Plakophilin 1 interferes with plakoglobin binding to desmoplakin, yet together with plakoglobin promotes clustering of desmosomal plaque complexes at cell-cell borders.

Desmosomes are adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. These junctions play an important role in providing strength to tissues that experience mechanical stress such as heart and epidermis. The basic structural elements of desmosomes are similar to those of the better-characterized adherens junctions, which anchor actin-containing microfilaments to cadherins at the plasma membrane. This linkage of actin to classic cadherins is thought to occur through an indirect mechanism requiring the associated proteins, alpha- and beta-catenin. In the case of desmosomes, both linear and lateral interactions have been proposed as playing an important role in formation of the plaque and linkage to the cytoskeleton. However, the precise nature of these interactions and how they cooperate in desmosome assembly are poorly understood. Here we employ a reconstitution system to examine the assembly of macromolecular complexes from components found in desmosomes of the differentiated layers of complex tissues. We demonstrate the existence of a Triton-soluble complex of proteins containing full length desmoplakin (DP), the arm protein plakoglobin, and the cytoplasmic domain of the desmosomal cadherin, desmoglein 1 (Dsg1). In addition, full length DP, but not an N-terminal plakoglobin binding domain of DP, co-immunoprecipitated with the Dsg1 tail in the absence of plakoglobin in HT1080 cells. The relative roles of the arm proteins plakoglobin and plakophilin 1 (PKP1) were also investigated. Our results suggest that, in the Triton soluble pool, PKP1 interferes with binding of plakoglobin to full length DP when these proteins are co-expressed. Nevertheless, both plakoglobin and PKP1 are required for the formation of clustered structures containing DP and the Dsg1 tail that ultrastructurally appear similar to desmosomal plaques found in the epidermis. These findings suggest that more than one armadillo family member is required for normal assembly and clustering of the desmosomal plaque in the upper layers of the epidermis.

The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. Implications for cutaneous disease.

The contribution of desmosomes to epidermal integrity is evident in the inherited blistering disorder associated with the absence of a functional gene for plakophilin-1. To define the function of plakophilin-1 in desmosome assembly, interactions among the desmosomal cadherins, desmoplakin, and the armadillo family members plakoglobin and plakophilin-1 were examined. In transient expression assays, plakophilin-1 formed complexes with a desmoplakin amino-terminal domain and enhanced its recruitment to cell-cell borders; this recruitment was not dependent on the equimolar expression of desmosomal cadherins. In contrast to desmoplakin-plakoglobin interactions, the interaction between desmoplakin and plakophilin-1 was not mediated by the armadillo repeat domain of plakophilin-1 but by the non-armadillo head domain, as assessed by yeast two-hybrid and recruitment assays. We propose a model whereby plakoglobin serves as a linker between the cadherins and desmoplakin, whereas plakophilin-1 enhances lateral interactions between desmoplakin molecules. This model suggests that epidermal lesions in patients lacking plakophilin-1 are a consequence of the loss of integrity resulting from a decrease in binding sites for desmoplakin and intermediate filaments at desmosomes.

Desmosomes: intercellular adhesive junctions specialized for attachment of intermediate filaments.

Cell-cell adhesion is thought to play important roles in development, in tissue morphogenesis, and in the regulation of cell migration and proliferation. Desmosomes are adhesive intercellular junctions that anchor the intermediate filament network to the plasma membrane. By functioning both as an adhesive complex and as a cell-surface attachment site for intermediate filaments, desmosomes integrate the intermediate filament cytoskeleton between cells and play an important role in maintaining tissue integrity. Recent observations indicate that tissue integrity is severely compromised in autoimmune and genetic diseases in which the function of desmosomal molecules is impaired. In addition, the structure and function of many of the desmosomal molecules have been determined, and a number of the molecular interactions between desmosomal proteins have now been elucidated. Finally, the molecular constituents of desmosomes and other adhesive complexes are now known to function not only in cell adhesion, but also in the transduction of intracellular signals that regulate cell behavior.

VE-cadherin and desmoplakin are assembled into dermal microvascular endothelial intercellular junctions: a pivotal role for plakoglobin in the recruitment of desmoplakin to intercellular junctions.

Vascular endothelial cells assemble adhesive intercellular junctions comprising a unique cadherin, VE-cadherin, which is coupled to the actin cytoskeleton through cytoplasmic interactions with plakoglobin, beta-catenin and alpha -catenin. However, the potential linkage between VE-cadherin and the vimentin intermediate filament cytoskeleton is not well characterized. Recent evidence indicates that lymphatic and vascular endothelial cells express desmoplakin, a cytoplasmic desmosomal protein that attaches intermediate filaments to the plasma membrane in epithelial cells. In the present study, desmoplakin was localized to intercellular junctions in human dermal microvascular endothelial cells. To determine if VE-cadherin could associate with desmoplakin, VE-cadherin, plakoglobin, and a desmoplakin amino-terminal polypeptide (DP-NTP) were co-expressed in L-cell fibroblasts. In the presence of VE-cadherin, both plakoglobin and DP-NTP were recruited to cell-cell borders. Interestingly, beta-catenin could not substitute for plakoglobin in the recruitment of DP-NTP to cell borders, and DP-NTP bound to plakoglobin but not beta-catenin in the yeast two-hybrid system. In addition, DP-NTP colocalized at cell-cell borders with alpha-catenin in the L-cell lines, and endogenous desmoplakin and alpha-catenin colocalized in cultured dermal microvascular endothelial cells. This is in striking contrast to epithelial cells, where desmoplakin and alpha -+catenin are restricted to desmosomes and adherens junctions, respectively. These results suggest that endothelial cells assemble unique junctional complexes that couple VE-cadherin to both the actin and intermediate filament cytoskeleton.

The expression of desmoglein isoforms in cultured human keratinocytes is regulated by calcium, serum, and protein kinase C.

Three desmoglein (Dsg) isoforms are expressed in a differentiation-specific fashion in the epidermis, with Dsg2 being basal, Dsg3 (pemphigus vulgaris antigen) basal and spinous, and Dsg1 (pemphigus foliaceus antigen) predominantly granular. To better understand the mechanism(s) regulating Dsg isoform expression, we examined the expression pattern of Dsg1, Dsg2, and Dsg3 in normal human epidermal keratinocytes (NHEKs), the immortalized, nontumorigenic HaCaT cell line, and several squamous cell carcinoma cell lines (SCC-9, SCC-12F, SCC-13, and SCC-25). In all cells, the accumulation of high Dsg protein levels required calcium and was not observed in low calcium (0.05-0.07 mM) media. NHEKs expressed Dsg1 in all media tested, consistent with their normal differentiation capacity. HaCaT and SCC-25 also expressed Dsg1; however, the presence of serum in the media dramatically decreased Dsg1 protein levels. Serum also inhibited Dsg1 mRNA levels in HaCaT cells. Dsg1 was not detected in extracts from SCC-9, SCC-12F, and SCC-13 under any conditions. Since activation of protein kinase C (PKC) is involved in keratinocyte differentiation, we evaluated the effects of PKC down-regulation on Dsg isoform expression. Long-term treatment with either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or bryostatin 1 inhibited levels of Dsg1 and Dsg3, but not Dsg2 in NHEKs and HaCaT cells. Chronic TPA also decreased Dsg1 and Dsg3 mRNA levels in NHEKs, further supporting a role for PKC activation in the expression of the suprabasal Dsg1 and Dsg3. These results identify several regulatory mechanisms by which the differentiation-specific pattern of desmosomal cadherins is established in the epidermis.

The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell-cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell-cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin-plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

Localization of fibronectin matrix assembly sites on fibroblasts and endothelial cells.

Polymerization of soluble fibronectin into extracellular matrix fibers occurs through the interaction between the amino terminus of fibronectin contained within a 70 kDa fragment and 'matrix assembly sites' on the cell surface. The present studies were performed to localize the 'matrix assembly sites' (defined by 70 kDa binding sites) on newly adherent cells and on cells containing preformed fibronectin matrix. Matrix nucleation sites on newly spread cells were visualized using Texas Red conjugated 70 kDa fragment and were found to colocalize with vinculin and substrate fibronectin fibrils. Cells plated onto vitronectin coated coverslips did not exhibit any 70 kDa binding sites although these cells were well-spread with fully developed focal adhesions. Time course studies indicated that 70 kDa binding sites could be detected on newly adherent cells within 30-40 minutes following cell plating onto fibronectin coated coverslips, prior to the reorganization of substrate fibronectin into fibrils. Similarly, exogenous fibronectin conjugated with Texas Red was also colocalized with vinculin when added to newly adherent cells. The disruption of actin filaments with cytochalasin D both prevented the expression of 70 kDa binding sites and also resulted in the loss of established 70 kDa binding sites on newly spread cells. After 3 days in culture, cells organized an extensive fibronectin matrix and 70 kDa was colocalized with two distinct types of matrix fibronectin fibers: fine linear cell-associated fibers which co-stained with the beta1 integrin and coarse extracellular fibers which did not stain for the beta1 integrin. There was also a third type of fibronectin fiber which was organized into a meshwork structure. There was no localization of either beta1 or 70 kDa to these structures. Treatment of 3-day cells with cytochalasin D resulted in the disruption of cell-matrix fibers and cell-associated 70 kDa binding sites. In contrast, the coarse extracellular matrix fibers as well as the meshwork fibers were unaffected by cytochalasin. In the presence of cytochalasin D, 70 kDa bound to sites which colocalized with the coarse extracellular matrix fibers. These data suggest that de novo assembly of fibronectin matrix occurs at sites of focal adhesion and as fibronectin polymerization proceeds, matrix nucleation sites colocalize along cell associated fibronectin fibers. At later times 70 kDa is localized to a subset of more mature fibronectin-containing fibers. These results suggest that there are at least three morphologically distinct 70 kDa binding sites on adherent cells: one which colocalizes with beta1 to focal adhesions, a second which colocalizes with beta1 and fibronectin in matrix contacts, and a third which localizes to extracellular matrix fibers.

Analysis of desmosomal cadherin-adhesive function and stoichiometry of desmosomal cadherin-plakoglobin complexes.

Desmosomes are intercellular adhesive junctions that associate with the intermediate filament cytoskeleton. The two major classes of transmembrane desmosomal glycoproteins, desmogleins and desmocollins, are widely considered to function as adhesion molecules. This assumption is based in part on their homology to the cadherin family of calcium-dependent homophilic adhesion molecules. In addition, autoantibodies from pemphigus patients bind directly to desmoglein family members and are thought to cause epidermal blistering by inhibiting the function of these cadherins. To directly test the ability of the desmosomal cadherins to mediate adhesion, desmoglein-1 (Dsg1), desmocollin-2 (Dsc2a) and plakoglobin were expressed in mouse L cell fibroblasts. Similar to catenin:classical cadherin complexes, plakoglobin:Dsc2a complexes exhibited an approximately 1:1 stoichiometry; however, plakoglobin:Dsg1 complexes exhibited a 6:1 stoichiometry. When L cells expressing the desmosomal cadherins were tested for the ability to aggregate in suspension, L cells expressing E-cadherin exhibited extensive aggregation, but L cells expressing Dsg1 or Dsc2a did not aggregate. In addition, L cells co-expressing Dsg1, Dsc2a, and plakoglobin failed to aggregate. The cytoplasmic domain of E-cadherin is thought to play a central role in the adhesive function of E-cadherin by providing a link to the actin cytoskeleton. Therefore, two chimeric cadherins comprising the cytoplasmic domain of E-cadherin and the extracellular domain of either Dsg1 or Dsc2a were expressed in L cells. Both chimeras formed a complex with alpha- and beta-catenin. Nevertheless, neither of these chimeras supported aggregation of L cells when expressed individually or when co-expressed. These data suggest that the extracellular domains of the desmosomal cadherins exhibit functional properties distinct from those of the classical cadherins, such as E-cadherin.

Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1.

To identify regions of the Desmoglein-1 (Dsg1) extracellular domain that are targeted by pemphigus antisera, cDNA sequences encoding various regions of the Dsg1 extracellular domain were ligated to sequences encoding the E-cadherin extracellular anchor and transmembrane and cytoplasmic domains. These constructs were then expressed in mammalian cells, and pemphigus sera were tested for the ability to recognize the Dsg1 extracellular domains. When analyzed by immunoblot, very few pemphigus foliaceus sera recognized the Dsg1 domains. To determine whether pemphigus sera recognize non-denatured Dsg1 domains, constructs were expressed in cultured cells and tested for reactivity with pemphigus sera using live-cell immunofluorescence. The pemphigus foliaceus sera reacted strongly with the Dsg1 extracellular domain by live-cell immunofluorescence and recognized predominantly the amino-terminal region of the Dsg1 extracellular domain. In addition, sera from patients with pemphigus vulgaris also demonstrated strong reactivity with the Dsg1 extracellular domain when tested using live-cell immunofluorescence. In contrast, sera from normal human controls and sera from bullous pemphigoid patients did not react with the Dsg1 extracellular domain. These data indicate that both pemphigus foliaceus and pemphigus vulgaris sera react with conformationally sensitive epitopes in the amino-terminal region of Dsg1.

Posttranslational regulation of plakoglobin expression. Influence of the desmosomal cadherins on plakoglobin metabolic stability.

Desmosomes are adhesive intercellular junctions that act as cell surface attachment sites for intermediate filaments. The desmosomal glycoproteins, desmogleins and desmocollins, are members of the cadherin family of adhesion molecules. In addition, desmoglein has been shown to coimmunoprecipitate with the junctional protein plakoglobin. To characterize further the interaction between plakoglobin and the desmosomal cadherins, stable mouse fibroblast (L-cells) cell lines were generated that express plakoglobin, desmoglein and plakoglobin, or desmocollin and plakoglobin. L-cell lines transfected with a plasmid encoding human plakoglobin expressed plakoglobin mRNA but very little plakoglobin protein. However, plakoglobin protein was expressed at high levels in L-cells coexpressing either desmoglein or desmocollin. In addition, both desmocollin and desmoglein were found to coimmunoprecipitate with plakoglobin. The transient expression of desmoglein in L-cell lines expressing plakoglobin mRNA resulted in the formation of a complex between plakoglobin and desmoglein and in the accumulation of plakoglobin protein. Furthermore, the rate of plakoglobin protein degradation was decreased by 15-20-fold in cell lines expressing either desmoglein or desmocollin. These results demonstrate that the desmosomal cadherins posttranslationally regulate plakoglobin expression by decreasing the rate of plakoglobin degradation.

Structure and function of desmosomal transmembrane core and plaque molecules.

Desmosomes are intercellular junctions that function in cell-cell adhesion and attachment of intermediate filaments (IF) to the cell surface. Desmogleins and desmocollins are the major components of the transmembrane adhesion complex, whereas desmoplakins (DPs) are the most prominent components of the cytoplasmic plaque. Based on sequence similarity, desmogleins and desmocollins are related to the calcium-dependent homophilic adhesion molecules known as cadherins. Like the classical cadherins, the desmosomal cadherins contain four homologous extracellular domains bearing putative calcium-binding sites, a single transmembrane spanning domain, and a C-terminal cytoplasmic tail. Molecules in the desmoglein subclass contain a unique C-terminal extension within which is found a repeating motif that is predicted to form two beta-strands and two turns. Stable cell lines expressing desmoglein 1 have been generated from normally non-adherent L cell fibroblasts, to study the contribution of this cadherin to desmosomal adhesion. The predicted sequence of desmoplakin (DP) I suggests it will form homodimers comprising a central alpha-helical coiled-coil rod and two globular end domains. The C-terminus contains three regions with significant homology, each of which is made up of a 38-residue motif also found in two other molecules involved in organization of IF, bullous pemphigoid antigen and plectin. Ectopically expressed polypeptides including the C-terminus of DP I specifically align with keratin and vimentin IF in cultured cells, whereas those lacking this domain do not align with IF. The last 68 amino acids of DP are required for alignment along keratin but not vimentin IF, and residues 48-68 from the C-terminal end are critical for this interaction. These results suggest that the C-terminus of DP plays a role in the attachment of IF to the desmosome and that a specific site is necessary for interaction with keratin IF. A sequence at the most N-terminal end of DP appears to be required for efficient incorporation into the desmosomal plaque. Interestingly, this region has not been reported to be present in the homologous bullous pemphigoid antigen or plectin molecules and may represent a desmosomal targeting sequence.

Basolateral distribution of fibronectin matrix assembly sites on vascular endothelial monolayers is regulated by substratum fibronectin.

Endothelial cells exhibit binding sites for the amino terminus of fibronectin that participate in subendothelial fibronectin matrix assembly. These binding sites, termed matrix assembly sites, are localized on the basolateral surface of confluent endothelial monolayers (Kowalczyk et al. Blood, 75:2335, 1990). The present study investigates the role of cell-cell and cell-substratum interactions in the localization of matrix assembly sites to the basal surface of endothelial cells. Cells were cultured in Transwell culture inserts and matrix assembly sites were detected by binding assays using an iodinated 70 Kd amino-terminal fibronectin fragment. Integrity of intercellular junctions was monitored by measuring protein flux across Transwell filters. Time course experiments demonstrated that matrix assembly site expression on the basolateral cell surface preceded intercellular junction formation. Transfer of confluent monolayers to calcium-free medium resulted in the loss of junctions and in an increase in 125I-70 kD binding from the apical medium. The increased 125I-70 kD binding resulted from increased access of 125I-70 kD to basolateral matrix assembly sites and not from the relocation of binding sites to the apical membrane. To determine the effect of matrix composition on matrix assembly site expression and localization, cells were seeded onto vitronectin- or fibronectin-coated substrates. Fibronectin increased the expression of matrix assembly sites on the apical surface within 24 hours. By 48 hours, matrix assembly sites were located only on the basolateral surface. Vitronectin had no effect on the expression or localization of matrix assembly sites. These results indicate that the expression and localization of matrix assembly sites on the surface of vascular endothelial cells can be regulated by substratum fibronectin.

Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation.

Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I-fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.

Eosinophilic pustular folliculitis.

Eosinophilic pustular folliculitis was first described by Ofuji et al in 1970. It is characterized by pruritic circinate plaques that are studded with follicular papules and pustules. Lesions are located chiefly on the face, trunk, and arms. Biopsies of lesions demonstrate an infiltrate of eosinophils and neutrophils within hair follicles, dermis, and epidermis. Peripheral leukocytosis and eosinophilia are common.

Blepharochalasis syndrome.

Patients with chronic forms of blepharochalasis often develop eyelid deformities characterized by blepharoptosis and prolapse of the orbital fat and lacrimal gland. Some individuals have an acquired form of blepharophimosis, secondary to the dehiscence of the canthal tendons. In this late stage of the condition, the tendons still adhere to the periosteum of the orbital rims and loss of fixation occurs at the distal attachment between the tendons and the eyelid tissues. This results in a horizontally shortened palpebral fissure and a rounded deformity of the lateral canthal angle. Surgery remains the primary treatment.

Pilomatrix carcinoma with pulmonary metastasis. Report of a case.

A 67-year-old white man developed a locally recurrent pilomatrix carcinoma of the back. Within a 4-year period bilateral pulmonary metastases developed. This to the knowledge of the authors is the first reported case of a metastasizing pilomatrix carcinoma in the medical literature.