MICROSCOPY RESEARCH AND TECHNIQUE 38:343–352 (1997) Roles of E- and P-Cadherin in the Human Skin FUKUMI FURUKAWA,1* KIMIO FUJII,2 YUJI HORIGUCHI,2 NORIHISA MATSUYOSHI,2 MAYUMI FUJITA,2 KEN-ICHI TODA,2 SADAO IMAMURA,2 HISASHI WAKITA,1 SHIGEHO SHIRAHAMA,1 AND MASAHIRO TAKIGAWA1 1Department 2Department of Dermatology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431–31, Japan of Dermatology, Graduate School of Medicine, Kyoto University, Shogoin, Sakyo, Kyoto 606 Japan KEY WORDS cadherin; soluble E-cadherin; cell adhesion molecules; cancer; pemphigus; cell culture ABSTRACT The Ca21-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. Microsc. Res. Tech. 38:343–352, 1997. r 1997 Wiley-Liss, Inc. INTRODUCTION Cell adhesion mechanisms play a major role in vital processes such as embryogenesis, tissue and organ pattern formation, and maintenance of specific tissue architecture. Alternations in cell adhesion molecules (CAM) have been implicated in the loss of control of cell proliferation and neoplasia. Various classes of cell-cell adhesion molecules are differentially expressed in the skin during development and epidermal cell-cell adhesion is involved, directly or indirectly, in the induction process. Cadherins, which are transmembranous polypeptides that connect one cell to another through calcium ion-dependent homophilic binding (Nagafuchi et al., 1987; Nose et al., 1988; Takeichi, 1988; Takeichi, 1991), are subdivided into several subclasses such as E (epithelial)-cadherin, N (neural)-cadherin, P (placental)cadherin, and L-CAM (liver cell adhesion molecule). Using genetic technology, more than 20 cadherins have been described in the central nervous system, on liver and vascular endothelial cells and in other tissues and organs (Matsuyoshi, 1993a; Suzuki et al., 1991). Thus, the cadherin family of adhesion molecules is much larger than previously thought. r 1997 WILEY-LISS, INC. Recent studies on cadherins have focused on cytoplasmic adhesion mechanisms. Sequence comparison of the classic cadherins and the heterotypic expression studies of mutant proteins led to the identification of cytoplasmic proteins of 102, 88, and 80 kDa, which complex with cadherins via a specific cytoplasmic binding domain. These proteins, termed a-, b-, and g-catenin respectively, are crucial for cadherin function. Catenins link cadherins to the actin filamentous network and to other transmembrane and cytoplasmic proteins. a-catenin is homologous to vinculin, and b-catenin is homologous to the product of the Drosophila gene armadillo, while g-catenin seems to be identical to plakoglobin. Structural and functional analyses of catenins have indicated that cytoplasmic anchorage is important in mediating adhesive interactions. In addition, the anchorage of the cadherin-catenin complex to the cytoskeleton possibly depends upon tyrosine phosphorylation (Kemler, 1993; Takeichi et al., 1992). *Correspondence to: Fukumi Furukawa, MD, Department of Dermatology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431–31, Japan. Received 18 November 1994; Accepted in revised form 16 March 1995 344 FURUKAWA ET AL. TABLE 1. Classification of the cadherin superfamily Cadherins are essential for the mutual association of vertebrate cells, and through their homophilic binding interactions they play a role in cell-sorting mechanisms and confer adhesion specificities to cells (Takeichi, 1991; Larue et al., 1994). Cadherins are thus considered to be important regulators of morphogenesis in various organs, including the skin. Cadherins present in the epidermal cells are involved not only in maintaining the arrangement of these cells but also in dermal condensation (Hirai et al., 1989). Recent pathological examinations suggest that the down-regulation of cadherin expression is associated with increased invasiveness of tumor cells (Birchmeier et al., 1993). Furthermore, the role of soluble E-cadherin is the subject of much discussion (Katayama et al., 1994). In this review, we describe the roles of E- and P-cadherins in human skin development, cultured keratinocytes, and skin cancers. ies on the pathogenesis of autoimmune bullous diseases, especially pemphigus (Amagai, 1995; Amagai et al., 1991; Stanley, 1995). The second family of cadherins has multi-repeated extracellular domains and unique intracellular domains. The third family has the enzyme activity of tyrosine phosphorylation. Other classifications schemes for the cadherin family have been proposed. Pouliot (1992) reported the result of phylogenic analyses of the cadherin superfamily. He studied the first extracellular domain of cadherin which is the site responsible for mediating adhesive interaction, and showed that cadherins can be classified into three groups with distinct structural features. Group I includes N-, E-, P-, B(brain)-, R(retinal)-cadherin, and L-CAM(liver cell adhesion molecule). Group II is M(muscle)-cadherin. Group III is T(truncated)-cadherin. Phylogenically, N-cadherin shows very little sequence divergence between species, whereas all other cadherin subtypes show more substantial divergence, suggesting that selective pressure upon the first domain may be greater for N-cadherin than for the other cadherins. The protocadherin family, which is characterized by a distinct cadherin repeat in the extracellular domain, was proposed by Sano et al. (1993). Rimm and Morrow (1994) pointed out the relatedness of different cadherins based on the molecular cloning of human E-cadherin. The gene encoding full-length human E-cadherin has been cloned and sequenced from liver and colon cDNA libraries. The derived amino acid sequence of the extracellular domain of human Ecadherin displays strong homologies with all other vertebrate cadherins, including the presence of the EC (extracellular) 1 domain. The sequence of the cytoplasmic domain defines it as a particular subtype of cadherin. In addition, the cytoplasmic domain contains two distinct <30–35 amino acid sequence motifs that are conserved in several but not all cadherins. Referring to these motifs as cadherin homology domains ‘‘two’’ (CH2) and ‘‘three’’ (CH3), the authors suggest a sub-classification of the cadherins that is cognizant of the divergence in the cytoplasmic domain, and identify two sequence motifs that may represent functional cassettes variably used in some of the cadherins and possibly other proteins as well. CLASSIFICATION OF CADHERINS A classification of the cadherin superfamily, based on their homology of extracellular domains (Table 1), was proposed by Matsuyoshi (1993). The first family, which has 4-repeated extracellular domains, includes the classic cadherin, desmosomal cadherins, and cadherins without intracellular domains. Classic cadherins can be subdivided further into adherens junction cadherin, unidentified cadherin, and several others. The E- and P-cadherins of adherens junction cadherin are expressed on cell surfaces of the epidermis. Cadherin 5 localized on human and mouse vascular endothelial cells, provides new insights into vascular morphogenesis and regulation (Matsuyoshi et al., 1997). Desmosomal cadherins include pemphigus vulgaris antigen (PVAg, desmoglein III), desmoglein I, II, and desmocollin I, II, III (Amagai et al., 1991; Buxton et al., 1993; Collins et al., 1991; Koch et al., 1990). Identification of these desmosomal cadherins opened the door for stud- CADHERIN EXPRESSION IN HUMAN SKIN DEVELOPMENT Among these cadherins, E- and P-cadherins have been investigated thoroughly in relation to mouse skin morphogenesis. In mouse skin, E-cadherin is expressed in all cells of the embryo at the implantation stage. During development, E-cadherin disappears from some cell layers in the mesoderm. In older embryos, essentially all proliferating epithelial cells derived from the ectoderm and the endoderm express E-cadherin, although some terminally differentiated and/or keratinized cells lose E-cadherin. P-cadherin was first detected in the extraembryonic layers of early embryos, such as the ectoplacental cone and the visceral endoderm, at the stage of implantation. Around the neurula stage, various tissues begin to express P-cadherin. In the fetal epidermis, P- and E-cadherin are differentially expressed; the germinative basal layer has P-cadherin as A. 4-repeated family 1. Classic cadherin (1) Adherens junction cadherin N-cadherin R-cadherin P-cadherin E-cadherin (uvomorulin) B-cadherin L-CAM EP-cadherin U-cadherin XB-cadherin (2) Unidentified localization sites cadherin 6, 8, 9, 10, 11 (3) Others M-cadherin (muscle cadherin) Cadherin 5 (VE-cadherin) (endothelial cadherin) 2. Cadherins without intracytoplasmic domains T-cadherin 3. Desmosome cadherin desmolgein I, II, III desmocollin I, II, III (IV) B. Multi-repeated cadherin Fat, PC42, PC43 C. Others c-Ret This classification is based on the report of Matsuyoshi (1993) and Buxton et al. (1993) CADHERINS IN THE SKIN Fig. 1. Photographs of immunofluorescence localization of E- and P-cadherins in the sole of a 5-year-old infant. Specimens were fixed in periodate-lysine-paraformaldehyde (PLP) solution and 6 µm cryosectioned specimens were incubated with a monoclonal antibody (mAb) to human E-cadherin (HECD-1) or a mAb to human P-cadherin (NCCCAD-299). FITC-conjugated goat anti-mouse IgG was used as the second antibody. Detailed procedures are described in our previous well as E-cadherin and the middle layer has only E-cadherin (Takeichi, 1988). As for the hair, P-cadherin is expressed in proliferating regions such as the basal layer, the outer root sheath, and the hair matrix, whereas E-cadherin is expressed in both proliferating and non-proliferating regions (Hirai et al., 1989). In human adult and infant skin, E-cadherin is expressed on the cell surfaces of all epidermal layers including skin appendages, whereas P-cadherin is expressed only on cells of the basal layers and the outer layers of skin appendages (Fig. 1). These latter observations suggest that P-cadherin is associated with the proliferating cell compartment (Fujita et al., 1992). Ultrastructurally, E-cadherin is distributed on all of the plasma membranes of keratinocytes, but not on the dermal surface of basal cells. Dense deposits of Ecadherin are found in the intercellular space of desmosomes (Fig. 2), and P-cadherin is localized on the surfaces of basal cells, but not on the dermal surface (Horiguchi et al., 1994). In human organs E-cadherin is expressed in almost all epithelial tissues, while the distribution of P-cadherin is restricted to the basal or lower layers of stratified epithelia in which both cadherins are co-expressed (Shimoyama et al., 1989). In human fetal skin, the patterns of E- and Pcadherin expression are generally similar to those found in the adult (Fujita et al., 1992; Furukawa et al., 1989). Embryonic skin at 7–8 weeks of estimated gestational age (EGA) consists of basal and peridermal cell layers. Basal cells have both E- and P-cadherin on their surfaces, whereas some of the periderm cells have only E-cadherin. After 9 weeks of EGA the intermediate cells have only E-cadherin, and the periderm cells have neither of the cadherins. When hair follicles develop after 12 weeks of EGA their associated structures—the outer root sheath, the outer-most layer of the hair matrix, and the sebaceous gland—express both E- and P-cadherins. P-cadherin temporarily shows a unique spatiotemporal expression pattern in the developing 345 report (Reproduced with permission from Fujita, M., Furukawa, F., Fujii, K., Horiguchi, Y., Takeichi, M., and Imamura, S. (1992) Expression of cadherin cell adhesion molecules during human skin development: morphogenesis of epidermis, hair follicles and sweat ducts. Arch. Dermatol. Res., 284:159–166. rSpringer-Verlag GmbH & Co. KG). Bar: 40 µm. Fig. 2. Photograph of ultrastructural localization of E-cadherin in adult skin (38,000). Samples were fixed in PLP solution and cryosections (6 µm) were allowed to react with HECD-1. The sections were incubated with biotinylated anti-mouse IgG and then with soluble avidin-biotin-peroxidase complex. Other details were described recently (Horiguchi et al., 1994). BC: basal cell, De: dermis. An arrow indicates E-cadherin on the desmosome. Bar: 1 µm. sweat ducts. During this stage, P-cadherin accumulates on cells in the epidermal ridges that are distinct from the proliferating cells detected by Ki-67. The expression of P-cadherin is spatiotemporally controlled, and may be closely related to the segregation of the basal layers and the arrangement of epidermal cells into eccrine sweat ducts. 346 FURUKAWA ET AL. Fig. 3. Immunofluorescence photograph of E-cadherin localization in an early lesion of pemphigus vulgaris. Reduced E-cadherin expression is observed at the acantholytic cells. Bar: 40 µm. CADHERIN EXPRESSION IN SKIN DISEASES Autoimmune Bullous Diseases Of special interest is the E-cadherin expression pattern in skin diseases since E-cadherin plays an essential role in cell-cell adhesion. The desmosome cadherins listed in (Table 1) play essential roles in the pathogenesis of autoimmune bullous diseases. E-cadherin, one of the adherens junction cadherins, accumulates at desmosomes and exhibits, in vivo and in vitro, the distribution pattern described above (Horiguchi et al., 1994). In early lesions of pemphigus, the immunofluorescence intensity of E-cadherin is markedly reduced especially on the acantholytic cells, whereas at the non-acantholysis sites E-cadherin shows an intercellular distribution with a near uniform immunofluorescence intensity (Fig. 3) (Furukawa et al., 1990; Furukawa et al., 1989). Similar staining patterns were observed in our previous report of Darier’s disease (Furukawa et al., 1991), for which the gene mapped to chromosome 12q (Bashir et al., 1993; Buxton, 1993; Craddock et al., 1993). However, the fully developed bullae in PV show an intercellular distribution of E-cadherin with a near uniform immunofluorescence intensity for this molecule in both the suprabasal cell layer and the separated roof epidermis (Furukawa et al., 1994). The epitope(s) of E-cadherin was considered to be different from those of pemphigus vulgaris antigens (PVA) (Furukawa et al., 1989). Amagai, Stanley and co-workers (1991) cloned the cDNA encoding pemphigus vulgaris antigen (PVA) using antibodies obtained from patients. They demonstrated by sequence analysis that PVA belongs to the cadherin family. PVA is now recognized as desmoglein III, but PVA lacks the biochemical properties characteristic of classical cadherins (Plott et al., 1994). Differences are observed in catenin binding and protection of the extracellular domain by calcium from degradation by trypsin. These results suggest that PVA and classical cadherins may not subserve identical biological functions. Skin Cancers Cancers are characterized by their non-regulated cell growth, loss of polarity, and invasion or metastasis. Several recent studies have suggested that the loss of E-cadherin may be associated with tumor progression and E-cadherin acts principally as a suppressor of invasive ability (Behrens et al., 1989; Birchmeier et al., 1993; Navarro et al., 1991). In fact, reduced amounts of cadherins have been immunohistochemically described in human cancers from various organs such as esophagus, stomach, liver, breast, head, neck, prostate, and gingiva. Several mechanisms of impaired function of cadherin are now hypothesized (Fig. 4) (Matsuyoshi, 1993; Takeichi, 1991). In murine skin squamous cell carcinoma induced by chemical carcinogens, the association between cadherin expression and tumor progression has been investigated by Cano and colleagues (Navarro et al., 1991). They studied the expression of E- and P-cadherin in various mouse epidermal keratinocyte cell lines that are representative of different stages of epidermal carcinogenesis. Complete absence of E-cadherin mRNA and protein was found in the epithelioid type and fibroblastoid type of epidermal keratinocyte cell lines, which all grew as fast as tumors in nude mice. In contrast, well differentiated squamous cell carcinoma expressed considerable amounts of E-cadherin. Pcadherin was detected in all cell lines irrespective of cell differentiation. The introduction of an exogeous Ecadherin cDNA into epithelioid type cells leads to a partial suppression of the tumorigenicity. This finding CADHERINS IN THE SKIN Fig. 4. Proposed mechanisms of cadherin dysfunction. suggests that E-cadherin expression is associated with the inhibition of tumor growth in some cases of experimentally induced mouse skin carcinomas. Reduced E-cadherin expression in aggressive squamous cell carcinoma is suggested to be associated with a redistribution or absence of sodium, potassium-adenosine triphosphatase (Na1, K1-ATPase), a marker of cell polarity (Ruggeri et al., 1992), in the combined function of catenin complex (Nathke et al., 1994) and membranecytoskeleton (Marrs et al., 1993). Impaired cadherin functions have been proposed in human skin cancers. For example, the transformed keratinocyte cell line HSC-1 (Kondo and Aso, 1981), originally derived from human squamous cell carcinoma shows a reduced and dot-like distribution of E-cadherin (Furukawa et al., 1994). E-cadherin expression by basal cell carcinoma (BCC) is preserved in superficial and nodular types but reduced in infiltrative BCC, which suggests E-cadherin is related to the growth pattern and the local aggressive behaviour of BCC (Pizarro et al., 1994). A lower or unstable expression of E-cadherin is also found in squamous cell carcinoma and Paget’s disease (Shirahama et al., 1996). Expressions of E- and P-cadherins are correlated with the degree of differentiation of squamous cell carcinoma and a marked reduction of E-cadherin is observed with the appearance of metastatic squamous cell carcinoma cells in lymph nodes (Furukawa et al., 1994; Shirahama et al., 1996). In addition, immunohistochemical studies indicate that epidermal cells of solar keratosis show unstable expression of cadherins. The expression of P-cadherin in malignant melanoma is associated with tumor proliferation and progression. These findings 347 support the hypothesis that E-cadherin and P-cadherin are involved in invasion or metastasis in vivo. The significance of tyrosine phosphorylation in the action of E-cadherin has also been intensely investigated. V-src oncogene is a potent effector of epithelial differentiation and invasiveness, and promotes the dedifferentiation through phosphorylation of the Ecadherin/catenin complex (Behrens et al., 1994). Matsuyoshi et al. (1992) observed that direct or indirect v-src-mediated tyrosine phosphorylation perturbs cadherin function in metastatic fibroblasts. In addition, they demonstrated that the inhibition of tyrosine phosphorylation restores cadherin action to its normal state. Mutations of the E-cadherin gene in human gastric carcinoma cell lines were recently reported (Oda et al., 1994; Becker et al., 1994). E-cadherin-mediated cellcell adhesion potential may be hampered by mutations in the structural region of the E-cadherin gene. Similar approaches need to be applied to skin cancers, and the contribution of other CAM family molecules to cancer should be investigated. Based on experimental data, E- and P-cadherin are now considered to be key adhesion molecules in the primary event-neoplastic cell detachment from tumor mass. For the next step in metastasis formation, neoplastic cells have to cope with the basement membrane, which requires digestion of and interaction with extracellular matrix components. Both of these interactions involve the integrin family in association with altered cadherin function (Brichmeier and Behrens, 1994). CADHERIN FUNCTION IN CULTURED KERATINOCYTES It is well known that calcium switching induces keratinocyte differentiation or stratification. When keratinocytes are cultured in low Ca21 medium, they do not form any desmosomes. However, once they are transferred to normal Ca21 medium, they start to make cell-cell contact and to reproduce desmosomes. In controlling the stratification of keratinocytes, the adherens junctions, like desmosomes, play an essential role (O’Keefe et al., 1987). Thus, since cadherin function is calcium dependent, cadherins are one of the keys in controlling keratinocyte differentiation or stratification. Normal human keratinocytes (NHK), cultured to 50–60% confluency in low Ca21 medium (0.03 mM), show no obvious cell-cell interaction and only faint localization of E-cadherin. NHK in 0.6 mM Ca21containing medium show a well defined meshwork pattern of intercellular distribution of E-cadherin (Fig. 5) (Furukawa et al., 1990; Furukawa et al., 1994; Horiguchi et al., 1994). At 90–100% confluency, after culturing with high Ca21 (1.0 mM), a uniform distribution of E-cadherin is observed and its expression is much higher than that seen with low Ca21. In contrast, the expression of P-cadherin is reduced in high Ca21 medium (Furukawa et al., 1994). Ultrastructural studies reveal that E-cadherin distributes all around the free surface of the plasma membrane of NHK in low Ca21 medium, whereas high Ca21 induces its accumulation at desmosomes (Fig. 6) (Horiguchi et al., 1994). With an immunoblot analysis no apparent differences 348 FURUKAWA ET AL. Fig. 5. Immunofluorescence micrographs showing E-cadherin localization in normal human cultured keratinocytes in KGM (keratinocyte growth medium) with 0.6 mM (A) and 0.1 mM (B) calcium. Cultured keratinocytes were fixed in acetone, incubated with diluted HECD-1 and then reacted with FITC-conjugated anti-mouse IgG. Bar: 50 µm. (Reproduced, with permissions, from Horiguchi, Y., Furukawa, F., Fujita, M., and Imamura, S.: Ultrastructural localization of Ecadherin cell adhesion molecule on the cytoplasmic membrane of keratinocytes in vivo and in vitro, Journal of Histochemistry and Cytochemistry, 42:1333–1340, 1994). are seen in the expression pattern of E-cadherin in NHK cultured in the various Ca21 concentrations (Fig. 7). These results suggest that under physiological conditions E-cadherin on the plasma membrane of NHK shifts to the desmosomes where it expresses an adhesion function. Recently Hodivala and Watt (1994) reported that cadherins play a role in the down-regulation of integrin expression that occurs during keratinocyte terminal differentiation. Thus, a cadherin-catenin complex and other cell adhesion molecules appear to be involved in keratinocyte differentiation. Cadherin functions have been assayed using cell aggregation assays (Takeichi, 1977). Briefly, cell suspensions, harvested by trypsin solution in the presence of calcium, are incubated to allow cell aggregation on a gyratory shaker, and then the degree of cell aggregation is determined and expressed by the aggregation index. Using this aggregation assay the generation of cell-cell contacts can be determined. Cells treated with purified pemphigus vulgaris antibody showed much lower aggregation rates than cells treated with purified normal human IgG (Fig. 8). Pemphigus vulgaris antibody produced an extra band of E-cadherin in Western blot analysis (Takeichi et al., 1990). These results suggest that pemphigus vulgaris antibody possibly induces the degradation of E-cadherin through protease action. It is well known that in the process of blister formation in pemphigus vulgaris, induction of plasminogen activator synthesis, secretion of plasminogen activator, and activation of plasminogen to plasmin occur (Hashimoto et al., 1983). These reactions might trigger the dysfunction of E-cadherin and/or P-cadherin. E-cadherin is involved in the persistence of Langerhans cells in epidermis (Tang et al., 1993). Fresh murine Langerhans cells express cadherins and Langerhans cells adhere to keratinocytes in vitro through E-cadherin. Cultured Langerhans cells express lower levels of E-cadherin and exhibit decreased affinity for keratinocytes. These findings suggest that E-cadherin mediates adhesion of epidermal Langerhans cells to keratinocytes. A monoclonal anti-E-cadherin antibody slightly reduces the cell-cell contact of cultured NHK and also the increases intracytoplasmic Ca21 concentration of these cells under certain conditions (Wakita et al., manuscript in preparation). Anti-E-cadherin plus anti-Pcadherin inhibit the formation of adherens junctions CADHERINS IN THE SKIN 349 Fig. 6. Immunoelectron microscopic view of normal human cultured keratinocytes in 0.1 mM calcium (A,B) and 0.6 mM calcium (C,D) calcium containing medium. Cultured cells on LabTek chamber slides were washed with PBS supplemented with the same calcium concentration, and fixed with the PLP solution. Cells were incubated with diluted HECD-1 using the immunoperoxidase method. Resinembedded immunoperoxidase-stained cultured cells were detached from the glass slides. Details were given in the report of Horiguchi et al. (1994). Fig. A & C: vertical sections, Fig. B & D: horizontal sections, Kc: keratinocyte, arrow heads or arrows: E-cadherin. Bars: A,C 5 1 µm; B,D 5 0.1 µm. (Reproduced, with permissions, from Horiguchi, Y., Furukawa, F., Fujita, M., and Imamura, S., Ultrastructural localization of E-cadherin cell adhesion molecule on the cytoplasmic membrane of keratinocytes in vivo and in vitro. Journal of Histochemistry and Cytochemistry, 42:1333–1340, 1994). and desmosomes, prevent reorganization of the cytoskeleton, and block stratification (Lewis et al., 1994). Cadherin function is required for calcium-induced intercellular junction organization. Balda et al. (1993) found a promotive role for diacylglycerol in the assembly of the tight junctions of MDCK cells. These results suggest the importance of activated signaling pathways in E-cadherin-mediated cell-cell adhesion. SOLUBLE E-CADHERIN IN SKIN DISEASES Soluble E-cadherins were first identified as 80–84 kDa peptides released from MCF-7 human carcinoma cells (Damsky et al., 1991). These peptides retain their functional activities of disrupting cell-cell contact in cultured epithelial cells, and antibodies against them induce disruption of the mutual adhesion of target cells 350 FURUKAWA ET AL. Fig. 7. Immunoblot analysis of E- and P-cadherins in normal human cultured keratinocytes in KGM supplemented with various calcium concentration. Detailed procedures were based on the report of Matsuyoshi et al. (1992). (Wheelock et al., 1987; Wheelock and Jensen, 1992). sE-cadherin is probably a degradation product of the 120 kDa form of intact E-cadherin and is generated by a Ca21-dependent proteolytic action. Thus, sE-cadherin should be a good indicator for monitoring the regeneration of E-cadherin in vivo. sE-cadherin is elevated in several skin diseases including bullous pemphigoid, pemphigus vulgaris, psoriasis, and other inflammatory diseases (Matsuyoshi et al., 1995), but not in collagen diseases or in burn patients. The elevated levels of sE-cadherin in autoimmune bullous diseases decrease after therapy. The blister fluids of bullous pemphigoid patients contain two times more sE-cadherin than is found in their serum, which itself is twice as high as the levels in normal serum. The levels of sE-cadherin in skin lesions correlate with the levels in the circulation in both normal controls and bullous pemphigoid patients. In psoriasis there is a weak, but significant, association between sE-cad levels and PASI score, which suggests that dysregulation of epidermal cell growth might be related to the increase of sE-cadherin. In cases of skin cancer, several patients showed elevated levels of sEcadherin (Furukawa et al., 1994). Such patients with squamous cell carcinoma, Paget’s disease and malignant melanoma had metastasis or widely distributed lesions. Katayama et al. (1994) reported increased circulating levels of sE-cadherin in patients with gastric carcinoma or hepatocellular carcinoma. Since sE- Fig. 8. Aggregation assay of E-cadherin function using cultured transformed human epidermal cells (HSC-1) treated with pemphigus vulgaris antibody. Cells were incubated with purified IgG (5–10 mg/ml) for 8 or 24 hours. The culture medium was DMEM supplemented with 1% bovine serum albumin. IgG was purified with a protein A sepharose affinity column. IgG-treated cell suspensions were obtained with 0.01% trypsin in PBS supplemented with 1 mM calcium, and then prepared for the cell aggregation assay. The results are expressed as the aggregation index. X axis represents the incubation time. A higher index indicates a lower binding ability of cadherin. The basic protocol is similar to that reported by Matsuyoshi et al. (1992). cadherin disrupts cell-cell junctions in vitro (Wheelock et al., 1987), it is thought that the presence of sEcadherin up-regulates the invasiveness of tumor cells. These results suggest that sE-cadherin is a useful marker of various diseases. Although the role and origin of sE-cadherin remain controversial, the elevated levels of sE-cadherin would inhibit cell-cell adhesion and thus favor bullous formation or carcinoma invasion. CONCLUSION Cadherins are involved in vital processes such as embryogenesis, tissue and organ pattern formation, and maintenance of specific organ architecture. Cadherin dysfunction may induce various skin diseases including bullous diseases and metastatic or invasive cancers. Investigations into the family of cadherin molecules will provide new and exciting insight into cutaneous biology. The focus in cadherin research has recently changed from extracellular binding to cytoplasmic control of cell adhesions by the cadherin-catenin complex. We believe that in the near future the control CADHERINS IN THE SKIN systems regulating cadherin molecules will be more clearly and completely understood. ACKNOWLEDGMENT This work was supported by grants from the Japanese Ministry of Welfare and Health, and the Japanese Ministry of Science, Culture and Education. REFERENCES Amagai, M. (1995) Adhesion molecules. 1. keratinocyte-keratinocyte interactions; cadherins and pemphigus. J. Invest. Dermatol., 104: 146–152. Amagai, M., Kalus-Kovtun, V., and Stanley, J.R. (1991) Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. Cell, 67:869–877. Balda, M.S., Gonzalez-Mariscal, L., Matter, K., Cereijido, M., and Anderson, J.M. (1993) Assembly of the tight junction: the role of diacylglycerol. J. Cell Biol., 123:293–302. Bashir, R., Munro, C.S., Mason, S., Stephenson, A., Rees, J.L., and Strachan, T. (1993) Localisation of a gene for Darier’s disease. Hum. Molecular Genet., 2:1937–1939. Becker, K-F., Atkinson, M.J., Reich, U., Becker, I., Nekarda, H., Siewert, J.R., and Hofler, H. (1994) E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res., 54:3845–3852. Behrens, J., Mareel, M.M., van Roy, F.M., and Birchmeier, W. (1989) Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion. J. Cell Biol., 108:2435–2447. Behrens, J., Vakaet, L., Friis, R., Winterhager, E., Roy, F.V., Mareel, M.M., and Birchmeier, W. (1993) Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/b-catenin complex in cells transformed with a temperature-sensitive v-src gene. J. Cell Biol., 120:757–766. Birchmeier, W., and Behrens, J. (1994) Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Biochim. Biophys. Acta, 1198:11–26. Birchmeier, W., Weidner, K.M., Hulsken, J., and Behrens, J. (1993) Molecular mechanisms leading to cell junction (cadherin) deficiency in invasive carcinomas. Semi. Cancer Biol., 4:231–239. Buxton, R.S. (1993) Yet another skin defect, Darier’s disease, maps to chromosome 12q. Hum. Molecular Genet., 2:1763–1764. Buxton, R.S., Cowin, P., Franke, W.W., Garrod, D.R., Green, K.J., King, I.A., Koch, P.J., Magee, A.I., Rees, D.A., Stanley, J.R., and Steinberg, M.S. (1993) Nomenclature of the desmosomal cadherins. J. Cell Biol., 121:481–483. Collins, J.E., Legan, P.K., Kenny, T.P., MacGarvie, J., Holton, J.L., and Garrod, D.R. (1991) Cloning and sequence analysis of desmosomal glycoprotein 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogenous cytoplasmic domains. J. Cell Biol., 113:381–391. Craddock, N., Dawson, E., Burge, S., Parfitt, L., Mant, B., Roberts, Q., Daniels, J., Gill, M., McGuffin, P., Powell, J., and Owen, M. (1993) The gene for Darier’s disease maps to chromosome 12q23-q24.1. Hum. Molecular Genet., 2:1941–1943. Damsky, C.H., Richa, J., Solter, D., Knudsen, K., and Buck, C.A. (1991) Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue. Cell, 34:455–466. Fujita, M., Furukawa, F., Fujii, K., Horiguchi, Y., Takeichi, M., and Imamura, S. (1992) Expression of cadherin cell adhesion molecules during human skin development: morphogenesis of epidermis, hair follicles and sweat ducts. Arch. Dermatol. Res., 284:159–166. Furukawa, F., Fujii, K., Matsuyoshi, N., Horiguchi, Y., and Imamura, S. (1990) The cadherins: cell-cell adhesion molecules possibly control pathogenesis of pemphigus. J. Invest. Dermatol., 94:526 (Abstract). Furukawa, F., Kanauchi, H., and Imamura, S. (1989) Localization of E-cadherin in human skin tissue. Conn. Tissue, 21:3–4. Furukawa, F., Miyachi, Y., Wakai, Y., and Imamura, S. (1991) Localization of cadherin cell-adhesion molecules in the skin lesion of Darier disease. Int. J. Dermatol., 30:599–600. Furukawa, F., Takigawa, M., Matsuyoshi, N., Shirahama, S., Wakita, H., Fujita, M., Horiguchi, Y., and Imamura, S. (1994) Cadherins in cutaneous biology. J. Dermatol., 21:802–813. Hashimoto, K., Shafran, K., Webber, P., Lazarus, G.S., and Singer, K.H. (1983) Anti-cell surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells: a mecha- 351 nism for the loss of epidermal cohesion and blister formation. J. Exp. Med. 157:259–272. Hirai, Y., Nose, A., Kobayashi, S., and Takeichi, M. (1989) Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. skin morphogenesis. Development, 105:271–277. Hodivala, K.J., and Watt, F.M. (1994) Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation. J. Cell Biol., 124:589–600. Horiguchi, Y., Furukawa, F., Fujita, M., and Imamura, S. (1994) Ultrastructural localization of E-cadherin cell adhesion molecule on the cytoplasmic membrane of keratinocytes in vivoand in vitro. J. Histochem. Cytochem., 42:1333–1340. Katayama, M., Hirai, S., Kamihagi, K., Yasumoto, M., and Kato, I. (1994) Soluble E-cadherin fragments increased in circulation of cancer patients. Br. J. Cancer, 69:580–585. Kemler, R. (1993) From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. T.I.G., 9:317–321. Koch, P.J., Walsh, M.J., Schmelz, M., Goldschmidt, M.D., Zimbdmann, R., and Franke, W.W. (1990) Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules. Eur. J. Cell Biol., 53:1–12. Kondo, S., and Aso, K. (1981) Establishment of a cell line of human skin squamous cell carcinoma in vitro. Br. J. Dermatol., 105:125– 132. Larue, L., Ohsugi, M., Hirchenhain, J., and Kemler, R. (1994) Ecadherin null mutant embryos fail to form a trophectoderm epithelium. Proc. Natl. Acad. Sci., USA, 91:8263–8267. Lewis, J.E., Jensen, P.J., Wheelock, M.J. (1994) Cadherin function is required for human keratinocytes to assemble desmosomes and stratify in response to calcium. J. Invest. Dermatol., 102:870–877. Marrs, J.A., Napolitano, E.W., Murphy-Erdosh, C., Mays, R.W., Reichardt, L.F., and Nelson, W.J. (1993) Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na1, K1-ATPase distribution in polarized epithelia. J. Cell Biol., 123:149–164. Matsuyoshi, N. (1993a) Cadherin family and adhesion in cancer cells. Biotherapy, 7:1158–1165 (in Japanese). Matsuyoshi, N., Hamaguchi, M., Taniguchi, S., Nagafuchi, A., Tsukita, S., and Takeichi, M. (1992) Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts. J. Cell Biol., 118:703–714. Matsuyoshi, N., Toda, K-I., Horiguchi, Y., Tanaka, T., Nakagawa, S., Takeichi, M., and Imamura, S. (1997) In vivo evidence of the critical role of cadherin-5 in murine vascular integrity. Proc. Ass. Am. Physicians 109: (in press) Matsuyoshi, N., Tanaka, T., Toda, K-I., Okamoto, H., Furukawa, F., and Imamura, S. (1995) Soluble E-cadherin: a novel cutaneous disease marker. Br. J. Dermatol, 132:745–749. Nagafuchi, A., Shirayoshi, Y., Okazaki, K., Yasuda, K., and Takeichi, M. (1987) Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature, 329:341–343. Nathke, I.S., Hinck, L., Swedlow, J.R., Papkoff, J., and Nelson, W.J. (1994) Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells. J. Cell Biol., 125:1341– 1352. Navarro, P., Gomez, M., Pizarro, A., Gamallo, C., Quintanilla, M., and Cano, A. (1991) A role for the E-cadherin cell-cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis. J. Cell Biol., 115:517–533. Nose, A., Nagafuchi, A., and Takeichi, M. (1988) Expressed recombinant cadherins mediate cell sorting in model systems. Cell 54:993– 1001. Oda, T., Kanai, Y., Oyama, T., Yoshiura, K., Shimoyama, Y., Birchmeier, W., Sugimura, T., and Hirohashi, S. (1994) E-cadherin gene mutations in human gastric carcinoma cell lines. Proc. Natl. Acad. Sci. USA, 91:1858–1862. O’Keefe, E.J., Briggaman, R.A., and Herman, B. (1987) Calciuminduced assembly of adherens junctions in keratinocytes. J. Cell Biol., 105:807–817. Pizarro, A., Benito, N., Navarro, P., Palacios, J., Cano, A., Quintanilla, M., Contreras, F., and Gamallo, C. (1994) E-cadherin expression in basal cell carcinoma. Br. J. Cancer, 69:157–162. Plott, R.T., Amagai, M., Udey, M.C., and Stanley, J.R. (1994) Pemphigus vulgaris antigen lacks biochemical properties characteristic of classical cadherins. J. Invest. Dermatol., 103:168–172, 1994. Pouliot, Y. (1992) Phylogenetical analysis of the cadherin superfamily. Bio. Essays 14:743–748. Rimm, D.L., and Morrow, J. (1994) Molecular cloning of human E-cadherin suggests a novel subdivision of the cadherin superfamily. Biochem. Biophys. Res. Comm., 200:1754–1761. Ruggeri, B., Caamano, J., Slaga, T.J., Conti, C.J., Nelson, W.J., and 352 FURUKAWA ET AL. Klein-Szanto, A.J.P. (1992) Alterations in the expression of uvomorulin and Na1, K1-adenosine triphosphatase during mouse skin tumor. Am. J. Pathol., 140:1179–1185. Sano, K., Tanihara, H., Heimark, R.L., Obata, S., Davidson, M., St. John, T., Taketani, S., and Suzuki, S. (1993) Protocadherins: a large family of cadherin-related molecules in central nervous system. EMBO J., 12:2249–2256. Shimoyama, Y., Hirohashi, S., Hirano, S., Noguchi, M., Shimosato, Y., Takeichi, M., and Age, O. (1989) Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas. Cancer Res., 49:2128– 2133. Shirahama, S., Furukawa, F., Wakita, H., and Takigawa, M. (1996) Eand P-cadherin expression in tumor tissues and soluble E-cadherin levels in sera of patients with skin cancer. J. Dermatol. Sci., 13:30–36. Stanley, J.R. (1995) Autoantibodies against adhesion molecules and structures in blistering skin diseases. J. Exp. Med., 181:1–4. Suzuki, S., Sano, K., and Tanihara, H. (1991) Diversity of the cadherin family: evidence for eight new cadherins in nervous system. Cell Regulation, 2:261–270. Takeichi, M. (1977) Functional correlation between cell adhesive properties and some cell surface protein. J. Cell Biol., 75:464–474. Takeichi, M. (1988) The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development, 102:639–655. Takeichi, M. (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science, 251:1451–1455. Takeichi, M., Hirano, S., Matsuyoshi, N., and Fujimori, T. (1992) Cytoplasmic control of cadherin-mediated cell-cell adhesion. Cold Spring Harbor Symposium on Quantitative Biology, LVII: 327–334. Takeichi, M., Matsuyoshi, N., Fujii, K., Furukawa, F., and Imamura, S. (1990) Roles of cadherin in the development of pemphigus. Annual report of rare and intractable disease research committee. ed. by Imamura, S., Japanese Ministry of Health and Welfare, Tokyo, pp. 185–187. (in Japanese). Tang, A., Amagai, M., Granger, L.G., Stanley, J.R., and Udey, M.C. (1993) Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature 361:82–85. Wheelock, M.J., Buck, C.A., Bechtol, K.B., and Damsky, C.H. (1987) Soluble 80-kd fragment of cell-CAM 120/80 disrupts cell-cell adhesion. J. Cell. Biochem., 34:187–202. Wheelock, M.J., and Jensen, P.J. (1992) Regulation of keratinocyte intercellular junction organization and epidermal morphogenesis by E-cadherin. J. Cell Biol., 117:415–425.