Desmosomal Cadherins and Disease

During the past 10–15 years, a wide range of studies revealed roles for desmosomal cadherins in human diseases, particularly in disorders affecting the heart and the skin. Mutations in three of the desmoglein isoforms cause inherited disorders. Loss of DSG4 is associated with defective hair-follicle differentiation (Kljuic et al. 2003), whereas DSG1 haploinsufficiency leads to striate palmoplantar keratoderma, an epidermal-thickening disease (Rickman et al. 1999). The localized impact of these mutations in DSG1 and DSG4 on the skin is consistent with the tissue expression patterns of these genes (Fig. 3). In contrast, mutations in DSG2 result in arrhythmogenic right ventricular cardiomyopathy (ARVC) (Awad et al. 2006; Pilichou et al. 2006).

In this case, heart manifestation of disease most likely reflects a strict requirement for Dsg2 function in resisting mechanical stresses associated with cardiac contraction. To date, no examples of DSG3 mutations have been reported. However, Simpson and colleagues recently reported the first case of a human mutation in a desmocollin (DSC2) and its association with autosomal recessive ARVC (Simpson et al. 2008). Similar to the mouse genetic models described previously, these human disorders further reveal functions for desmosomal cadherins in both tissue integrity and differentiation.

In addition to inherited disorders, desmosomal cadherins are also targets of autoimmune disease. Pemphigus is a class of diseases in which autoantibodies target the desmosomal cadherins, predominantly Dsg1 and Dsg3. The term “pemphigus” is derived from the Greek word “pemphix,” which means blister. These pathologies include pemphigus foliaceus (PF) and pemphigus vulgaris (PV) (Prajapati and Mydlarski 2008). PV is a potentially fatal autoimmune skin disease characterized by circulating autoantibodies (IgG) against Dsg3 and sometimes Dsg1 (Amagai et al. 1991; Amagai 1996; Amagai 1999; Amagai 2002; Payne et al. 2004). PV is characterized histologically by suprabasal acantholysis (loss of cell–cell adhesion) and clinically by mucous membrane erosion and epidermal blisters (Stanley and Amagai 2006). In contrast, PF patients show antibodies against Dsg1 and present clinically with superficial blisters of the epidermis and without mucous membrane involvement. The difference in the clinical presentation in PV and PF is thought to be because of the tissue distribution of Dsg1 and Dsg3 (Mahoney et al. 1999). Nonetheless, in both cases, autoantibodies directed against the desmosomal cadherins are known to cause loss of keratinocyte adhesion (Amagai et al. 2006).

Pemphigus is relatively rare, but these diseases have proven important in revealing functions of desmogleins as well as the mechanisms by which desmosome function is regulated. For example, the fact that IgG against Dsg3 and Dsg1 causes loss of epidermal integrity substantiates a key role for these proteins in epithelial cell–cell adhesion strength. Furthermore, advances in understanding the molecular mechanisms of pemphigus led Stanley and Amagai to correctly predict that bullous impetigo, a common infection caused by a staphylococcal bacterium, causes epidermal blistering by targeting Dsg1. The bacterium produces exfoliative toxin, a serine protease that cleaves the Dsg1 extracellular domain. The cleavage results in blistering in the granular layer that is histologically identical to the acantholysis caused by Dsg1 autoantibodies in PF patients (Amagai et al. 2000; Stanley and Amagai 2006). Thus, epidermal integrity is compromised by either autoantibodies or bacterial toxins that target desmogleins.

Although great strides have been made in identifying the pemphigus antigens, it remains unclear exactly how pemphigus antibodies cause loss of adhesion. One likely explanation is that antibody binding to the Dsg extracellular domain simply blocks adhesion by steric hindrance. However, this hypothesis is called into question by several observations. For example, PV IgG are unable to induce acantholysis in plakoglobin-null cells (Caldelari et al. 2001). In addition, human keratinocytes treated with PV IgG at 4°C do not show loss of cell–cell adhesion until the cells are shifted to 37°C, suggesting that keratinocyte responses are required in order for the antibodies to cause loss of adhesion (Calkins et al. 2006). Other studies have implicated a role for signal transduction in mediating PV-induced acantholysis. Pemphigus IgG binding has been shown to cause activation of numerous cell signaling pathways.

Work by the Rubenstein laboratory has illustrated that PV IgG binding induced phosphorylation of heat shock protein 27 (HSP27) via p38 mitogen-activating protein kinase (p38MAPK) (Berkowitz et al. 2006; Berkowitz et al. 2007; Berkowitz et al. 2008). p38MAPK has also been shown to be involved in phosphorylation of Dsg3 in response to PV IgG (Kawasaki et al. 2006). Furthermore, inhibition of p38MAPK activity prevents keratin retraction, actin reorganization, and formation of epidermal blisters in a mouse model (Berkowitz et al. 2005; Berkowitz et al. 2006). Similarly, signaling through the c-myc pathway has also been implicated in PV pathogenesis (Williamson et al. 2006). These studies suggest that either the activation of signaling pathways on autoantibody binding to the desmosomal cadherins causes loss of adhesion, or alternatively, that manipulation of intracellular signaling pathways can bolster baseline adhesion strength and prevent blistering caused by pemphigus IgG. Either scenario implies that pemphigus patients could be treated by targeting these regulatory networks.

A number of observations have also illustrated that pemphigus IgG binding may interfere with the normal turnover of the desmogleins. Early studies found that PV IgG were internalized after binding to the surface of keratinocytes (Patel et al. 1984; Iwatsuki et al. 1989; Sato et al. 2000). Work from our lab has shown that PV IgG binding results in clathrin independent endocytosis of Dsg3 and subsequent routing of the cadherin to a lysosomal compartment for degradation (Calkins et al. 2006; Delva et al. 2008). Furthermore, blocking Dsg3 endocytosis (Delva et al. 2008) or up-regulating Dsg3 biosynthesis by exogenously expressing Dsg3 prevents keratinocyte loss of adhesion in response to PV IgG (JM Jennings and A. Kowalczyk, unpubl.).

Work performed by Payne and Kitajima also lends support to the impact of PV IgG on desmosomal assembly, as PV IgG causes internalization of newly synthesized pools of Dsg3 (Sato et al. 2000; Mao et al. 2008). Altogether, these findings suggest that pemphigus IgG disrupt the normal turnover and assembly of desmosomes. In fact, a growing body of evidence suggests that regulation of post Golgi trafficking of cadherins is a key mechanism by which cell adhesion is regulated during development and disease (Mosesson et al. 2008; Delva and Kowalczyk 2009).

Plakophilins

The other armadillo members represented in the desmosome are the plakophilins (PKPs). Based on the number and organization of the central armadillo repeat domain, the plakophilins are considered to be members of the p120-catenin subfamily of armadillo proteins. In contrast to p120-catenin, the plakophilins are predominantly found at desmosomes instead of adherens junctions, with PKP1 and 2 also localizing to the nucleus. Plakophilins 1–3 share approximately 55% sequence similarity within the armadillo repeats and approximately 50% sequence similarity with the p120-catenin arm domain (Hatzfeld 2007). Both PKP1 and 2 exist as two isoforms, each generating a short “a” form and a longer “b” form, differing in the addition of 21 amino acids in arm repeat three (PKP1) and the addition of 44 amino acids in arm repeat four (PKP2) (Mertens et al. 1996; Schmidt et al. 1997). Based on structural analysis, the plakophilins contain 9 arm repeat domains (Fig. 4) (Choi and Weis 2005).

Although the armadillo domain of plakoglobin, β-catenin, and other p120-catenin family members all function as a scaffold for numerous binding partners, plakophilin binding partners that have been identified to date associate with the plakophilin amino-terminal head domain. Thus, the precise role of the central armadillo domain of the plakophilins remains mysterious.

Similar to the desmosomal cadherins, the plakophilins show tissue and differentiation-specific patterns of expression. PKP1 is more highly expressed in the suprabasal layers of stratified epithelia, whereas PKP2 is expressed in simple epithelia, the lower layers of stratified epithelia, and in nonepithelial tissues such as cardiac muscle and lymph nodes (Heid et al. 1994; Mertens et al. 1996; Schmidt et al. 1997; Mertens et al. 1999; Franke et al. 2007). PKP3 resides in simple and stratified epithelia, showing uniform expression throughout the epidermis (Fig. 3) (Bonne et al. 1999; Schmidt et al. 1999). It is unclear how these expression patterns are integrated with potential differences in functions of the various plakophilin isoforms.

An obvious morphological difference between desmosomes and adherens junctions is the remarkably electron-dense and highly organized disk-shaped plaque that characterizes the desmosome. Plakophilins are likely to play a key role in the clustering that drives the formation of this highly ordered structure. This function is particularly evident in PKP1. The amino-terminal head domain of PKP1 associates with Dsg1, desmoplakin, and keratin intermediate filaments (Hatzfeld et al. 2000). The binding of the PKP1 head domain to desmoplakin appears particularly robust, and this binding event drives desmoplakin recruitment to cell–cell junctions (Kowalczyk et al. 1999b; Hatzfeld et al. 2000; Wahl 2005).

Based on these interactions, a model has been proposed in which plakophilins play a role in the lateral interactions between desmosomal plaque complexes (Kowalczyk et al. 1999b). In support of this model, PKP1 is highly expressed in the suprabasal layers of the epidermis where desmosomes are larger and more numerous than in the lower layers of the tissue. In contrast, PKP3 overexpression does not result in larger desmosomes or an increase in desmosome number (Bonne et al. 1999; Schmidt et al. 1999; Bonne et al. 2003). However, PKP3 interacts with the largest number of desmosomal proteins, including desmoplakin, plakoglobin, all three desmoglein proteins, Dsc 3a and 3b, and Dsc 1a and 2a (Hatzfeld 2007). PKP2 is also less efficient than PKP1 at recruiting desmoplakin and other desmosomal proteins to the plasma membrane (Chen et al. 2002), suggesting differences in plakophilin isoform function. Recent studies from the Green lab indicate that PKP2 plays an important role in transport of desmoplakin to the plasma membrane during desmosome assembly. In this model system, PKP2 functions as a scaffold for PKC-α and thereby regulates desmoplakin association with intermediate filaments (Godsel et al. 2005; Bass-Zubek et al. 2008; see also Green et al. 2009). Additional studies will be needed to determine whether PKP1 and PKP3 also regulate assembly through PKC-α.

The importance of plakophilins in maintaining desmosomal integrity is underscored by various diseases and/or defective cellular processes that are associated with mutations in each of the plakophilins. For example, patients who suffer from a severe autosomal recessive ectodermal dysplasia and skin fragility syndrome were found to have mutations in PKP1 (McGrath et al. 1997; McGrath et al. 1999).

The dysplasia suggests that in addition to its role in regulating desmosome stability, PKP1 may also regulate epidermal morphogenesis. Because PKP2 is the only isoform expressed in the heart, mutations associated with this particular plakophilin cause ARVC (Gerull et al. 2004). In most cases, the mutations are found within the carboxy-terminus of the PKP2 protein, although other mutations have been observed. Based on experiments in which the gene encoding PKP2 is ablated in mice, the effects seen in ARVC patients are likely the result of mechanical fragility. These mice display mid-gestational embryonic lethality caused by cardiac patterning defects and fragility of the myocardium (Grossmann et al. 2004). PKP2-null mice also display retraction of intermediate filaments from the plasma membrane, further illustrating the importance of plakophilins in desmoplakin recruitment and intermediate filament tethering to the desmosome plaque. Although no human pathologies are associated with mutations in PKP3, conditional ablation of PKP3 in mouse epidermis resulted in defective hair follicle morphogenesis, increased keratinocyte proliferation, and desmoplakin mislocalization. These mice are also more susceptible to dermatitis (skin inflammation) and secondary alopecia (hair loss) (Sklyarova et al. 2008).

Emerging evidence indicates that plakophilins also function outside of the desmosome. Plakophilins, especially PKP1, show striking nuclear localization, although it is currently unclear what functions these proteins may play in gene regulation and/or nuclear structure (Mertens et al. 1996; Hatzfeld et al. 2000).

In addition, PKP2 was found in RNA polymerase III complexes and PKP3 in cytoplasmic particles containing RNA-binding proteins (Mertens et al. 2001; Hofmann et al. 2006). Additional studies are clearly warranted to understand how these associations and nuclear localization are integrated with function in the desmosome, and more broadly, with epithelial cell contributions to tissue architecture.