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Differential Effects of Desmoglein 1 and Desmoglein 3 on Desmosome Formation

2002; Elsevier BV; Volume: 119; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2002.19648.x

1523-1747

Yasushi Hanakawa, Yuji Shirakata, Yoko Yahata, Sho Tokumaru, Kenshi Yamasaki, Mikiko Tohyama, Koji Sayama, Koji Hashimoto, Masayuki Amagai,

Wnt/β-catenin signaling in development and cancer

The desmoglein plays an important part in the formation of desmosomes. We constructed recombinant adenoviruses containing desmoglein 1 and desmoglein 3 derivatives partly lacking the extracellular domain (desmoglein 1ΔEC and desmoglein 3ΔEC, respectively), and full-length desmoglein 1 and desmoglein 3 and studied the involvement of desmoglein 1 and desmoglein 3 in desmosome formation. During low-level expression of desmoglein 3ΔEC in transduced HaCaT cells, keratin insertion at cell–cell contact sites was only partially inhibited and desmoplakin was partially stained at cell–cell contact sites. Low-level expression of desmoglein 1ΔEC, however, resulted in complete inhibition of keratin insertion at the cell–cell contact sites, and desmoplakin was stained in perinuclear dots. These results indicate the dominant-negative effect of desmoglein 1ΔEC on desmosome formation was stronger than that of desmoglein 3ΔEC. Desmoglein 1ΔEC coprecipitated plakoglobin to approximately the same extent as desmoglein 3ΔEC. Therefore, we conclude that the dominant-negative effect of desmoglein 1ΔEC is not simply due to plakoglobin sequestration. On the other hand, during low-level expression of full-length desmoglein 3 and desmoglein 1, they both colocalized with desmoplakin. During high-level expression, however, keratin insertion at cell–cell contact sites was inhibited in desmoglein 1 but not in desmoglein 3, and desmoplakin was stained at cell–cell contact sites in desmoglein 3 but not in desmoglein 1. These data suggest desmoglein 1 and desmoglein 3 expressed at low level were incorporated into desmosome but at high-level expression, desmoglein 1 disrupted desmosomes but desmoglein 3 did not. Our findings provide biologic evidence that desmoglein 1 and desmoglein 3 play a different functional role in cell–cell adhesion of keratinocytes. The desmoglein plays an important part in the formation of desmosomes. We constructed recombinant adenoviruses containing desmoglein 1 and desmoglein 3 derivatives partly lacking the extracellular domain (desmoglein 1ΔEC and desmoglein 3ΔEC, respectively), and full-length desmoglein 1 and desmoglein 3 and studied the involvement of desmoglein 1 and desmoglein 3 in desmosome formation. During low-level expression of desmoglein 3ΔEC in transduced HaCaT cells, keratin insertion at cell–cell contact sites was only partially inhibited and desmoplakin was partially stained at cell–cell contact sites. Low-level expression of desmoglein 1ΔEC, however, resulted in complete inhibition of keratin insertion at the cell–cell contact sites, and desmoplakin was stained in perinuclear dots. These results indicate the dominant-negative effect of desmoglein 1ΔEC on desmosome formation was stronger than that of desmoglein 3ΔEC. Desmoglein 1ΔEC coprecipitated plakoglobin to approximately the same extent as desmoglein 3ΔEC. Therefore, we conclude that the dominant-negative effect of desmoglein 1ΔEC is not simply due to plakoglobin sequestration. On the other hand, during low-level expression of full-length desmoglein 3 and desmoglein 1, they both colocalized with desmoplakin. During high-level expression, however, keratin insertion at cell–cell contact sites was inhibited in desmoglein 1 but not in desmoglein 3, and desmoplakin was stained at cell–cell contact sites in desmoglein 3 but not in desmoglein 1. These data suggest desmoglein 1 and desmoglein 3 expressed at low level were incorporated into desmosome but at high-level expression, desmoglein 1 disrupted desmosomes but desmoglein 3 did not. Our findings provide biologic evidence that desmoglein 1 and desmoglein 3 play a different functional role in cell–cell adhesion of keratinocytes. desmoglein multiplicity of infection Desmosomes are cell–cell adhesion complexes that provide mechanical integrity to keratinocytes by linking to keratin intermediate filaments. Desmosomes are composed of two major transmembrane proteins, desmoglein (Dsg) and desmocollin (Amagai, 1996aAmagai M. Pemphigus: autoimmunity to epidermal cell adhesion molecules.Adv Dermatol. 1996; 11: 319-352PubMed Google Scholar;Kowalczyck et al., 1999Kowalczyck A.P. Bornslaeger E.A. Norvell S.M. Palka H.L. Green K.J. Desmosomes. intercellular adhesive junctions specialized for attachment of intermediate filaments.Int Rev Cytol. 1999; 185: 237-302Crossref PubMed Google Scholar;Green and Gaudry, 2000Green K.J. Gaudry C.A. Are desmosomes more than tethers for intermediate filaments?.Nat Rev Mol Cell Biol. 2000; 1: 208-216Crossref PubMed Scopus (309) Google Scholar). In humans, three desmoglein isoforms have been identified: Dsg1, Dsg2, and Dsg3. They are encoded by individual genes and differentially distributed in tissue. Dsg2 is expressed in all desmosome-containing tissues, including simple epithelium and myocardium. In contrast, Dsg1 and Dsg3 are expressed in stratified squamous epithelia. Dsg3 is found in the basal and suprabasal layers of stratifying epithelia, whereas Dsg1 is dominantly expressed in the differentiated upper layers of epithelia (Arnemann et al., 1993Arnemann J. Sullivan K.H. Magee A.I. King I.A. Buxton R.S. Stratification-related expression of isoforms of the desmosomal cadherins in human epidermis.J Cell Sci. 1993; 104: 741-750Crossref PubMed Google Scholar;Shimizu et al., 1995Shimizu H. Masunaga T. Ishiko A. Kikuchi A. Hashimoto T. Nishikawa T. Pemphigus vulgaris and pemphigus foliaceus sera show an inversely graded binding pattern to extracellular regions of desmosomes in different layers of human epidermis.J Invest Dermatol. 1995; 105: 153-159Crossref PubMed Scopus (87) Google Scholar;Amagai et al., 1996bAmagai M. Koch P.J. Nishikawa T. Stanley J.R. Pemphigus vulgaris antigen (desmoglein 3) is localized in the lower epidermis, the site of blister formation in patients.J Invest Dermatol. 1996; 106: 351-355Crossref PubMed Scopus (181) Google Scholar). Desmogleins play important parts in the formation and maintenance of desmosomes. Autoantibodies against desmogleins lead to impairment of epidermal tissue integrity. Pemphigus vulgaris is a disease caused by autoantibodies directed against Dsg3 in which skin lesion biopsies exhibit suprabasilar acantholysis (Udey and Stanley, 1999Udey M.C. Stanley J.R. Pemphigus—diseases of antidesmosomal autoimmunity.JAMA. 1999; 282: 572-576Crossref PubMed Scopus (82) Google Scholar). On the other hand, pemphigus foliaceus is caused by autoantibodies directed against Dsg1 and in this case, lesion biopsies exhibit subcorneal acantholysis. Dsg3 knockout mice phenotypically mimicked pemphigus vulgaris patients (Koch et al., 1997Koch P.J. Mahoney M.G. Ishikawa H. et al.Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris.J Cell Biol. 1997; 137: 1091-1102Crossref PubMed Scopus (363) Google Scholar) in that they displayed oral erosions and loss of intercellular adhesion of suprabasal layers of the mucosal epithelium and epidermis. Several studies have suggested that both the extracellular and cytoplasmic domains of desmogleins are critical for normal desmosome formation. N-terminally truncated Dsg3 caused dominant-negative effects on desmosome formation in HaCaT cells (Hanakawa et al., 2000Hanakawa Y. Amagai M. Shirakata Y. Sayama K. Hashimoto K. Different effects of dominant negative mutants of desmocollin and desmoglein on the cell–cell adhesion of keratinocytes.J Cell Sci. 2000; 113: 1803-1811Crossref PubMed Google Scholar). Expression of chimeric molecules containing the transmembrane domain of connexin and cytoplasmic domain of Dsg1 disrupted desmosomes in A431 cells (Troyanovsky et al., 1993Troyanovsky S.M. Eshkind L.G. Troyanovsky R.B. Leube R.E. Franke W.W. Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage.Cell. 1993; 72: 561-574Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Similarly, a chimeric molecule containing the extracellular domain of E-cadherin and cytoplasmic domain of Dsg1 (Ecad-Dsg1) disrupted desmosomes in A431 cells (Norvell and Green, 1998Norvell S.M. Green K.J. Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells.J Cell Sci. 1998; 111: 1305-1318Crossref PubMed Google Scholar). In contrast, a recent report indicated that a chimeric molecule of the extracellular domain of E-cadherin and cytoplasmic domain of Dsg3 (Ecad-Dsg3) was incorporated into desmosomes of A431 cells (Andl and Stanley, 2001Andl C.D. Stanley J.R. Central role of the plakoglobin-binding domain for desmoglein 3 incorporation into desmosomes.J Invest Dermatol. 2001; 117: 1068-1074Crossref PubMed Google Scholar). This difference between Ecad-Dsg1 and Ecad-Dsg3 indicates functional differences of cytoplasmic domains of Dsg1 and Dsg3. In addition, full-length Dsg1 disrupted desmosome when the expression level was high; however, full-length Dsg1 was incorporated into desmosome when the expression level was low in A431 cells (Norvell and Green, 1998Norvell S.M. Green K.J. Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells.J Cell Sci. 1998; 111: 1305-1318Crossref PubMed Google Scholar). These data suggest that Dsg1 and Dsg3 expression have different effects on desmosome formation and integrity. To characterize further and define these differences, we constructed full-length and N-terminally truncated mutants of Dsg1 and Dsg3, and introduced them into cultured keratinocytes using an adenovirus vector. Taking advantage of the adenovirus vector to control expression level by changing multiplicity of infection (MOI), we evaluated the effects of expression levels of these isoforms on the integrity of a desmosome–keratin structured complex. The human embryonic kidney cell line 293 was obtained from American Type Tissue Culture (ATCC; Rockville, MD). The human naturally immortalized keratinocyte HaCaT cell line was a kind gift from Dr Norbert Fusenig (German Cancer Research Center, Heidelberg, Germany). These cells were cultured in Dulbecco modified Eagle's medium supplemented with 10% fetal bovine serum. The full-length cDNA encoding human Dsg1 was a kind gift from Dr Kathleen Green (North-western University, Chicago, IL). The construction of the Dsg3 mutant, in which a large part of the extracellular domain was deleted and a seven c-myc tag was inserted flanking the C-terminal end, has been described previously (Hanakawa et al., 2000Hanakawa Y. Amagai M. Shirakata Y. Sayama K. Hashimoto K. Different effects of dominant negative mutants of desmocollin and desmoglein on the cell–cell adhesion of keratinocytes.J Cell Sci. 2000; 113: 1803-1811Crossref PubMed Google Scholar). A mutant of Dsg1 (Dsg1ΔEC), with a large part of the extracellular domain deleted and with a seven c-myc tag flanking the C-terminal end, was also constructed. Dsg1 DNA fragments (nucleotides 13–402 and 1726–3360) were generated by polymerase chain reaction using primers DG1F15′ (5′-TTTTAGGGTGGGGATCCAGAC-3′), DG1F13′ (5′-GGGGTACCGTCTTCACCTTCACGACAGGC-3′, KpnI site at the 3′ end), DG1F25′ (5′-GGGGTACCGGCAGACAAGAAAGTACT-3′, KpnI site at the 5′ end), and DG1F23′ (5′-CGGAATTCCTTGCTATAT-TGCACGGT-3′, EcoRI site at the 3′ end). The polymerase chain reaction products were subcloned into pcDNA1–7myc/Amp to generate pcDsg1ΔEC. pcDNA1–7myc/Amp was a kind gift from Dr Kathleen Green (North-western University;Roth et al., 1991Roth M.B. Zahlerm A.M. Stolkm J.A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription.J Cell Biol. 1991; 115: 587-596Crossref PubMed Scopus (262) Google Scholar). The cosmid cassette pAxCAw, pAxCALNLw (Kanegae et al., 1996Kanegae Y. Takamori K. Sato Y. Lee G. Nakai M. Saito I. Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase.Gene. 1996; 181: 207-212Crossref PubMed Scopus (102) Google Scholar), the loxP-NeoR-loxP unit under a CA promoter consisting of a cytomegalovirus enhancer and chicken β-actin promoter (Niwa et al., 1991Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4424) Google Scholar), the nuclear localizing signal-tagged Cre recombinase-expressing adenovirus (AxCANCre), the control adenovirus Ax1w, and the parent virus Ad5-dLX (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (781) Google Scholar) were all kind gifts from Dr Izumu Saito (Tokyo University, Japan). Fragments of Dsg1ΔEC were subcloned into the adenovirus cosmid cassette pAxCALNLw. Adenovirus containing CALNL and Dsg1ΔEC (AxCALNLDsg1ΔEC) were generated by the COS-TPC method (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (781) Google Scholar). The cosmid DNA was mixed with the EcoT22I-digested DNA-terminal protein complex of Ad5-dLX, and used to cotransfect 293 cells. Recombinant viruses were generated through homologous recombination in 293 cells. Virus stocks were prepared by a standard procedure (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (781) Google Scholar). Concentrated, purified virus stocks were prepared by the CsCl gradient method and the virus titer was checked using the plaque formation assay. HaCaT cells were doubly infected with AxCANCre and Ax1w, AxCALNLDsg1ΔEC, or AxCALNLDsg3ΔEC. HaCaT cells were infected with AxCANCre at a MOI of 5 and Ax1w, AxCALNLDsg1ΔEC, or AxCALNLDsg3ΔEC at a MOI of 5 (low expression) or were infected with AxCANCre at a MOI of 15 and Ax1w, AxCALNLDsg1ΔEC, or AxCALNLDsg3ΔEC at a MOI of 15 (high expression). Adenovirus expressing mouse Dsg1-FLAG and mouse Dsg3-FLAG have already been shown elsewhere (Amagai et al., 2002Amagai M. Yamaguchi T. Hanakawa Y. Nishifuji K. Sugai M. Stanley J.R. Staphylococcal exfoliative toxin B specifically cleaves desmoglein 1.J Invest Dermatol. 2002; 118: 845-850Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Briefly, cDNA encoding mouse Dsg1-FLAG and mouse Dsg3-FLAG were subcloned into the adenovirus cosmid cassette pAxCAw. Adenovirus containing CA promoter and cDNA encoding mouse Dsg1-FLAG and mouse Dsg3-FLAG (AxmDsg1F and AxmDsg3F) were generated by the COS-TPC method (terminal protein complex). Purified, concentrated, and titer-checked viruses were infected to HaCaT cells at a MOI of 10 and 30. The following monoclonal antibodies were used: AE1 and AE3 (murine anti-keratin intermediate filament; PROGEN, Heidelberg, Germany); 9E10.2 (murine anti-myc; American Type Tissue Culture); anti-β-catenin (murine; Transduction Laboratories, Lexington, KY); DPI/II (murine anti-desmoplakin; PROGEN); PG5.1 (murine anti-plakoglobin; PROGEN); PP1-5C2 (murine anti-plakophilin 1; PROGEN); anti-plakophilin 2 (murine; PROGEN); anti-involucrin (Abcam, Cambridge, U.K.); and 6H6 (murine anti-Dsg3). The following rabbit anti-sera were used: Z622 (anti-pan-keratin; DAKO, Copenhagen, Denmark); anti-myc (a kind gift from Dr John Stanley, University of Pennsylvania, PA); anti-keratin 1 (CONVENCE, Richmond, CA); anti-loricrin (CONVENCE); and anti-FLAG (Abcam). Fluorescein isothiocyanate-conjugated goat anti-mouse or goat anti-rabbit antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and rhodamine-conjugated goat anti-rabbit antibody (BioSource, Camarillo, CA) were used. Cells grown on Laboratory-Tech 4-well culture slides (Nalge-Nunc, Napierville, IL) were fixed with methanol at –20°C for 20 min and permeabilized with 0.05% Triton X-100 in Tris-buffered saline containing 1 mM CaCl2 (TBS-Ca2+) at room temperature for 5 min. After incubation with 1% bovine serum albumin in TBS-Ca2+ for 20 min at room temperature, the cells were incubated with various antibodies at the appropriate dilution in 1% bovine serum albumin in TBS-Ca2+ for 1 h at room temperature. The samples were further incubated with fluorescein isothiocyanate- or rhodamine-conjugated secondary antibodies at the appropriate dilution in 1% bovine serum albumin in TBS-Ca2+ for 1 h at room temperature. Stained cells were examined using a confocal laser microscope (Zeiss, Oberkachen, Germany). For immunoblotting, cells were lyzed in sodium dodecyl sulfate sample buffer (62.5 mM Tris–HCl, pH 7.5, 1% sodium dodecyl sulfate, 0.0025% bromophenol blue, 10% glycerol, 2.5% 2-mercaptoethanol) and subjected to immunoblotting using enhanced chemifluorescence according to the manufacturer's protocol (Amersham Pharmacia Biotech, Uppsala, Sweden). For soluble and insoluble fractionation, the cells were scraped in 1% Nonidet P-40 and 1% Trion-X-100 in TBS-Ca2+ on ice. Following centrifugation at 15,000 r.p.m. (30,000g) for 10 min at 4°C, the supernatant was collected as the soluble fraction. The pellet was dissolved in sodium dodecyl sulfate sample buffer as the insoluble fraction. Immunoprecipitation was carried out on adenovirus-infected cells extracted with 1% Nonidet P-40, 1% Triton X-100, 2 mM CaCl2, and 150 mM NaCl in 10 mM Tris–HCl, pH 7.4, in the presence of protease inhibitors (2 μg aprotinin per ml, 2 μg leupeptin per ml, phenylmethylsulfonyl fluoride 1 mM). Aliquots of cell lysates were preadsorbed with normal mouse IgG or normal rabbit IgG and protein G-Sepharose (Amersham Pharmacia Biotech), then precipitated with various antibodies and protein G-Sepharose. Immunoprecipitates were eluted from protein G-Sepharose with sodium dodecyl sulfate sample buffer and subjected to immunoblotting. The immunoblots were analyzed by using ImageQuantTM (Amersham Pharmacia Biotech) software to determine the relative ratio in the immunoprecipitated complex. A Dsg1 mutant was constructed by deleting a large part of the extracellular domain and adding a seven c-myc tag (Dsg1ΔEC; Figure 1). The corresponding Dsg3 mutant has been previously described (Hanakawa et al., 2000Hanakawa Y. Amagai M. Shirakata Y. Sayama K. Hashimoto K. Different effects of dominant negative mutants of desmocollin and desmoglein on the cell–cell adhesion of keratinocytes.J Cell Sci. 2000; 113: 1803-1811Crossref PubMed Google Scholar; Figure 1). To prevent the expression of toxic, truncated products that interfered with the production of recombinant virus, the loxP gene sequence, disrupted by a neomycin-resistance cassette, was interposed between the CAG promoters and the coding regions in the mutants (Kanegae et al., 1996Kanegae Y. Takamori K. Sato Y. Lee G. Nakai M. Saito I. Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase.Gene. 1996; 181: 207-212Crossref PubMed Scopus (102) Google Scholar). Co-infection of keratinocytes (HaCaT cells) with adenovirus-expressing Cre recombinase removed the stuffer sequence and activated the expression of the mutant desmogleins. The expression of mutant proteins was detected at 6 h and reached a plateau between 24 and 36 h after infection (data not shown). Almost all cells expressed the mutant proteins when HaCaT cells were coinfected with adenovirus carrying mutant desmogleins and Cre recombinase, flanked with the nuclear localization signal (NCre) at a MOI of 30 (mutant: Cre=1:1) or a MOI of 10. The relative expression levels of both mutants were essentially the same when HaCaT cells were infected at the same MOI, with a MOI of 30 causing greater expression than MOI of 10 (Figure 2). No toxicity was apparent in cells infected with control adenovirus (Ax1w) or NCre adenovirus at MOI of 10 or 30 in phase microscope. We examined the effects of these different expression levels of mutant desmogleins on HaCaT cells, at 36 h after infection. There were no apparent morphologic changes in cells infected with Dsg1ΔEC adenovirus or Dsg3ΔEC adenovirus when compared with cells infected with the control adenovirus, using phase microscopy (data not shown). The effects of mutant desmogleins on the formation of adherens junctions were examined by immunofluorescence staining using double staining for β-catenin (a marker of adherens junctions) and myc. No differences were observed in β-catenin staining patterns 36 h after infection with control adenovirus, Dsg1ΔEC adenovirus, or Dsg3ΔEC adenovirus at a MOI of 30 (data not shown).Figure 2Expression of mutant desmogleins in HaCaT cells. Immunoblots using anti-myc antibody show the expression of Dsg1ΔEC and Dsg3ΔEC in HaCaT cells 36 h after adenovirus infection. The expression levels of Dsg1ΔEC and Dsg3ΔEC were stronger at a MOI of 30 than at a MOI of 10.View Large Image Figure ViewerDownload (PPT) We next studied desmosomal changes in HaCaT cells induced by each mutant by immunofluorescence localization of keratin and desmoplakin. Dsg1ΔEC and Dsg3ΔEC adenovirus infections effectively abolished keratin insertion and desmoplakin expression at cell–cell contact sites when the expression levels of the mutant were high (MOI = 30; Figure 3a,b,c,l,e,n). Keratin was found in perinuclear regions, and desmoplakin was seen in punctuate locations throughout the cytoplasmic region. No differences were observed in the keratin or desmoplakin staining patterns 36 h after infection with control adenovirus (Figure 3a,b,a,j). In contrast, when the expression level was low (MOI=10), Dsg3ΔEC adenovirus partially affected on keratin insertion and desmoplakin expression (Figure 3a,b,d,m), whereas Dsg1ΔEC adenovirus completely inhibited keratin insertion and desmoplakin expression at the cell–cell contact site (Figure 3a,b,b,k). In low-level expression, the Dsg3ΔEC mutant proteins were detected with anti-myc antibodies at cell–cell boundaries and in the cytoplasm (Figure 3b,h,q); however, the Dsg1ΔEC mutant proteins were detected mainly in the cytoplasm but not at cell–cell contact sites (Figure 3b,f,o). In high-level expression, both the Dsg3ΔEC and Dsg1ΔEC mutant proteins were detected at cell–cell boundaries and in the cytoplasm (Figure 3b,g,i,p,r). We combined immunoprecipitation with immunoblotting in order to detect plakoglobin bound to either Dsg1ΔEC or Dsg3ΔEC (Figure 4). Cell extracts from HaCaT cells expressing mutant desmogleins were immunoprecipitated with an anti-myc antibody, then subsequently immunoblotted with anti-myc and anti-plakoglobin antibodies. Plakoglobin coprecipitated with the Dsg1ΔEC and Dsg3ΔEC proteins expressed by the two mutants with similar efficiencies (Figure 4a). When the order in which the antibodies were used was reversed, i.e., immunoprecipitation with anti-plakoglobin followed by immunoblotting with anti-myc antibody, plakoglobin was again coprecipitated with Dsg1ΔEC and Dsg3ΔEC at similar levels (Figure 4b). We could not detect any association of plakophilin 1 and plakophilin 2 with Dsg1ΔEC or Dsg3ΔEC (data not shown). Several published data indicate that disruption of cell adhesion leads to altered differentiation in keratinocytes (Allen et al., 1996Allen E. Yu Q.C. Fuchs E. Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation.J Cell Biol. 1996; 133: 1367-1382Crossref PubMed Scopus (133) Google Scholar;Zhu and Watt, 1996Zhu A.J. Watt F.M. Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes.J Cell Sci. 1996; 109: 3013-3023Crossref PubMed Google Scholar). To test whether Dsg1ΔEC and Dsg3ΔEC might affect differentiation in HaCaT cells, we analyzed the differentiation marker of keratinocytes after transduction with Dsg1ΔEC and Dsg3ΔEC. Western blotting of Dsg1ΔEC and Dsg3ΔEC expressing HaCaT cell extracts stained with anti-keratin 1, anti-involucrin, and anti-loricrin antibody antibodies showed no change (data not shown), suggesting dominant-negative effects on desmosomes did not affect differentiation in our system. We also tried to find the functional difference in full-length Dsg1 and Dsg3 in desmosome formation. We constructed adenovirus containing full-length Dsg1 and Dsg3 with FLAG epitope tag on the C-termini and transduced HaCaT cells. Western blots stained with anti-FLAG antibody showed the relative expression levels of Dsg1 and Dsg3 were essentially the same when HaCaT cells were infected at the same MOI (Figure 5). Double immunofluorescence staining of anti-FLAG and anti-desmoplakin showed colocalization of Dsg1 and Dsg3 with desmoplakin at cell–cell contact site when the expression level was low, although in the merged picture red staining of anti-FLAG was stronger compared with desmoplakin staining (Figure 6). These results show the incorporation of Dsg1 and Dsg3 into desmosomes when their expression levels were low.Figure 6Full-length Dsg1 and Dsg3 were incorporated into desmosome at low-level expression. HaCaT cells were transduced with adenovirus containing full-length Dsg1 and Dsg3 at a MOI of 10. Double immunofluorescence staining of anti-FLAG (A,D) and anti-desmoplakin (B,E) showed colocalization of Dsg1 and Dsg3 with desmoplakin at cell–cell contact site. C Shows a merged picture of A and B, and F shows a merged picture of D and E.View Large Image Figure ViewerDownload (PPT) On the other hand, when the expression levels were high, Dsg1 and Dsg3 showed different effects on desmosome formation. In high level full-length expressing HaCaT cells, double immunofluorescence staining of anti-FLAG and anti-keratin showed insertion of keratin to cell–cell contact site in Dsg3 (Figure 7a,b). Disruption of insertion to cell–cell contact site and shrinking around nucleus of keratin, however, was observed in Dsg1. Double staining of FLAG and desmoplakin showed dot staining of desmoplakin at cell–cell contact site in Dsg3, suggesting Dsg3 is still incorporated into desmosomes at high-level expression. In contrast, high-level expression of Dsg1 changed staining of desmoplakin to a diffuse cytoplasmic pattern. These results suggest that when full-length protein expression levels were high, Dsg1 disrupts desmosome formation but Dsg3 is still incorporated into desmosomes. In high-level expression, full-length Dsg3 localized mainly at cell–cell contact site (Figure 7a,c); however, Dsg1 localized both cell–cell contact site and cytoplasmic region (Figure 7e,g). Although full-length Dsg1 in cytoplasm showed reticular pattern, we cannot explain the reason for such a staining pattern. We investigated the roles of Dsg1 and Dsg3 in desmosome formation in HaCaT keratinocytes with adenovirus vectors containing dominant-negative mutants and full-length forms of Dsg1 and Dsg3. When the mutant desmoglein expression levels were high, both Dsg1ΔEC and Dsg3ΔEC inhibited the formation of desmosomes. When expression levels were low, however, Dsg3ΔEC only partially inhibited the formation of desmosomes, whereas Dsg1ΔEC completely inhibited the formation of desmosomes. These findings indicate that the dominant-negative effect on desmosome formation of the mutated Dsg1 was stronger than that of mutated Dsg3. On the other hand, when the full-length desmoglein expression levels were low, both Dsg1 and Dsg3 incorporated into desmosomes. In contrast, when expression levels were high, Dsg1 inhibited the formation of desmosomes, whereas Dsg3 did not. These findings indicate that the functional difference between the full-length form of Dsg1 and Dsg3 at least in less differentiated cells in proliferating in culture. Why is the effect of the Dsg1ΔEC mutant stronger than that of Dsg3ΔECΔ One possibility is that mutated desmoglein interacts with desmosome components. By combining immunoprecipitation with subsequent immunoblotting, we found that the cytoplasmic domains of Dsg1ΔEC and Dsg3ΔEC interacted with plakoglobin to the same extent. Recently it has been reported that the binding ratio of plakoglobin/full-length Dsg1 exhibited less than 2:1 but was still higher compared with Dsg3 (Bannon et al., 2001Bannon L.J. Cabrera B.L. Stack M.S. Green K.J. Isoform-specific differences in the size of desmosomal cadherin/catenin complexes.J Invest Dermatol. 2001; 117: 1302-1306Crossref PubMed Scopus (16) Google Scholar). Sequestration of plakoglobin by the mutant desmoglein proteins would make it inaccessible to native desmoglein and might partly explain the dominant-negative effect. The idea of differential sequestering of plakoglobin, however, is contraindicated by the finding that there were no differences in plakoglobin-binding capacity between Dsg1ΔEC and Dsg3ΔEC. It seems likely that molecules other than plakoglobin contribute to the formation of desmosomes. Another armadillo gene family member of plakophilin (Hatzfeld et al., 1994Hatzfeld M. Kristjansson G.I. Plessmann U. Weber K. Band 6 protein, a major constituent of desmosomes from stratified epithelia, is a novel member of the armadillo multigene family.J Cell Sci. 1994; 107: 2259-2270Crossref PubMed Google Scholar;Heid et al., 1994Heid H.W. Schmidt A. Zimbelmann R. et al.Cell type-specific desmosomal plaque proteins of the plakoglobin family: plakophilin 1 (band 6 protein).Differentiation. 1994; 58: 113-131Crossref PubMed Scopus (158) Google Scholar;Mertens et al., 1996Mertens C. Kuhn C. Franke W.W. Plakophilins 2a and 2b. constitutive proteins of dual location in the karyoplasm and the desmosomal plaque.J Cell Biol. 1996; 135: 1009-1025Crossref PubMed Scopus (237) Google Scholar;Bornslaeger et al., 2001Bornslaeger E.A. Godsel L.M. Corcoran C.M. Park J.K. Hatzfeld M. Kowalczyk A.P. Green K.J. Plakophilin 1 interferes with plakoglobin binding to desmoplakin, yet together with plakoglobin promotes clustering of desmosomal plaque complexes at cell–cell borders.J Cell Sci. 2001; 114: 727-738Crossref PubMed Google Scholar) is now thought to be a major desmosomal plaque protein; however, we could not detect binding of plakophilin 1 or plakophilin 2 to Dsg1ΔEC or Dsg3ΔEC (data not shown). We cannot rule out the possibility that indirect interactions occur between the mutant cadherins and plakophilin 1 and plakophilin 2. The other possibility for the disruption of desmosome is instability of desmosomes at cell–cell contact sites. Exfoliative toxin from Staphylococcus aureus cleaves the extracellular domain of Dsg1 and causes dysfunction of Dsg1 (Amagai et al., 2001Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.Nat Med. 2001; 6: 1275-1277Google Scholar). Keratinocytes in neonatal mice injected with exfoliative toxin A or B showed internalization of Dsg1 and resultant blister formation in the upper layers of the epidermis. We speculate that cleaved Dsg1 might be unstable in the desmosome because it does not bind its partner, and such unstabilized desmosomes could be easily internalized; however, these observed internalizations only happened in living epidermis but were not seen in the cryosection of epidermis (Amagai et al., 2002Amagai M. Yamaguchi T. Hanakawa Y. Nishifuji K. Sugai M. Stanley J.R. Staphylococcal exfoliative toxin B specifically cleaves desmoglein 1.J Invest Dermatol. 2002; 118: 845-850Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). It is probable that the dynamics of a living cell are a prerequisite for desmosome internalization after the dysfunction of desmogleins. These data are consistent with our findings of a retracted, perinuclear staining of keratin filament network in HaCaT cells expressing high amounts of mutant desmogleins. The reason for the disruption of the desmosome with full-length Dsg1 might be similar. We speculate that Dsg1 might associate with more desmosomal molecules than Dsg3, based on our results with HaCaT cells with regard to basal layer keratinocytes in the epidermis. When Dsg1 cannot find enough amount partners to stabilize on the cell surface, Dsg1 might be easily internalized with already interacted desmosomal proteins. A recent report that Dsg2 efficiently incorporated into desmosomes but Dsg1 did not in MDCK and A431 cells (Ishii et al., 2001Ishii K. Norvell S.M. Bannon L.J. Amargo E.V. Pascoe L.T. Green K.J. Assembly of desmosomal cadherins into desmosomes is isoform dependent.J Invest Dermatol. 2001; 117: 26-35Crossref PubMed Google Scholar) support the idea that cell and differentiation specific capacity for isotype dependent incorporation of desmogleins into desmosome. This study has shown a functional difference between Dsg1 and Dsg3 on desmosome formation in keratinocytes; however, why does the human body need different isoforms of Dsg1 and Dsg3 in stratified epithelia? Dsg3 knockout mice showed erosions in the oral mucosa and in areas subject to mechanical irritation (Koch et al., 1997Koch P.J. Mahoney M.G. Ishikawa H. et al.Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris.J Cell Biol. 1997; 137: 1091-1102Crossref PubMed Scopus (363) Google Scholar), although the epidermis was intact in nonmechanically stressed areas. This result suggests that Dsg1 may compensate functionally for a deficiency in Dsg3 (Mahoney et al., 1999Mahoney M.G. Wang Z. Rothenberger K. Koch P.J. Amagai M. Stanley J.R. Explanations for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris.J Clin Invest. 1999; 103: 461-468Crossref PubMed Scopus (384) Google Scholar;Wu et al., 2000Wu H. Wang Z.H. Yan A. et al.Protection against pemphigus foliaceus by desmoglein 3 in neonates.N Engl J Med. 2000; 343: 31-35Crossref PubMed Scopus (105) Google Scholar). If this hypothesis is correct, small amounts of Dsg1 in the lower part of the epidermis are enough to produce desmosomes, although normal desmosomal function may be compromised in such situations. Although expression levels of Dsg1 are lower than Dsg3, Dsg1 may play an important part in desmosome formation in basal layer keratinocytes in epidermis and in certain circumstances, compensate for the lack of Dsg3 function. The distribution of Dsg1 and Dsg3 is different between the epidermis and mucous membrane (Shirakata et al., 1998Shirakata Y. Amagai M. Hanakawa Y. Nishikawa T. Hashimoto K. Lack of mucosal involvement in pemphigus foliaceus may be due to low expression of desmoglein 1.J Invest Dermatol. 1998; 110: 76-78Crossref PubMed Scopus (141) Google Scholar). In mucous membranes, Dsg1 is less expressed in upper layers compared with epidermis. Recently, involucrin promoter driven transgenic expression of Dsg3 in mouse showed altered stratum corneum and increased transepidermal water loss in epidermis (Elias et al., 2001Elias P.M. Matsuyoshi N. Wu H. Lin C. Wang Z.H. Brown B.E. Stanley J.R. Desmoglein isoform distribution affects stratum corneum structure and function.J Cell Biol. 2001; 153: 243-249Crossref PubMed Scopus (107) Google Scholar). These data suggest that Dsg1 and Dsg3 contribute not only to the formation of desmosomes but also to stratification. Strictly organized expression of Dsg1 and Dsg3 might be needed to architect the difference in epidermis and mucous membrane, which have different functions, such as water loss protection in epidermis and water absorption in mucous membrane. In summary, the discovery of the difference in effects on desmosome by Dsg1ΔEC and Dsg3ΔEC mutants and by full-length forms of Dsg1 and Dsg3 support the hypothesis of functional differences in desmoglein activities during desmosome formation in keratinocytes. Further investigations are needed in order to clarify the precise roles of different desmoglein isotopes in human tissue. We especially thank Dr John Stanley for insightful discussion and critical reading of this manuscript. We thank Ms Teruko Tsuda and Mrs Akiko Kon for expert technical assistance. We also thank Dr Izumu Saitou for the adenovirus expression system and adenovirus Cre-loxP system, Dr June-ichi Miyazaki for the CAG promoter, Dr Norbert Fusenig for the HaCaT cells, Dr Kathleen Green for the pcDNA1–7myc/Amp and cDNA encoding human Dsg1, and Dr John Stanley for the polyclonal anti-myc antibody. This work was supported by a Health Sciences Research Grant for Research on Specific Diseases from the Ministry of Health, labor and Welfare of Japan and a Grant-in-Aid of Scientific Research from the Ministry of Education, Cluture, Sports, Science and Technology of Japan.