We have demonstrated previously that pemphigus vulgaris (PV)-IgG induces activation of phospholipase C (PLC), production of inositol 1,4,5-trisphosphate, and a rapid transient increase in [Ca2+]i in cultured human keratinocytes, leading to secretion of plasminogen activator and cell-cell detachment in cell culture. In the current study, to examine the involvement of protein kinase C (PKC) in the mechanism of blister formation in PV, we studied the PV-IgG-induced translocation of PKC isozymes from the cytosol to the particulate/cytoskeleton (p/c) fractions and the activation of PKC in human keratinocytes. Cells cultured in Eagle's minimum essential medium were incubated with PV-IgGs for 30 s, 1 min, 5 min, or 30 min. PV-IgG binding to the cell surface antigen (desmoglein III) induced translocation of PKC-alpha from the cytosol to the p/c fractions within 30 s, with a peak at 1 min that lasted at least 30 min. PKC-delta also was translocated within 1 min and reached a peak at 5 min but was reduced to basal levels at 30 min. Alternatively, PKC-eta translocation to the p/c fraction was induced slowly, taking more than 5 min, and was reduced to approximately half-maximum at 30 min, whereas PKC-zeta translocation reached a maximum at 30 s, rapidly returning to baseline by 5 min after PV-IgG stimulation. The total PKC activity in the p/c fraction also was increased after PV-IgG exposure, peaked at 1 min, and was sustained for at least 30 min. These findings suggest that a unique activation profile of PKC isomers may be involved in mediating the intracellular signaling events induced by PV-IgG binding to desmoglein III in cultured human keratinocytes.
The precise mechanism for acantholysis after pemphigus IgG binds to the cell surface is as yet unknown, although involvement of proteinases such as plasminogen activator (PA) has been suggested. We previously reported that pemphigus IgG, but not normal nor bullous pemphigoid IgGs, caused a transient increase in intracellular calcium ([Ca++]i) and inositol 1,4,5-trisphosphate (IP3) concentration in cultured DJM-1 cells (a squamous cell carcinoma line). To clarify whether phospholipase C is involved in this process after the antibody binds to the cell surface, we examined the effects of a specific phospholipase C inhibitor (U73122) on the pemphigus IgG-induced increase in [Ca++]i, IP3, PA secretion, and cell-cell detachment in DJM-1 cells. [Ca+2]i and IP3 contents were determined with or without 30-min pre-incubation with U73122 or an inactive analogue (U73343) with fura-2 acetoxymethylester and a specific IP3 binding protein, respectively. PA activity in the culture medium was measured after various incubation periods with pemphigus IgG by two-step amidolytic assay. The detachment of cell-cell contacts was examined by detecting the retraction of keratin filament bundle from cell-cell contact points to the perinuclear region by immunofluorescence microscopy using anti-keratin antibody. Pemphigus IgG immediately increased [Ca++]i and IP3 content. PA activity in the culture medium has also been increased at 24 h after pemphigus IgG was added in association with cell-cell detachment. However, pre-incubation with U73122 (1-10 microM), but not with U73343 (10 microM), dramatically reduced the pemphigus IgG-induced increases in [Ca++]i, IP3, and PA activity and inhibited the pemphigus IgG-induced cell-cell detachment. Both U73122 and U73343 caused no effects on cell viability and IgG binding to the cell surface. These results suggest that phospholipase C plays an important role in transmembrane signaling leading to cell-cell detachment exerted by pemphigus IgG binding to the cell surface.
It is still unclear what kinds of mechanisms are involved in blister formation after antibodies bind to the antigens in pemphigus and bullous pemphigoid. The effects of IgGs from pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid sera on intracellular calcium concentration ([Ca++]i) and inositol 1,4,5-trisphosphate were examined in a human squamous cell carcinoma cell line (DJM-1 cells) and in cultured human keratinocytes to clarify whether signal transduction via calcium is involved. IgGs were purified with protein A affinity column from the sera of five pemphigus vulgaris patients, three pemphigus foliaceus patients, eight bullous pemphigoid patients, and 14 normal volunteers. Keratinocytes were cultured in Eagle's minimum essential medium containing 1.8 mM Ca++ and loaded with fura-2/AM, followed by addition of the IgGs. Subsequently, [Ca++]i was determined by measuring the fluorescence ratio (F340/F360) with videomicroscopy. Pemphigus IgGs (seven of eight cases) induced a rapid and transient increase in [Ca++]i in both the cells, whereas a [Ca++]i increase was caused by very few IgGs from bullous pemphigoid (one of eight cases) and normal sera (two of 14 cases). The pemphigus IgG-induced transient [Ca++]i increase was not affected by chelating extracellular Ca++ with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetracetic acid. In addition, monoclonal antibodies acid. In addition, monoclonal antibodies against 180-kD and 230-kD antigens did not exert this change. Pemphigus IgGs that caused a [Ca++]i increase induced rapid and transient production of inositol 1,4,5-trisphosphate, peaking at 20 seconds. These findings suggest that IgG from pemphigus induces Ca++ mobilization by inositol 1,4,5-trisphosphate from internal stores, and that mechanisms of antibody-transmitted signaling in pemphigus may differ from those in bullous pemphigoid.
We previously found that the binding of pemphigus IgG to desmogleins caused marked activation of phospholipase C, a transient increase in inositol 1,4,5-trisphosphate production, and a concomitant increase in the intracellular calcium concentration in DJM-1 cells, a squamous cell carcinoma line. The binding of pemphigus IgG to cell membranes increased the activity of urokinase plasminogen activator in culture medium and induced subsequent cell-cell detachment in DJM-1 cells. Because urokinase plasminogen activator activates the conversion of plasminogen to plasmin by binding to urokinase plasminogen activator receptor evading inhibitors in serum, it is likely that plasmin is generated only in microenvironments adjacent to urokinase plasminogen activator receptor on the cell surface. It is not known whether pemphigus IgG causes acantholysis by inducing urokinase plasminogen activator receptor expression on the cell surface and secreting urokinase plasminogen activator in inhibitor-rich environments. We examined the effects of pemphigus IgG on urokinase plasminogen activator receptor expression in DJM-1 cells and normal keratinocytes by immunoblot analysis and immunofluorescence microscopy using antibodies to urokinase plasminogen activator receptor. IgG were obtained from serum samples from eight patients with bullous pemphigoid, five patients with pemphigus vulgaris, seven patients with pemphigus foliaceus, and eight normal subjects. Pemphigus vulgaris and pemphigus foliaceus IgG significantly increased the urokinase plasminogen activator receptor expression on the surface of DJM-1 cells and normal keratinocytes after 3- and 7-d incubation compared with normal IgG. These results suggest that enhanced urokinase plasminogen activator activity and urokinase plasminogen activator receptor expression activates plasmin in the limited cell surface of pemphigus IgG-bound keratinocytes and may contribute to the pathogenesis of differential acantholysis in pemphigus vulgaris and pemphigus foliaceus.
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