane cadherins and DrosophilaCad 102F. The classic cadherins themselves consist of four subgroups: Type I classic cadherins that include Epithelial Cadherin, Neuronal Cadherin, Placental Cadherin and Retinal Cadherin (Boggonet al., 2002).
The classic cadherin family members contain five cadherin domains and a conserved cytoplasmic tail that interacts with multiple proteins that functionally link cadherins to the underlying cytoskeleton (see Figure 1.8, page 41) (Ozawa et al., 1990b). It is the cytoplasmic region of the cadherin that binds to β-catenin and to p120 (Pokutta and Weis, 2007). β-catenin in turn binds toα-catenin, which has a number of binding part- ners, one of which is actin (Pokutta and Weis, 2007). The cadherin-catenin complex mediates adhesion but only when the adhesion involves classic cadherins (Nagafuchi and Takeichi, 1988; Ozawaet al., 1990a).
Neuronal Cadherin (N-Cadherin) was identified for the first time in 1982 (Grunwald
et al., 1982). It is a 130 kilo Dalton (kDa) molecule and was discovered in the chick neural retina that was protected from proteolysis by calcium (Grunwald et al., 1982). In 1984 a molecule called A-CAM was identified and shown to localise at the ad- herens junctions; A-CAM is now known as N-Cadherin (Volk and Geiger, 1984). The fibroblast growth factor receptor (FGFR) is known to be associated with the function of N-Cadherin. N-Cadherin has many functions in the nervous system with the key roles being regulation of the growth of axons, and guidance to synaptic formation and synaptic plasticity (Doherty and Walsh, 1996). Neurite outgrowth can be inhibited by many blocking buffers that inhibit FGFR function if the neurite growth is controlled by N-Cadherin (Williams et al., 1994). The cytoplasmic part of N-Cadherin is complexed with a multitude of regulators for cadherin such as the catenins p120 catenin,β-catenin and α-catenin. The binding with p120 and N-Cadherin is increased when the p120 is
1 INTRODUCTION
Figure 1.8: Domain structure of Classical Cadherins. The best understood cadherins are the “classical” cadherins of vertebrates, and the closely related desmosomal cad- herins. Classical and desmosomal cadherins have ectodomains composed of five extracellular cadherin (EC) repeats, a single transmembrane region, and a cyto- plasmic domain that interacts with eitherβ-catenin (classical cadherins),γ-catenin andα-catenin which then in turn binds to actin.
phosphorylated.
β-catenin is a protein that contains 781 amino acids and is very highly conserved. E-Cadherin binds to β-catenin on the arm domain of the protein whereas α-catenin binds just before the arm domain (Huber et al., 1997). The E-Cadherin binding site of the protein is also the binding site for other ligands and is known to be involved in Wnt signalling (Choi et al., 2006). P120 is a protein that binds to the cytoplasmic region of the cadherin (Daniel and Reynolds, 1995; Thoreson et al., 2000). Although the role of P120 remains unclear it is thought to play a role in controlling the regulators of the
1 INTRODUCTION
actin cytoskeleton (Anastasiadis and Reynolds, 2001).
α-catenin is a protein that is homologous to the focal adhesion molecule vinculin. There have been a lot of binding partners already identified forα-catenin but not all of them have been verified biochemically (Kobielak and Fuchs, 2004). α-catenin forms a homodimer, but when it binds to β-catenin it dissociates to form a 1:1 heterodimer (Koslov et al., 1997; Pokutta and Weis, 2000). It was previously believed that α- catenin links theβ-catenin-cadherin complex to actin, providing the stable linkage that is needed between the extracellular contact and the cytoskeleton, due to the ability of α-catenin to bind to bothβ-catenin and F-actin. However more recent reports suggest that this is not the case as purified recombinant proteins showed thatα-catenin cannot bind toβ-catenin and actin simultaneously. This is even the case when it is in the pres- ence of proteins that have been reported to bind both actin and α-catenin like native membranes or the cytosol (Yamada et al., 2005).
The adhesive function of cadherins is a dynamic process and cells responding to physiological and environmental cues regulate this (Yapet al., 1997). It is thought that in developing organisms, the levels of E-Cadherin based adhesion are increased (Vest- weber et al., 1987; Winkel et al., 1990), and the levels of C-cadherin are reduced by activin, and this decrease in the levels of adhesions enables all the intercellular move- ments (Brieher and Gumbiner, 1994).
Distinctive types of cell-cell junctions containing cadherins are formed in epithelial cells and are known to express equal levels of both N-Cadherin which is a type I cad- herin and VE cadherin which is a type II cadherin (Wheelock and Johnson, 2003). VE-cadherin is thought to localise to the junctions and N-Cadherin localises on the surface of the cell (Navarro et al., 1998). Although the role of N-Cadherin is not fully
1 INTRODUCTION
understood in endothelial cells it is known that it does not form part of the endothelial cell junction complex (Wheelock and Johnson, 2003). VE-cadherin however does form part of this complex along withβ-catenin, plakoglobin and p120 catenin that then con- nect to the actin cytoskeleton and form typical adherens junctions (Lampugnaniet al., 1995). Endothelial cells also have junctions that connect to the cytoskeleton, however the filaments are made up of vimentin rather than keratin. Endothelial cells do not contain desmosomal cadherin so the VE-cadherin is restricted to α-catenin and actin interactions (Kowalczyket al., 1998).
During early vertebrate development, polarised assembly of junctional proteins at the apical surface of the cell is essential (Yeamanet al., 1999). Epithelial cells maintain their polarity by the sorting of apical and basolateral proteins into various vesicles in the TGN, the proteins are then targeted to their specific membrane compartments (Whee- lock and Johnson, 2003). Cell polarity is established at the very early stages of devel- opment and this polarity is mediated by E-Cadherin. For polarisation to occur within the cell proper localisation of a number of proteins in a number of different cell types is needed. These include cadherins, scaffolding proteins and signalling molecules (see Figure 1.7, page 39) (Ohno, 2001).
E-Cadherin is the most studied of the cadherin family in tumourigenesis due to the high levels expressed in epithelial cells, which are the origin of most cancers. In vitro
and in vivo studies have proven that if you inhibit the activity of E-Cadherin normal epithelial cells change into invasive cells, and overexpression of E-Cadherin converts invasive cells to non-invasive cells (Perl et al., 1998). It is well established that E- Cadherin functions as a tumour suppressor with many studies describing and proving this in great detail (Ghadimiet al., 1999; Wheelocket al., 2001; Perretet al., 2002).
1 INTRODUCTION
Expression of N-Cadherin also causes epithelial cells (squamous and breast cells) to become more motile and more invasivein vitro and more metastaticin vivo(Nieman
et al., 1999; Hazan et al., 2000). It has also been shown that the effect of N-Cadherin on motility is mediated through the FGF receptor signalling pathway (Nieman et al., 1999). N-Cadherin is thought to directly interact with FGF receptor 1 and prevent the receptor from being internalised therefore increasing the receptor on the cell surface and enhancing downstream signalling (Cavallaro et al., 2001). N-Cadherin also inter- acts with FGF receptor 4 mediated by N-CAM, which is a cell-cell adhesion molecule (Suyama et al., 2002). Thus expression of N-Cadherin by tumour cells can influence cellular behaviour via ligand-dependent and ligand-independent interactions with FGF receptor (Hulitet al., 2007).
Epithelial to mesenchymal transitions occur when epithelial cells are converted into fibroblast-like motile cells, which is regularly observed during normal development. Similar actions are seen through the stages of carcinogenesis. Decreased expres- sion of E-Cadherin is a hallmark for epithelial-mesenchymal transitions (Perret et al., 2002).
Cadherin function and expression has major effects on the cell phenotype and be- haviour and expression levels are essential in development not only to form cell-cell junctions but to mediate cell signalling events and cell sorting that regulates develop- ment (Wheelock and Johnson, 2003).