Reciprocal altered expression of T-cadherin and P-cadherin in psoriasis vulgaris.

BACKGROUND: The most characteristic change in psoriasis vulgaris is markedly increased, persistent keratinocyte proliferation. The underlying mechanism of excessive epidermal growth is controversial. We previously found and reported that T-cadherin was expressed in keratinocytes and confined to the basal layer of mouse and human skin. Invasive cutaneous squamous cell carcinoma showed a loss of T-cadherin expression. Another study showed that T-cadherin was a negative growth regulator of epidermal growth factor in T-cadherin transfectant neuroblastoma cells. OBJECTIVES: To obtain insight into the role of T-cadherin in keratinocyte proliferation and to investigate further the pathogenesis of psoriasis vulgaris, we examined the expression of T-cadherin, as well as E- and P-cadherin, in psoriasis vulgaris. METHODS: Four untreated active psoriatic skin samples from psoriasis vulgaris patients and four normal human skin samples from plastic surgery were collected, cryosectioned and immunohistochemically stained by antihuman T-, P- and E-cadherin antibodies. Further, the immunofluorescence intensities of T- and P-cadherin on the basal layer of the epidermis were quantitatively measured by the histogram function of LSM 510 software installed in a Zeiss laser scanning confocal microscope. The data were statistically analysed by Student's t-test. RESULTS: It was observed that T-cadherin was weakly and discontinuously expressed on the basal layer of psoriatic skin, while it was intensively expressed on all basal keratinocytes in normal human skin. In contrast, P-cadherin was strongly expressed throughout the entire epidermal layer in psoriatic skin samples, although its expression is restricted to the basal cell layer in normal human skin. There were no obvious differences in E-cadherin expression between normal human skin and psoriatic skin. Statistical analyses showed that the immunofluorescence intensity of T-cadherin in the basal cell layer of psoriatic skin (35 +/- 9.08) was significantly decreased compared with that in normal human skin (131.75 +/- 3.49, P = 2.46 x 10(-6)). There was a significant increase (P = 0.00139) in the immunofluorescence intensity of P-cadherin in the basal layer of psoriatic skin (68.25 +/- 12.13) compared with normal human skin (26 +/- 4.90). CONCLUSIONS: The present study demonstrates that there is downregulation of T-cadherin expression and upregulation of P-cadherin expression in psoriatic skin, which are considered to be involved in the hyperproliferation of keratinocytes in psoriasis vulgaris.

The expression of nectin-1alpha in normal human skin and various skin tumours.

BACKGROUND: A novel cell-cell adhesion system that consists of nectin and afadin has been identified at cadherin-based cell-cell adherens junctions. Nectin is a Ca2+-independent homophilic and heterophilic cell adhesion molecule that belongs to the immunoglobulin superfamily. Nectin has recently been shown to serve as an alpha-herpesvirus entry and cell-cell spread mediator. In spite of the ubiquitous expression of nectin-1alpha, its detailed localization in human skin has not been examined so far. OBJECTIVES: To investigate the localization of nectin-1alpha in normal human skin and the alteration of its expression in malignant skin tumours. METHODS: Immunohistochemistry was employed to determine the expression of nectin-1alpha and other adhesion molecules. RESULTS: We detected nectin-1alpha in normal human epidermis, follicles and eccrine ducts. Nectin-1alpha was colocalized with E-cadherin at cell-cell adherens junctions of the epidermis. The concentration of the nectin-afadin system at cell-cell adherens junctions was reduced in the early stage of malignant transformation of keratinocytes, such as in basal cell carcinomas and squamous cell carcinomas, where the cadherin-catenin system was preserved. Nectin-1alpha at cell-cell adherens junctions was reduced in human epithelial cancer cells located at the advancing border of the tumour. CONCLUSIONS: Our results showed that nectin-1alpha is located at cell-cell adherens junctions in human skin and that reduction of nectin-1alpha at cell-cell adherens junctions may be involved in the invasion of squamous cell tumours.

Desmoglein isoform distribution affects stratum corneum structure and function.

Desmogleins are desmosomal cadherins that mediate cell-cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.

Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.

Exfoliative toxin A, produced by Staphylococcus aureus, causes blisters in bullous impetigo and its more generalized form, staphylococcal scalded-skin syndrome. The toxin shows exquisite specificity in causing loss of cell adhesion only in the superficial epidermis. Although exfoliative toxin A has the structure of a serine protease, a target protein has not been identified. Desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-cell adhesion, may be the target of exfoliative toxin A, because it is the target of autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue specificity and histology. We show here that exfoliative toxin A cleaved mouse and human Dsg1, but not closely related cadherins such as Dsg3. We demonstrate this specific cleavage in cell culture, in neonatal mouse skin and with recombinant Dsg1, and conclude that Dsg1 is the specific receptor for exfoliative toxin A cleavage. This unique proteolytic attack on the desmosome causes a blister just below the stratum corneum, which forms the epidermal barrier, presumably allowing the bacteria in bullous impetigo to proliferate and spread beneath this barrier.

Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1(+) cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34(-) to VE-cadherin+ PECAM-1(+) CD34(+) stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1(+) cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

Drug eruption due to Bufferin showing erythema exsudativum multiforme with a photo-recall-like phenomenon.

A 21-year-old woman who had been taking several kinds of analgesics to treat dysmenorrhea developed episodic attacks of a purpuric macular eruption and a burning sensation on unexposed areas of the upper chest and back where she had sustained severe sunburn eight months earlier. Target-like lesions developed on these areas after intake of Bufferin, a combination of aspirin and dialuminate. After the eruptions had abated following systemic administration of a corticosteroid agent, a challenge test was performed, using a quarter of a tablet of Bufferin. The patient developed a temporary burning sensation and a erythematous color on the previously sunburned skin. We diagnosed this case as a drug eruption due to Bufferin showing erythema exsudativium multiforme with a photo-recall-like phenomenon. In our case, skin tests would be useful to confirm the causal drug.

Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.

Totipotent murine ES cells have an enormous potential for the study of cell specification. Here we demonstrate that ES cells can differentiate to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm from other cell lineages was critical for hemopoietic cell differentiation, whereas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monitored easily in real time, this culture system will greatly facilitate the study of the mechanisms involved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the culture of ES cells, however, lineages and stages of differentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF receptor(alpha), VE-cadherin, CD45 and Ter119 was sufficient to define most intermediate stages during differentiation of ES cells to blood cells. Using this culture system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1-PDGFRalpha-), proximal lateral mesoderm (E-cadherin-Flk1+VE-cadherin-), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45-), hemopoietic progenitor (CD45+c-Kit+) and mature blood cells (c-Kit-CD45+ or Ter119+), though direct differentiation of blood cells from the Flk1+VE-cadherin- stage cannot be ruled out. Not only the VE-cadherin+CD45- population generated from ES cells but also those directly sorted from the yolk sac of 9.5 dpc embryos have a potential to give rise to hemopoietic cells. Progenitors with hemoangiogenic potential were identified in both the Flk1+VE-cadherin- and Flk1+VE-cadherin+ populations by the single cell deposition experiment. This line of evidence implicates Flk1+VE-cadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.

Roles of E- and P-cadherin in the human skin.

The Ca(2+)-dependent cell-cell adhesion molecules, termed cadherins, are subdivided into several subclasses. E (epithelial)- and P (placental)-cadherins are involved in the selective adhesion of epidermal cells. E-cadherin is expressed on the cell surfaces of all epidermal layers and P-cadherin is expressed only on the surfaces of basal cells. Ultrastructural studies have shown that E-cadherin is distributed on the plasma membranes of keratinocytes with a condensation in the intercellular space of the desmosomes. During human skin development P-cadherin expression is spatiotemporally controlled and closely related to the segregation of basal layers as well as to the arrangement of epidermal cells into eccrine ducts. In human skin diseases E-cadherin expression is markedly reduced on the acantholytic cells of tissues in pemphigus and Darier's disease. Cell adhesion molecules are now considered to play a significant role in the cellular connections of cancer and metastatic cells. Reduced expression of E-cadherin on invasive neoplastic cells has been demonstrated for cancers of the stomach, liver, breast, and several other organs. This reduced or unstable expression of E- and P-cadherin is observed in squamous cell carcinoma, malignant melanoma, and Paget's disease, but cadherin expression is conserved in basal cell carcinoma. Keratinocytes cultured in high calcium produce much more intense immunofluorescence of intercellular E- and P-cadherin than those cells grown in low calcium. E-cadherins on the plasma membrane of the keratinocytes are shifted to desmosomes under physiological conditions, and therein may express an adhesion function in association with other desmosomal cadherins. Soluble E-cadherins in sera are elevated in various skin diseases including bullous pemphigoid, pemphigus vulgaris, and psoriasis, but not in patients with burns. Markedly high levels in soluble E-cadherin are demonstrated in patients with metastatic cancers.

In vivo evidence of the critical role of cadherin-5 in murine vascular integrity.

Vascular endothelial cell-cell adhesion is crucial for the regulation of vascular functions and is associated with many circulatory disorders. We isolated a rat monoclonal antibody (VECD1) recognizing the mouse vascular endothelial cell adhesion molecule and found that it inhibited vascular endothelial cell-cell association. We sequenced a full-length cDNA of the antigen that was identical to mouse cadherin-5. L-cells transfected with its cDNA acquired cell-cell adhesiveness, and these transfectants reacted with VECD1 at cell-cell contact areas. We studied the role of mouse cadherin-5 in vascular functions. The addition of VECD1 antibody to a cultured vascular endothelial cell line (F-2) caused the detachment of each cell. Although normal F-2 cells formed tubular structures on Matrigel, VECD1 disturbed the tubulogenesis. VECD1 also increased the permeability through the F-2 cell layer. To clarify the in vivo function of mouse cadherin-5, we intraperitoneally injected the hybridomas producing VECD1 into adult mice. Severe venous stasis and subcutaneous hemorrhage were induced within several days after the injection, resulting in the early death of the animals. These findings are evidence of an essential role of cadherin-5 in the regulation of vascular endothelial cell-cell adhesion in vivo.

Multiple cadherins are expressed in human fibroblasts.

Although the cell-cell adhesiveness of fibroblasts is thought to be related to wound healing, the molecular basis of this adhesiveness is still unknown. We isolated five kinds of cadherin fragments from the cDNA of human fibroblasts by polymerase chain reaction (PCR). Two of the five were known cadherins: PC43, a protocadherin containing six cadherin repeats in the extracelluar domain, and human Fat, which is the human homologue of the Drosophila tumor suppressor Fat. The other three were novel cadherin fragments, and we named them cadherins FIB1, FIB2, and FIB3. The expressions of cadherins including E-, P-, and N-cadherin, PC43, human Fat, and cadherins FIB1, FIB2, and FIB3 were compared in human fibroblasts, human melanocytes, and human epidermal keratinocytes. The latter six cadherins were expressed in human fibroblasts, and cadherins FIB1 and FIB2 were fibroblast-specific. These results suggest that diverse cadherin molecules may contribute to the cell-cell adhesion in human fibroblasts.

Identification of novel cadherins expressed in human melanoma cells.

Cadherin molecules are essential for tissue morphogenesis and are also related to cancer invasion and metastasis. Although normal melanocytes express E- and P-cadherin, the activity and expression of E- and P-cadherin in melanoma cells are still unknown. We measured the homophilic adhesion activity of human normal epidermal melanocytes and the melanoma cell lines MeWo and A375. The melanoma cells showed stronger homophilic adhesion activity than did the melanocytes, despite the lower expression of E- and P-cadherin in the melanoma cells. This result suggested that melanoma cells expressed other types of homophilic adhesion molecules. Using degenerate primers to amplify multiple cadherin subtypes, we performed a polymerase chain reaction (PCR) with the first strand of cDNAs generated by reverse transcription of the mRNAs of the melanoma cells, and we isolated two known cadherin fragments, N-cadherin and PC42, and six novel cadherin fragments, cadherins ME1-ME6. The reverse transcriptase-PCR using specific primers of cadherins including E-, P-, and N-cadherins, PC42, and cadherins ME1-ME6 revealed that the melanoma cells expressed more kinds of cadherin molecules than did the melanocytes. Such cadherins may play an important role in melanoma cell-cell adhesion.

Werner's syndrome--chromosome analyses of cultured fibroblasts and mitogen-stimulated lymphocytes.

Two cases of Werner's syndrome are reported. Fibroblasts derived from both patients revealed reduced population doubling numbers. Chromosomal analyses for fibroblasts from both patients and lymphocytes from one patient revealed that chromosomal aberrations occur frequently and randomly. Although some of the chromosomal aberrations involved sites where tumour suppressor genes have been mapped, neither of our patients demonstrated malignancy. Chromosomal aberration at one critical site may not be sufficient to induce cancer or additional factors may be necessary.

Foot ulcer due to arteriovenous malformation: report of a case.

A 41-year-old woman had erosive eruptions surrounded by irregularly shaped pigmentation on the lateral aspect of her right foot, where she had noted gradually increasing warmth and pain for 10 years. The eruptions waxed and waned without complete healing, and an ulcer which had formed one year previously did not respond to topical treatments. Arteriography performed on the right lower extremity disclosed multiple diffuse arteriovenous malformations in the right lower leg and foot. The ulcer was treated by bed rest, surgical debridement, and topical application of bucladesine sodium ointment. After three months, the ulcer healed, leaving a shallow scar and pigmentation.

Establishment and characterization of a novel in vitro angiogenesis model using a microvascular endothelial cell line, F-2C, cultured in chemically defined medium.

The behavior of vascular endothelial cells (EC) is an important factor in the processes involved in angiogenesis, but the regulatory mechanisms of angiogenesis, especially underlying the tubulogenesis by EC are not yet clear. Although a number of in vitro experimental models of tubulogenesis have been developed by use of cultured EC, most of those models are too complex to be easily handled and further, the culture media are usually supplemented with serum, creating problems in interpretation of experimental results. To generate a simple in vitro angiogenesis study model under serum-free culture conditions, we adapted a murine microvascular endothelial cell line, F-2, to a chemically defined medium, Cos Medium 001, and successfully established a subline of F-2, designated F-2C, which revealed a unique growth pattern. In Cos Medium 001, F-2C proliferates in a cobblestone pattern at an early growth stage, but, at a late growth stage, spontaneously differentiates to form three-dimensional honeycomblike tubular structures without the supplementation of any specific factors. The cell aggregation activity of F-2C in the presence of Ca2+ was much greater than that of F-2. The amount of subendothelial matrix deposited by F-2C was significantly higher than that by F-2, and increased prominently after the F-2C cells reached the differentiating stage of tubulogenesis. These findings indicate that F-2C is a new EC line in which tubulogenesis is spontaneously induced by the marked deposition of basement membrane analog to the subendothelial matrix and by the enhancement of presumable cadherin activity. We suggest that this cell line, F-2C, represents a simple and useful in vitro angiogenesis model.

Keratin 9 point mutation in the pedigree of epidermolytic hereditary palmoplantar keratoderma perturbs keratin intermediate filament network formation.

Keratins form an intracellular keratin filament network in keratinocytes. Point mutations in the epidermal keratins could lead to the disruption of keratin filament formation, developing skin diseases such as epidermolytic hereditary palmoplantar keratoderma (EHPPK). We found a G to A transition in keratin 9 (K9) cDNA, resulting in the substitution of glutamine for arginine at 162, in all patients of a pedigree of EHPPK. Transfection into MDCK cells and DJM-1 cells revealed that the plasmid CMX vector containing normal keratin 9 cDNA showed normal keratin network formation, whereas the vector with a G to A point mutated keratin 9 cDNA showed disrupted keratin filaments with droplet formation in the cells. These results indicate that the point mutation seen in our patients had a dominant-negative effect on keratin network formation.

An acquired bullous dermatosis due to an autoimmune reaction against uncein.

A 59-year-old male showed acquired, mechanically induced, scarring blisters on the fingers, toes, scalp and abdomen, as well as in the oral cavity. Ultrastructural and immunohistochemical examination of the bullae revealed junctional epidermal-dermal separation and IgG deposits in the lamina lucida of the basement membrane zone (BMZ), where the reactivity of the 19-DEJ-1 monoclonal antibody was decreased. Anti-BMZ autoantibodies detected in his serum were reactive to the lower lamina lucida region of normal human skin. SDS-PAGE of affinity purified antigens from human keratinocytes with IgG from the patient's serum revealed three polypeptide bands at 165, 135 and 100 kDa, in reduced condition. The indirect immunofluorescence test of his serum was negative on skin cryosections from patients with lethal junctional epidermolysis bullosa. Pretreatment of normal human skin sections with the patient's serum, blocked the binding of 19-DEJ-1 monoclonal antibody but not that of the GB3 monoclonal antibody. This case is considered to be an acquired autoimmune bullous dermatosis due to an autoantibody reaction against uncein (19-DEJ-1 antigen), a component of anchoring filaments.

Ligand activation of overexpressed epidermal growth factor receptor results in colony dissociation and disturbed E-cadherin function in HSC-1 human cutaneous squamous carcinoma cells.

Various types of tumors show aberrant expression and overexpression of epidermal growth factor (EGF) receptor and the degree of receptor expression correlates with a malignant phenotype in many epithelial tumors. However, in vitro evidence supporting the advantageous role of receptor overexpression is deficient. In this study, we compared the effects of exogenous EGF on the cell colony morphology in monolayer and collagen gel culture between HSC-1 squamous carcinoma cells overexpressing EGF receptor and their revertant subline cells. These cells formed coherent cell colonies under routine culture conditions, but addition of EGF induced dissociation of cell colonies within 24 h in the parent HSC-1 cells, though not in the subline cells. Since the colony dissociation apparently involved loss of cell-cell adhesion, we also studied the effects of EGF on E-cadherin expression and its function. Cell aggregation assays showed that EGF reduced E-cadherin function dose-dependently in the parent cells, but not in the subline cells. However, immunoblotting analysis and ELISA showed the absence of downregulation or degradation of E-cadherin. Instead, EGF tyrosine phosphorylated cadherin/catenin complex components including beta-catenin and increased the detergent solubility of E-cadherin in the parent cells. These results suggest that EGF modified the functional association between E-cadherin and actin filament through tyrosine phosphorylation of the cadherin/catenin complex and thereby made the adhesion molecule incompetent. Our results indicate that the ligand activation of overexpressed EGF receptor impairs E-cadherin-mediated cell-cell adhesion and causes dissociation of the squamous carcinoma cell colonies, which facilitates tumor cell invasion in vivo. This might be relevant to the advantageous role of EGF receptor overexpression in malignant phenotype of epithelial tumor cells.

Bilateral inguinal scrofuloderma during steroid therapy in a patient with bullous pemphigoid.

This report described a case of scrofuloderma that developed in the bilateral inguinal regions during treatment of bullous pemphigoid with systemic corticosteroid. Analysis of the literature on scrofuloderma between 1978-1993 disclosed that the number of cases with extracervical involvement are increasing. Immunosuppression could disseminate tuberculous focuses, resulting in extracervical involvement of SD connected with the underlying extrapulmonary tuberculous lesions.