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ABSTRACT: Although desmocollins (Dscs) and desmogleins (Dsgs) are known to be bound to each other to form desmosomes, neither their interactions nor regulations that occur in human keratinocytes grown in low and high Ca2+medium has been determined. In this study, we investigated whether Dsc3 interacts with Dsg3 in a cell line of human squamous cell carcinoma keratinocytes (DJM-1) grown in low (0.05 mm) or high (1.27 mm) Ca2+ medium. Anti-Dsc3 monoclonal antibody did not co-immunoprecipitate Dsg3 nor plakoglobin with Dsc3 in low Ca2+ culture, whereas it co-immunoprecipitated plakoglobin already at 10 min and Dsg3 at 60 min after Ca2+ -switch in association with Dsc3 phosphorylation at serine residues. These results suggest that both the binding of Dsc3 to plakoglobin and Dsc3 phosphorylation are involved in Dsc3 binding to Dsg3 during Ca2+ -induced desmosome assembly.
Experimental Dermatology 05/2009; 18(4):404-8. · 3.54 Impact Factor
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ABSTRACT: We have shown that binding of bullous pemphigoid (BP)-patient IgG (BP-IgG) causes the internalization of BP180 from the cell membrane. This study examined whether BP-IgG treatment can deplete cultured keratinocytes of BP180, how it affects cellular levels of alpha6 and beta4 integrins (by western blot analysis using monoclonal antibodies to these antigens), and whether it reduces adhesion of cells to the culture dish (by a vibration detachment assay). All BP-IgG or BP sera with high values of BP180-ELISA from 18 BP patients before and after oral corticosteroid treatment showed dramatically decreased BP180 in cells after 6 hours of BP-IgG stimulation, whereas alpha6 and beta4 integrin levels were not decreased. Even IgG from patients in whom oral corticosteroid had suppressed active blistering could deplete cells of BP180, as long as sera retained a high value of BP180-ELISA. On the other hand, reduction of cell BP180 content increased detachment of cells from the dish. These results suggest that BP-IgG reduces hemidesmosomal BP180 content, weakening the adhesion of hemidesmosomes to the lamina densa. In the presence of BP180 deficiency, inflammation generated by BP180 immune-complex formation might then tear the weakened lamina lucida, and this could lead to generation of the BP-specific split at the lamina lucida.
Journal of Investigative Dermatology 02/2009; 129(4):919-26. · 6.31 Impact Factor
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ABSTRACT: We recently showed that p120 catenin (p120ctn), which is an armadillo family protein member that binds to E-cadherin (E-cad), is also localized to desmosomes by directly or indirectly binding to desmogleins (Dsg). We examined whether p120ctn is associated with Dsg1 and Dsg3, as compared with E-cad and plakoglobin (PG), in keratinocytes grown in high or low Ca2+, using a human squamous cell carcinoma cell line, DJM-1 cells. The cell lysate of DJM-1 cells grown in high- or low-Ca2+ media was immunoprecipitated with anti-Dsg1/2 and Dsg3 antibodies, and we examined whether p120ctn is associated with Dsg1 and Dsg3. Then, we observed the co-localization between Dsg3 and p120ctn in cells grown in high- or low-Ca2+ medium on double-staining immunofluorescence microscopy using anti-p120ctn and anti-Dsg3 antibodies. Immunoprecipitates with anti-Dsg1/2 and Dsg3 antibodies in cells grown in high-Ca2+ medium contained p120ctn. In contrast, in low-Ca2+ medium, p120ctn was co-immunoprecipitated with neither Dsg1 nor Dsg3, but was co-immunoprecipitated with E-cad in cells grown in both high- and low-Ca2+ media. Dsg3 was associated with PG in cells grown in both low- and high-Ca2+ media. On immunofluorescence microscopy, p120ctn and Dsg3 were independently observed in cells grown in low-Ca2+ medium; p120ctn, but not Dsg3, was observed in a linear pattern at the cell-cell boundary. However, they were co-localized at cell-cell contacts in cells grown in high-Ca2+ medium. Thus, these proteins are not co-localized in low Ca2+ medium. These results suggest that p120ctn plays an important role in Ca2+-induced desmosome formation.
The Journal of Dermatology 07/2008; 35(6):317-24. · 1.49 Impact Factor
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ABSTRACT: P120-catenin (p120ctn) is an armadillo-repeat protein that directly binds to the intracytoplasmic domains of classical cadherins. p120ctn binding promotes the stabilization of cadherin complexes on the plasma membrane and thus positively regulates the adhesive activity of cadherins. Using co-immunoprecipitation, we show here that p120ctn associates to desmogleins (Dsg) 1 and 3. To determine which region is involved in the association between Dsg3 and p120ctn, we constructed mutant Dsg3 proteins, in which various cytoplasmic subdomains were removed. The tailless Dsg3 constructs Delta IA:AA1-641Dsg3 and Delta 641-714Dsg3, which do not contain the intracellular anchor (IA) region, did not coprecipitate with p120cn, nor did they colocalize at the plasma membrane. Immunocytochemical analysis revealed that p120ctn does not localize to desmosomes, but colocalizes with Dsg3 at the cell surface. A biotinylation assay for Dsg3 showed that biotinylated Delta 641-714Dsg3 was turned over more rapidly than wild-type Dsg3. These results indicate that the membrane proximal region (corresponding to residues 641-714) in the IA region of Dsg3 is necessary for complex formation with p120ctn, and to maintain free Dsg3 at the cell surface before it is integrated into desmosomes. In summary, we show that p120ctn is a novel interactor of the Dsg proteins, and may play a role in desmosome remodeling.
Experimental Cell Research 06/2008; 314(8):1683-92. · 3.58 Impact Factor
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ABSTRACT: Although pemphigus vulgaris (PV)-IgG has been shown to activate urokinase plasminogen activator (uPA) in cultured keratinocytes, activation of uPA is thought to have no primary role in PV-acantholysis, because PV-IgG is still pathogenic in uPA- and tissue-PA-knockout mice.
To determine if PV-IgG-induced uPA activation is due to specific antibody against Dsg3, we examined whether or not pathogenic monoclonal anti-Dsg3 antibody can activate uPA, because PV-IgG is thought to contain antibodies against unknown antigens besides Dsg3.
We stimulated cultured normal human and DJM-1 keratinocytes with monoclonal anti-Dsg3 IgG1 antibodies (pathogenic AK23, AK19 and nonpathogenic AK18, AK20), negative control monoclonal mouse IgG1 and positive control PV-IgG. Cells were treated with IgGs over a time course of 24h, and uPA-protein content and activity in the culture medium were determined by enzyme-linked immunosorbent assay (ELISA) and chromogenic assay, respectively.
The uPA-protein content in samples treated with or without pathogenic, nonpathogenic, control monoclonal mouse IgG1s and PV-IgGs increased continuously up to 24h, with no differences between samples, suggesting a spontaneous secretion. In contrast, uPA activity in the culture medium of cells treated with PV-IgG increased dramatically, whereas that of cells treated with all AK-IgGs and control monoclonal mouse IgG1 did not increase at all.
These results suggest that PV-IgG-dependent uPA activation is not related to anti-Dsg3 antibody activity, which is an essential factor in PV-IgG acantholysis, and that it may be due to other antigens than Dsg3 or unknown factors contained in PV-IgG fraction.
Journal of Dermatological Science 09/2007; 47(2):119-25. · 3.72 Impact Factor
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ABSTRACT: Pemphigus vulgaris (PV) is an autoimmune blistering disease, characterized by the loss of cell-cell adhesion between epidermal keratinocytes and the presence of autoantibody against desmoglein 3 (Dsg3), which provides adhesive integrity to desmosomes between adjacent keratinocytes. We have previously shown that PV-IgG purified from patients depletes desmosomes of Dsg3. However, PV-IgG contains not only antibodies against a variety of different epitopes of Dsg3 but also against other unknown antigens. Therefore, we examined whether the Dsg3-depleting activity of PV-IgG is generated specifically by anti-Dsg3 activity in a human squamous cell carcinoma cell line (DJM-1) and normal human keratinocytes by using four different pathogenic and nonpathogenic monoclonal antibodies against Dsg3. We demonstrate that these monoclonal antibodies deplete cells and desmosomes of Dsg3, as PV-IgG does. Individual monoclonal anti-Dsg3 antibodies display characteristic limits to their Dsg3-depleting activity, which correlates with their pathogenic activities. In combination, these antibodies exert a cumulative or synergistic effect, which may explain the potent Dsg3-depleting capability of PV-IgG, which is polyclonal. Finally, although Dsg3-depletion activity correlated with AK-monoclonal antibody pathogenicity in mouse models, the residual level of Dsg3, when below approximately 50%, does not correlate with the adhesive strength index in the present study. This may suggest that although the Dsg3 depletion is not indicative for adhesive strength, the level of Dsg3 can be used as a read-out of pathogenic changes within the cell and that the Dsg3 depletion from desmosomes plays an important role in skin fragility or susceptibility to blister formation in PV patients.
Journal of Biological Chemistry 07/2007; 282(24):17866-76. · 4.77 Impact Factor
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ABSTRACT: Pemphigus vulgaris is an autoimmune blistering disease caused by antibodies against desmoglein (Dsg) 3. We previously reported that pemphigus vulgaris (PV)-IgG caused the formation of Dsg3-depleted desmosomes in normal human cultured keratinocytes and DJM-1, a human squamous cell carcinoma cell line. In the present study, we injected PV-IgG and normal human IgG into neonatal mice and examined the quantities of Dsg3 in the mouse skin. We showed that injection of PV-IgG into neonatal mice caused suprabasal blister formation and approximately 30% reduction of Dsg3 in mouse epidermal keratinocytes, compared to mice injected with normal human IgG. In addition, we showed that the quantity of Dsg3 in the skin of patients with PV did decrease, as compared to that in healthy volunteers. Our data suggests the reduction of Dsg3 might be relevant to blister formation. These results also suggest that even a partial depletion of Dsg3 may contribute to blistering in PV patients.
Archives for Dermatological Research 07/2007; 299(3):165-7. · 2.28 Impact Factor
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Journal of Dermatological Science 01/2006; 40(3):209-11. · 3.72 Impact Factor
