Decreased plakophilin-1 expression promotes increased motility in head and neck squamous cell carcinoma cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

Decreased Plakophilin-1 Expression Promotes Increased Motility in Head and Neck Squamous Cell Carcinoma Cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

Membrane-impermeable cross-linking provides evidence for homophilic, isoform-specific binding of desmosomal cadherins in epithelial cells

Desmosomes and adherens junctions are cadherin-based protein complexes responsible for cell-cell adhesion of epithelial cells. Type 1 cadherins of adherens junctions show specific homophilic adhesion that plays a major role in developmental tissue segregation. The desmosomal cadherins, desmocollin and desmoglein, occur as several different isoforms with overlapping expression in some tissues where different isoforms are located in the same desmosomes. Although adhesive binding of desmosomal cadherins has been investigated in a variety of ways, their interaction in desmosome-forming epithelial cells has not been studied. Here, using extracellular homobifunctional cross-linking, we provide evidence for homophilic and isoform-specific binding between the Dsc2, Dsc3, Dsg2, and Dsg3 isoforms in HaCaT keratinocytes and show that it represents trans interaction. Furthermore, the cross-linked adducts are present in the detergent-insoluble fraction, and electron microscopy shows that extracellular cross-linking probably occurs in desmosomes. We found no evidence for either heterophilic or cis interaction, but neither can be completely excluded by our data. Mutation of amino acid residues Trp-2 and Ala-80 that are important for trans interaction in classical cadherin adhesive binding abolished Dsc2 binding, indicating that these residues are also involved in desmosomal adhesion. These interactions of desmosomal cadherins may be of key importance for their ordered arrangement within desmosomes that we believe is essential for desmosomal adhesive strength and the maintenance of tissue integrity.     

Desmosomal cadherins

New evidence from blocking desmosomal adhesion with anti-adhesion peptides reveals a role for desmosomes in cell positioning in morphogenesis. Desmosomal adhesion is necessary for the stability of adherens junctions in epithelial cell sheets. Knockout and mis-expression of desmosomal cadherins in mice suggests that they may function directly or indirectly in regulating epidermal differentiation. Protein kinase C signalling and tyrosine phosphorylation appear to regulate desmosomal adhesion. There are new insights into the role of desmosomal cadherins in autoimmune, infectious and genetic disease.     

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG's effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.     

R-cadherin influences cell motility via Rho family GTPases

Classical cadherins are the transmembrane proteins of the adherens junction and mediate cell-cell adhesion via homotypic interactions in the extracellular space. In addition, they mediate connections to the cytoskeleton by means of their association with catenins. Decreased cadherin-mediated adhesion has been implicated as an important component of tumorigenesis. Cadherin switching is central to the epithelial to mesenchymal transitions that drive normal developmental processes. Cadherin switching has also been implicated in tumorigenesis, particularly in metastasis. Recently, cadherins have been shown to be engaged in cellular activities other than adhesion, including motility, invasion, and signaling. In this study, we show that inappropriate expression of R-cadherin in tumor cells results in decreased expression of endogenous cadherins (cadherin switching) and sustained signaling through Rho GTPases. In addition, we show that R-cadherin induces cell motility when expressed in epithelial cells and that this increased motility is dependent upon Rho GTPase activity.     

Endothelial cadherins in cancer

Cadherins are cell adhesion receptors that play important roles in embryogenesis and tissue homoeostasis. Endothelial cells express various members of the cadherin superfamily, in particular vascular endothelial (VE-) cadherin, which is the main adhesion receptor of endothelial adherens junctions and neural (N-) cadherin, which is normally localized outside the junctions and may mediate adhesion between endothelial cells and non-endothelial cells. Dysregulation of cadherin expression has been implicated in tumor progression, in particular the loss of epithelial (E-) cadherin expression or function and the gain of N-cadherin. Moreover, more recently, aberrant expression of VE-cadherin was observed in certain cancer types. In breast carcinoma, VE-cadherin was shown to promote tumor cell proliferation and invasion through enhancing TGF-β signaling. Thus, in breast cancer, the cadherin switch involves another player, vascular endothelial cadherin, which is part of an intricate interplay of classical cadherins in breast cancer progression.     

Plakophilins: Multifunctional proteins or just regulators of desmosomal adhesion?

Plakophilins 1-3 are members of the p120(ctn) family of armadillo-related proteins. The plakophilins have been characterized as desmosomal proteins, whereas p120(ctn) and the closely related delta-catenin, ARVCF and p0071 associate with adherens junctions and play essential roles in stabilizing cadherin mediated adhesion. Recent evidence suggests that plakophilins are essential components of the desmosomal plaque where they interact with desmosomal cadherins as well as the cytoskeletal linker protein desmoplakin. Plakophilins stabilize desmosomal proteins at the plasma membrane and therefore may function in a manner similar to p120(ctn) in the adherens junctions. The three plakophilins reveal distinct expression patterns, and although partially redundant in their function, mediate distinct effects on desmosomal adhesion. Besides a structural role, a function in signaling has been postulated in analogy to other armadillo proteins such as beta-catenin. At least plakophilins 1 and 2 are also localized in the nucleus, and all three proteins occur in a cytoplasmic pool. This review aims to summarize the current knowledge of plakophilin function in the context of cell adhesion, signaling and their putative role in diseases.     

Induction of hyper-adhesion attenuates autoimmune-induced keratinocyte cell-cell detachment and processing of adhesion molecules via mechanisms that involve PKC

In confluent keratinocyte monolayers, desmosomal adhesion gradually becomes calcium-independent and this is associated with an increase in the strength of intercellular adhesion (hyper-adhesion). In this study, we investigated the functional and molecular significance of hyper-adhesion in a system challenged by autoimmune sera from patients with Pemphigus Vulgaris (PV), a disease primarily targeting desmosomal adhesion. The results show that keratinocytes with calcium-independent desmosomes are resistant to disruption of intercellular contacts (acantholysis) in experimental PV. Furthermore, both the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 and the adherens junction protein E-cadherin were decreased in confluent keratinocytes at Day 1, but not in hyper-adhesive cells (Day 6) after incubation with PV serum. Pharmacological induction of the hyper-adhesive state with the PKC inhibitor Go6976 reduced both the acantholysis rate and the processing of cell adhesion molecules induced by PV serum. When the establishment of the hyper-adhesive state was prevented by cell adhesion recognition (CAR) peptides that perturbed desmosomal interactions, Go6976 could still partially attenuate PV acantholysis. Taken together, these data demonstrate that keratinocyte hyper-adhesion decreases the morphological, functional and biochemical dys-cohesive effects of PV serum via mechanisms that involve, at least in part, the function of PKC. This suggests that reinforcing keratinocyte adhesion may be a promising way to inhibit the effects of this most debilitating disorder.     

Desmosomes and disease: an update

Desmosomes play a critical role in the maintenance of normal tissue architecture. Skin blistering can occur when desmosomal adhesion is compromised by antibodies in autoimmune diseases such as pemphigus. Inherited mutations in genes encoding desmosomal constituents can adversely affect the skin, and result in heart abnormalities. Desmosomes may have a tumour suppressor function: expression of desmosomal components is reduced in some human cancers, and desmosomal cadherins have the capacity to suppress the invasiveness of cells in culture. Transgenic animal research has provided important insights into the role of these junctions in normal epithelial morphogenesis and disease.     

The differentiation-dependent desmosomal cadherin desmoglein 1 is a novel caspase-3 target that regulates apoptosis in keratinocytes

Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17- and 140- kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.     

Desmosomal adhesion regulates epithelial morphogenesis and cell positioning

Desmosomes are intercellular junctions of epithelia and are of widespread importance in the maintenance of tissue architecture. We provide evidence that desmosomal adhesion has a function in epithelial morphogenesis and cell-type-specific positioning. Blocking peptides corresponding to the cell adhesion recognition (CAR) sites of desmosomal cadherins block alveolar morphogenesis by epithelial cells from mammary lumen. Desmosomal CAR-site peptides also disrupt positional sorting of luminal and myoepithelial cells in aggregates formed by the reassociation of isolated cells. We demonstrate that desmosomal cadherins and E-cadherin are comparably involved in epithelial morphoregulation. The results indicate a wider role for desmosomal adhesion in morphogenesis than has previously been considered.     

E-, P-, and N-cadherin are co-expressed in the nasopharyngeal carcinoma cell line TW-039

The cadherin/catenin complex plays a key role in the initiation of cell-cell recognition, and adhesion, and the elaboration of structural and functional organization in multicellular tissues and organs. It is associated with tumor metastasis and also acts as an "invasion suppressor" of cancer cells. Nasopharyngeal carcinoma (NPC) is notorious for its highly metastatic nature. The expression of the E-cadherin/catenin complex is down-regulated in NPC tumor specimens. To obtain better insight into the intercellular adhesive property of NPC cells, we used immunofluorescence microscopy, immunoprecipitation, and immunoblot analysis to examine the expression of the classical cadherins and beta-catenin in a NPC cell line, TW-039. The results demonstrate a change in the distribution of E-cadherin from cytosolic flakes to cell-cell contacts with increasing time in culture. Between days 1 and 5 after plating, the detergent-insoluble fraction of E-cadherin increased from 20% to 37% of total E-cadherin, and that for P-cadherin increased from 33% to 40%. By contrast, the values for beta-catenin remained unchanged (26% and 25%). Both immunofluorescence and immunoblot studies suggested that P-cadherin may be involved in pioneer contact adhesion of TW-039 cells. Interestingly, E-, P-, and N-cadherin are co-expressed in this cell line. Immunoprecipitation studies also showed that other members of the cadherin family may be involved in the contact adhesion of TW-039 cells.     

The relationships among adhesion, stratification and differentiation in keratinocytes

Epidermis is a self-renewing, multilayered tissue composed primarily of keratinocytes. The epidermal keratinocyte follows a terminal differentiation pathway that under normal circumstances is tightly linked to its position within the epidermis and culminates in the formation of the protective barrier (stratum corneum) that constitutes the outermost layer of skin. Strong but pliant adhesive mechanisms are essential for normal functioning of the epidermis. In the epidermis, adhesion is mediated primarily by four structures: hemidesmosomes and focal adhesions, which function in cell-matrix adhesion, and desmosomes and adherens junctions, which function in cell-cell adhesion. In this review we concentrate on the transmembrane components of these structures, which are thought to mediate directly the adhesive function. Members of the integrin family of adhesion molecules comprise the transmembrane components of hemidesmosomes and focal adhesions, although hemidesmosomes also have a second, unrelated transmembrane molecule known as 'bullous pemphigoid antigen 2'. Members of the cadherin family are the transmembrane constituents of desmosomes and adherens junctions. Desmosomes consistently contain two types of cadherins (desmoglein and desmocollin), while adherens junctions may contain only one type of cadherin (E- or P-cadherin). Expression of most of the transmembrane components varies with the position of the keratinocyte within the epidermis and thus may reflect the degree of epidermal differentiation. All of the integrin subunits have been localized predominantly to the basal layer. In contrast, the cadherins show very complex expression patterns throughout the epidermis. Desmogleins and desmocollins (the desmosomal cadherins) are each encoded by three genes, and the expression of each gene is limited to certain epidermal layers. With respect to the cadherins of the adherens junction, it has been shown that E-cadherin is present throughout the epidermis, while P-cadherin is limited to the basal layer. Interestingly, these complex expression patterns of integrins and cadherins within the epidermis may not simply be passive events in differentiation; rather, evidence is accumulating that adhesion molecules can exert a dynamic role in epidermal differentiation/stratification. For example, decreased adhesion to extracellular matrix, induced by changes in one or more integrins, appears to be a signal that induces certain differentiation-related events. Even more profound effects on epidermal morphogenesis have been demonstrated for the cadherins. E- and/or P-cadherin is required not only to initiate normal intercellular junction formation but also for the subsequent development of a stratified epithelium. Thus, the findings to date with both integrins and cadherins suggest that adhesion molecules may function not just as direct mediators of adhesion, but also as regulators of epidermal stratification, differentiation, and morphogenesis.     

E‐, P‐, and N‐cadherin are co‐expressed in the nasopharyngeal carcinoma cell line TW‐039

The cadherin/catenin complex plays a key role in the initiation of cell‐cell recognition, and adhesion, and the elaboration of structural and functional organization in multicellular tissues and organs. It is associated with tumor metastasis and also acts as an “invasion suppressor” of cancer cells. Nasopharyngeal carcinoma (NPC) is notorious for its highly metastatic nature. The expression of the E‐cadherin/catenin complex is down‐regulated in NPC tumor specimens. To obtain better insight into the intercellular adhesive property of NPC cells, we used immunofluorescence microscopy, immunoprecipitation, and immunoblot analysis to examine the expression of the classical cadherins and β‐catenin in a NPC cell line, TW‐039. The results demonstrate a change in the distribution of E‐cadherin from cytosolic flakes to cell‐cell contacts with increasing time in culture. Between days 1 and 5 after plating, the detergent‐insoluble fraction of E‐cadherin increased from 20% to 37% of total E‐cadherin, and that for P‐cadherin increased from 33% to 40%. By contrast, the values for β‐catenin remained unchanged (26% and 25%). Both immunofluorescence and immunoblot studies suggested that P‐cadherin may be involved in pioneer contact adhesion of TW‐039 cells. Interestingly, E‐, P‐, and N‐cadherin are co‐expressed in this cell line. Immunoprecipitation studies also showed that other members of the cadherin family may be involved in the contact adhesion of TW‐039 cells. J. Cell. Biochem. 76:161–172, 1999. © 1999 Wiley‐Liss, Inc.     

Tumor progression: the role of cadherins and integrins

Established cadherin and integrin-containing adherens junctions maintain the integrity of normal tissues. Signalling via adhesion-molecule systems is an important factor in the control of cellular growth and differentiation. In transformed cells, destructive changes in the adhesion systems lead to abnormal relationships among neighbouring cells and the extracellular matrix. Adhesion molecules may prevent tumour progression by firmly attaching cells to each other, and by anchoring them in the extracellular matrix. In addition, cadherins and integrins may have a direct role in tumour suppression by participating in growth control. Dissemination of cancer cells, i.e. invasion and metastasis, requires movement of cells, as well as adhesion to extracellular matrices and to other cells. Particular integrins have been implicated in several aspects of this multistep process. In this article, the data on the possible roles of cadherins and integrins in tumor progression are summarized.     

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg¹-Thr¹⁷⁰) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca²⁺-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca²⁺-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion.