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Phosphatidylcholine-specific phospholipase C, but not phospholipase D, is involved in pemphigus IgG-induced signal transduction Received: 20 May 1999 / Accepted: 13 August 1999
Abstract The precise mechanism of the acantholysis after pemphigus IgGs bind to desmoglein (Dsg) 3 and/or Dsg 1 on the cell surface is as yet unknown. We have previously reported that pemphigus IgG (P-IgG) causes a transient increase in intracellular calcium and inositol 1,4,5-trisphosphate concentration, and subsequent activation of protein kinase C (PKC) in DJM-1 cells, a squamous cell carcinoma line. In order to see whether phosphatidylcholine (PC)-specific phospholipase C (PLC) or phospholipase D (PLD) is involved in the PIgG-induced signaling process, the production of 1,2diacylglycerol (DAG) and phosphatidylbutanol (PBut), a potential marker for the determination of PLD activity in the presence of butanol, was determined in DJM1 cells. A biphasic accumulation of DAG, which consisted of a first transient phase and a second sustained phase, was observed. The second phase of DAG accumulation was profoundly inhibited by pretreatment with D609, a selective inhibitor of PC-PLC, but not by propranolol, an inhibitor of phosphatidate phosphohydrolase. Pemphigus serum after preadsortion of antibodies to Dsg 3 and Dsg 1 with recombinant Dsg 3 and Dsg 1 did not show formation of DAG. PBut was not generated following the addition of P-IgG. In addition,
M. Seishima (Y) Department of Dermatology, Ogaki Municipal Hospital, Minaminokawa-cho 4-86, Ogaki, 503–8502, Japan Tel.: +81-584-813341, Fax: +81-58-2956472 Y. Iwasaki-Bessho · Y. Kitajima Department of Dermatology, Gifu University School of Medicine, Tsukasamachi 40, Gifu 500-8705, Japan Y. Itoh · Y. Nozawa Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi 40, Gifu 500-8705, Japan M. Amagai Department of Dermatology, Keio University School of Medicine, Shinanomachi 35, Shinjuku, Tokyo 160-8582, Japan
the levels of [3H]phosphocholine, a direct metabolite of PC-PLC, were elevated after the addition of P-IgG. These results suggest that the PC-PLC pathway plays a major role in P-IgG-induced transmembrane signaling by causing prolonged generation of DAG, which may lead to long-term activation of PKC. Key words Bullous disease · Diacylglycerol · Keratinocyte Abbreviations BP bullous pemphigoid · DAG 1,2-diacylglycerol · Dsg desmoglein · EDTA ethylenediamineteraacetic acid · HBSS Hank’s balanced salt solution · IP3 inositol 1,4,5-trisphosphate · PA phosphatidate · PBut phosphatidylbutanol · PC phosphatidylcholine · PI phosphatidylinositol · PIP2 phosphatidylinositol bisphosphate · PF pemphigus foliaceus · PKC protein kinase C · PLC phospholipase C · PLD phospholipase D · PV pemphigus vulgaris · rDsg3-His the chimeric molecule of the extracellular domain of Dsg 3-fused E tag and His tag at the carboxyl terminus · rDsg1-His the chimeric molecule of the extracellular domain of Dsg 1-fused E tag and His tag at the carboxyl terminus · TLC thin-layer chromatography
Introduction Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases characterized by the presence of autoantibodies against desmogleins (Dsg) 3 and Dsg 1, respectively [1–3]. Dsg 1 and Dsg 3 are desmosomal proteins [1, 4] that form protein complexes by association with plakoglobin . These antigens are expressed both in normal keratinocytes and cells of the squamous cell carcinoma cell line DJM-1 [6, 7] as well as 180 kDa and 230 kDa bullous pemphigoid antigens [8, 9]. Although the precise mechanism for cutaneous blistering induced by the binding of pemphigus IgG to keratinocytes has not been elucidated, pemphigus acantholysis may be caused either by proteinases from keratinocytes , such
as plasminogen activator [11, 12], or by the direct binding of PV IgG to desmosomal molecules, which disrupts cellcell attachment . In this regard, we have previously demonstrated that desmosome formation induced by switching the incubation medium from a low Ca2+ content (0.09 mM) to high Ca2+ content (> 1.0 mM) is not inhibited by the binding of PV IgG to PV antigens on the cell surface . However, these newly formed desmosomal connections are subsequently dissociated within 24 to 48 h by PV IgG treatment. This observation suggests that this cell-cell detachment may have been caused by proteinases, rather than by direct inhibition of desmosome formation . In this context, it is of importance to study the precise cell responses after the binding of pemphigus IgG to the antigens, focusing on the antibody-induced transmembrane signaling leading to blistering in pemphigus . It is now well known that one of the intracellular signaling pathways is mediated by phosphatidylinositol (PI)specific phospholipase C (PLC), by which PI bisphosphate (PIP2) produces two second messengers: inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). The former induces an increase in intracellular calcium concentration, and the latter acts as an endogenous activator of protein kinase C (PKC) . We have previously demonstrated that the binding of pemphigus IgG to the surface of DJM-1 cells induces a transient increase in PI-PLC activity  and increases IP3 production, with a concomitant increase in the intracellular calcium concentration . Furthermore, we have found that these events lead to activation of PKC . In the current study, we examined DAG formation induced by pemphigus IgG in DJM-1 cells, and the involvement of phosphatidylcholine (PC)-specific PLC or phospholipase D (PLD) in DAG generation after the binding of pemphigus IgG to the pemphigus antigens.
eight BP patients by immunoblot analysis. IgG fractions were isolated from serum using a HiTrap protein A affinity column (Pharmacia AB, Uppsala, Sweden) and the purity of isolated IgG was checked by immunoelectrophoresis. Immunoadsorption of PV serum with recombinant Dsg 3 and Dsg 1 The serum from a typical PV patient was preincubated with rDsg3His and rDsg1-His solution (approximate concentration, 10 µg/ml) to adsorb the antibodies to Dsg 3 and Dsg 1, respectively . Then the titers of antibodies to Dsg 3 and Dsg 1 in serum with or without preadsorption with rDsg3-His and rDsg1-His solution were measured by ELISA. ELISA scores against Dsg 3 and Dsg 1 were obtained using a previously described method with slight modification . A single PV and PF serum sample was selected as the standard for each respective ELISA. An index value was defined as follows: Index value = (ODsample – ODnegative)/(ODpositive – ODnegative) × 100 A cutoff value was defined as the average value plus 3 × SD of the value of normal control serum (9.4 for Dsg 3 ELISA, 9.1 for Dsg 1 ELISA). Each sample was run in duplicate. The DAG contents of DJM-1 cells were measured after incubation with the serum with or without preadsorption. Cell culture An isolated cell line (DJM-1 cells) from human skin squamous cell carcinoma [6, 19] was cultured in 1.8 mM Ca2+ medium containing Eagle’s minimum essential medium (MEM), 10% fetal calf serum, 100 µg/ml of streptomycin and 100 units/ml of penicillin. Normal human keratinocytes obtained from foreskin (Kurabo, Osaka, Japan) were cultured in keratinocyte-SFM (modified MCDB medium supplemented with 5 ng/ml of epidermal growth factor, 50 µg/ml bovine pituitary extract, 50 µg/ml gentamicin, and 0.25 µg/ml amphotericin B; Life Technologies, Grand Island, N.Y.). The medium was changed every 2 to 3 days. After reaching confluence, the cells were treated with Hank’s balanced salt solution (HBSS) containing 0.02% trypsin and 0.02% EDTA for 15 min at 37 °C, and were then resuspended in the medium. The third passage of human keratinocytes was cultured at 37 °C in a humidified atmosphere comprising 5% CO2/95% air.
Materials and methods Determination of mass content of DAG Reagents The quantitative assay kits for DAG and [9,10-3H]palmitic acid (52.4 Ci/mmol) were obtained from Amersham (Buckinghamshire, UK), [methyl-3H]choline chloride (85 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, Mo.), silica gel 60 plates were from Merck (Darmstadt, Germany), and silica gel LK60 was from Whatman Chemical Separation (Clinton, N.J.). All other chemicals were of reagent grade. Serum Serum from six PV, three PF, and eight bullous pemphigoid (BP) patients and five normal volunteers without any skin diseases were used for experiments. All serum samples were kept at –80 °C until use. These diseases were diagnosed by clinical examination, direct and indirect immunofluorescence microscopy and immunoblot analysis using ethylenetetraacetic acid (EDTA)-separated normal human epidermal extracts as substrates. Intercellular antibody in the epidermis was detected in all six PV and three PF patients by immunofluorescence microscopy. Antibody to PV antigen (130 kDa) was detected in the serum from all six PV patients and antibody to PF antigen (160 kDa) was detected in the serum from all three PF patients by immunoblot analysis. Antibodies to both 180 kDa and 230 kDa peptides were detected in the serum from all
Cultured keratinocytes were washed twice with HBSS and were then treated with 1 mg/ml of pemphigus, BP or normal IgG at 37 °C for the indicated times. These cells had been pretreated with 200 µM propranolol, an inhibitor of phosphatidate (PA) phosphohydrolase (Sigma Chemical Co., St. Louis, Mo.), 0.3% butanol, an inhibitor of PLD, 10 µM D609, an inhibitor of PC-PLC (Kamiya Biochemical Company, Seattle, Wash.)  or vehicle for 10 min to examine the effects of these agents on pemphigus IgG-induced DAG formation. The reaction was terminated by adding cold methanol and the cells were transferred to silicon-treated glass tubes containing chloroform/methanol (4 : 5, v/v). Lipids were then extracted essentially as described by Bligh and Dyer , except that 0.2 M KCl/5 mM EDTA was used instead of 2 M KCl. The DAG level was measured according to the procedure of Preiss et al.  using DAG kinase. Briefly, the DAG in lipid extracts prepared from cells was converted quantitatively into [32P]PA by DAG kinase in the presence of [32P]ATP. After extraction steps to remove [32P]ATP, [32P]PA was separated by thin-layer chromatography (TLC) (silica gel 60) with the solvent system chloroform/ methanol/acetic acid (12 : 3 : 1, v/v/v). The area corresponding to [32P]PA was located by autoradiography and then scraped off the plates, and the radioactivities were determined using a liquid scintillation counter (Beckman LS 7500). To examine the inhibitory effect of butanol on DAG production, the cells were preincubated with 0.3% butanol in HBSS for 10 min prior to the addition of IgG.
608 Analysis of PLD activity PLD activity was determined by measuring the formation of [3H]phosphatidylbutanol (PBut), a specific product formed via transphosphatidylation activity in the presence of butanol (0.3%, v/v), before and after pemphigus IgG treatment as previously described. DJM-1 cells were subcultured on 60-mm-diameter tissue culture dishes at 1 × 106 cells/dish and grown for 2 days. The cells were labeled for the last 3 h with [3H]palmitic acid (5 µCi/dish) and then washed twice with HBSS . The labeled cells were preincubated in HBSS at 37 °C for 10 min with the indicated reagents in the presence of 0.3% butanol (v/v). Cells were then stimulated with pemphigus, BP or normal IgGs at 37 °C for 10 min. The reaction was terminated by aspiration of the buffer, followed by immediate addition of 1 ml of an ice-cold phosphatebuffered saline/methanol (2 : 5, v/v) mixture to the culture dishes. Cells were scraped off the dishes with a rubber policeman, and dishes were washed with an additional 1 ml PBS/methanol mixture. The lipid extraction was performed according to the method of Bligh and Dyer . [3H]PBut was separated by one-dimensional TLC with silica gel LK60 plates in a solvent system with an upper phase of ethyl acetate/2,2,4-trimethyl-pentane/acetic acid/ water (13 : 2 : 3 : 10, by volume) . The area corresponding to [3H]PBut, identified by comigration with PBut standard, was scraped off the plate, and the radioactivity was determined in a liquid scintillation counter (Beckman LS 6500). The amount of [3H]PBut formed was expressed as a percentage of total radioactivity recovered from the TLC plate.
Fig. 1 Biphasic DAG formation induced by PV IgG. DJM-1 cells were incubated with pemphigus vulgaris IgG (PV-IgG, closed circles) or normal IgG (N-IgG, open circles) for the indicated times. Values are the means ± SD (bars) of duplicate determinations from three separate experiments
Extraction and analysis of choline metabolites from DJM-1 cells labeled with [3H]choline DJM-1 cells were exposed to 0.5 µCi/ml [3H]choline for 48 h. These labeled cells were rinsed twice with MEM, incubated in HBSS for 1 h, and then incubated in HBSS with or without 10 µM D609, an inhibitor of PC-PLC, before treatment with pemphigus, BP or normal IgGs (1 mg/ml) in HBSS. The incubation was arrested with chloroform/methanol (1 : 2, v/v), and the aqueous and lipid phases were separated as described by Bligh and Dyer . The aqueous phase was dried by centrifuging under vacuum and resuspended in 50 µl of 50% (v/v) ethanol, and then separated on a silica gel LK60 plate, using the solvent system chloroform/0.5% NaCl/28% NH3 (50 : 50 : 1, v/v/v), according to the method of Yavin . Authentic standards of choline and phosphocholine were also run and identified with I2 vapor. Data analysis Statistical analysis of the data was carried out by Student’s t-test.
Results Biphasic DAG formation was induced by pemphigus IgG The mass content of DAG was determined to examine the effect of pemphigus IgG on DAG formation in DJM-1 cells. Normal IgG had no effect on DAG formation (Fig. 1). After the addition of PV IgG, a biphasic accumulation of DAG was observed: a first transient phase peaking at 30 s and a second broad plateau which was sustained for at least 20 min. DAG formation at 30 s and 10 min was determined after the treatments with the IgGs from PV, PF, BP or normal serum. There were no significant differences in the DAG level between the normal IgG and the control solution after 30 s incubation (normal IgG, 19.8 ± 8.7 pmol/106 cells, n = 5; control, 15.3 ± 6.8 pmol/
Fig. 2 Increased DAG generation by pemphigus vulgaris (PV) or pemphigus foliaceus (PF) IgGs, but not by bullous pemphigoid IgGs (BP), compared with that by normal IgGs (N). DJM-1 cells were treated with PV-, PF-, BP- or N-IgGs or elution buffer as a control (Cont) for 30 s or 10 min. Each dot indicates the mean value of an individual case obtained from three independent experiments (*P < 0.005, **P < 0.001)
Fig. 3 A–D The second phase of pemphigus vulgaris IgG (PVIgG)-induced DAG formation (A) was inhibited by pretreatment with 10 µM D609, a PC-PLC inhibitor, for 10 min (B). On the other hand, 10 min pretreatment with 0.3% butanol (a PLD inhibitor) (C) or 200 µM propranolol (a PA phosphohydrolase inhibitor) (D) did not alter this phase. The first peak of DAG generation was not altered by pretreatment with these three agents. Values are means ± SD (bars) of duplicate determination from three separate experiments
106 cells, n = 3) or after 10 min incubation (normal IgG, 23 ± 12.1 pmol/106 cells, n = 5; control, 21.6 ± 6.5 pmol/ 106 cells, n = 3; Fig. 2). In contrast, DAG formation was significantly increased by treatment with PV IgG (146.2 ± 58 pmol/106 cells, n = 6) and PF IgG (174 ± 28.4 pmol/106 cells, n = 3) compared with normal IgG treatment (P < 0.005 and P < 0.001, respectively) 30 s after the addition of the IgGs. In addition, DAG formation was markedly increased after 10 min incubation with PV IgG (159.4 ± 65.4 pmol/106 cells, n = 6), and with PF IgG (202.1 ± 19 pmol/106 cells, n = 3) compared with normal IgG (23 ± 12.1 pmol/106 cells, n = 5; P < 0.005 and <0.001, respectively). The DAG content in cells incubated with BP IgG for 30 s (21.5 ± 1.46 pmol/ 106 cells, n = 5) or 10 min (23.5 ± 5.6 pmol/106 cells, n = 5) was consistent with that in cells incubated with normal IgG (Fig. 2). No significant differences were observed in DAG content between cells treated with PV IgG and those treated with PF IgG. There were no significant correlations between DAG contents and the titers of pemphigus antibodies detected by indirect immunofluorescence study. In addition, there were no differences in DAG content between cells treated with 5 mg/ml or with 1 mg/ml of the same PV IgG at 30 s or at 10 min. Furthermore, the biphasic accumulation of DAG was also observed in cultured
normal human keratinocytes after the addition of PV or PF IgGs but not after the addition of BP or normal IgGs. Immunoadsorption of PV serum with recombinant Dsg 3 and Dsg 1 inhibited DAG formation Following immunoadsorption with rDsg3-His and rDsg1His, antibodies to Dsg 3 and Dsg 1 were profoundly reduced in PV serum (Table 1). The DAG formation induced by PV serum was significantly inhibited by PV serum preadsorbed with recombinant Dsg 3 and Dsg 1 (P < 0.005; Table 1). Table 1 PV serum-induced DAG production inhibited by preadsorption of antibodies to Dsg 3 and Dsg 1 (ND not done)
* P < 0.005 vs non-adsorption a Index value = (OD sample – ODnegative)/(ODpositive – ODnegative) × 100. Cutoff values were 9.4 for Dsg 3 ELISA and 9.1 for Dsg 1 ELISA. Values are means of duplicate determinations b Values are means ± SD of duplicate determinations from two separate experiments c PV serum after adsorption of antibodies to Dsg 3 and Dsg 1 with rDsg3-His and rDsg1-His d PV serum without adsorption of antibodies to Dsg 3 and Dsg 1 eIncubation buffer for adsorption with rDsg3-His and rDsg1-His
D609, a PC-PLC inhibitor, reduced the second phase of DAG formation In order to examine the involvement of PC-PLC or PLD in DAG formation induced by PV IgG, cells were treated with the PC-PLC inhibitor, D609, the PLD inhibitor, butanol, or the PA-phosphohydrolase inhibitor, propranolol. The first peak of DAG formation was not affected by these three inhibitors. In sharp contrast, the second sustained phase was markedly reduced by 10 µM D609. However, neither propranolol (200 and 500 µM) nor butanol (0.3 and 0.5%) changed the second phase of DAG formation (Fig. 3). The time-course of DAG formation induced by IgG from one typical PV IgG is shown in Fig. 3. Similar results were obtained from other experiments using other PV and PF IgGs. Absence of PBut formation following butanol treatment in pemphigus IgG-treated cells
Fig. 4 No phosphatidylbutanol (PBut), a marker of PLD activation, formed by pemphigus vulgaris (PV) and pemphigus foliaceus (PF) IgGs. DJM-1 cells labeled with [3H]palmitic acid which were pretreated with 0.3% butanol for 10 min were incubated with PV, PF, bullous pemphigoid (BP) or normal (N) IgGs for 10 min. The amount of [3H]PBut formed was calculated as a percentage of the total radioactivity recovered from the TLC plate, and was then expressed as a ratio to the control value. Dots indicate the results of individual cases Fig. 5 [3H]Phosphocholine production induced by pemphigus vulgaris IgG (PV-IgG) in DJM-1 cells labeled with [3H]choline. The cells were incubated with PV-IgG (closed circles) or normal IgG (N-IgG, open circles) for the indicated times. The levels of [3H]phosphocholine, a metabolite of the PC-PLC pathway, were elevated within 5 min, peaked at 10 min, and then gradually decreased. The [3H]choline level showed a gradual increase after 10 min. Values are means ± SD (bars) of duplicate determination from three separate experiments
To examine whether the second phase of DAG production induced by PV IgG was due to PLD activation followed by PA hydrolase, generation of PBut, a marker of PLD activation was examined. It was not formed after 10 min incubation with any of the IgGs (six PV, three PF, three BP and three normal; Fig. 4).
611 Fig. 6 Inhibitory effects of D609, a PC-PLC inhibitor, on the increases in [3H]phosphocholine and [3H]choline production induced by pemphigus IgG. The increases in [3H]phosphocholine and [3H]choline (PV-IgG, closed circles) were inhibited by preincubation with 10 µM D609 for 10 min (closed squares). Values are means ± SD (bars) of duplicate determination from three separate experiments
Phosphocholine, a metabolite of the PC-PLC pathway, was increased by PV IgG To differentiate the PLC and PLD pathways for the prolonged phase of DAG formation, we analyzed the choline metabolites derived from PC hydrolysis in DJM-1 cells incubated with PV IgG. Cells were labeled to equilibrium with [3H]choline (0.5 µCi/ml) for 48 h. More than 90% radioactivity was distributed in the three major metabolites, phosphocholine, choline, and glycerophosphocholine, in unstimulated cells. There were no significant changes in the levels of [3H]glycerophosphocholine in the cells treated with PV IgG or normal IgG or in control cells. Normal IgG treatment did not change the level of [3H]phosphocholine, a metabolite of the PC-PLC pathway, or that of [3H]choline. After the addition of PV IgG, however, the levels of [3H]phosphocholine were elevated within 5 min, peaked at 10 min, and then gradually decreased (Fig. 5). The increase in [3H]phosphocholine following PV IgG addition (108 ± 19.5 pmol/106 cells and 96.2 ± 17.6 pmol/106 cells, n = 3, 10 and 20 min after PV IgG addition, respectively) was inhibited by preincubation with 10 µM D609 (38.1 ± 7.1 pmol/106 cells and 32.4 ± 6.3 pmol/106 cells, n = 3, P < 0.005; Fig. 6). On the other hand, the [3H]choline level showed a gradual increase after 10 min and did not reach a plateau within 20 min (Fig. 5). The increase in [3H]choline 20 and 30 min after PV IgG addition (12.5 ± 3.6 pmol/106 cells and 14.5 ± 2.8 pmol/106 cells, n = 3,
respectively) was also inhibited by 10 µM D609 (6.0 ± 1.6 pmol/106 cells and 8.9 ± 1.9 pmol/106 cells, n = 3, P < 0.05; Fig. 6). These experiments determining choline metabolites were performed using IgG from one typical PV and similar results were obtained from duplicate determinations in other experiments with another PV IgG and another PF IgG.
Discussion DAG is known to activate PKC in signal transduction events involved in keratinocyte differentiation [26–29], and also to be produced via PLC-mediated hydrolysis of PIP2  with concurrent production of IP3 as second messengers. In addition, receptor-mediated activation of PC-hydrolyzing PLD [30, 31] and PLC [31–33] has been widely demonstrated in various types of cell. The hydrolysis of PC by PLD yields PA under physiologic conditions, whereas in the presence of primary alcohols such as ethanol and butanol, the enzyme produces corresponding nonmetabolizable phosphatidylalcohols, the unambiguous products of PLD, at the expense of PA generation [30, 34]. The PA produced is an intermediate in DAG formation via the action of PA phosphohydrolase, as in the biosynthetic pathway. In the present study, PV and PF IgG induced a biphasic DAG generation, a first phase with a peak at 30 s and a second phase which was sustained for
at least 20 min. Although PI hydrolysis induced by P-IgG was not checked in this study, the first peak of DAG formation is likely to have been derived from PI-hydrolysis by PI-PLC, because pemphigus IgG causes a concurrent production of IP3 peaking at 20 s  and choline metabolites were unchanged at least within 2 min (Fig. 5). Both phases were inhibited by preadsorption of antibodies to Dsg 3 and Dsg 1 with recombinant peptides, suggesting that DAG formation is induced by these antibodies. In order to investigate the precise pathway of the second sustained phase of DAG formation, choline metabolites were analyzed after the addition of PV IgG. PV IgG increased the levels of [3H]phosphocholine, a metabolite of the PC-PLC pathway, with a peak at 10 min after the PV IgG addition, followed by a gradual decrease. On the other hand, the levels of [3H]choline, a metabolite of the PLD pathway, showed a gradual increase after 10 min. These data suggest that the second sustained phase of DAG production is derived mainly from PC through the PC-PLC-catalyzed pathway. PBut, a marker of PLD activity in the presence of butanol, was not formed by pemphigus IgG and the second phase of DAG generation was not inhibited by pretreatment with butanol. Furthermore, D609, an inhibitor of PC-PLC, reduced significantly, although not completely, the second sustained phase of DAG formation and the levels of [3H]phosphocholine. These results suggest that the PC-PLC, but not the PLD, pathway contributes to the second sustained phase of DAG formation in the signaling mechanism after the binding of pemphigus IgG to Dsg. However, it cannot be completely excluded that the PLD pathway might contribute to the late part in the second sustained phase of DAG formation, because the choline level increased after 10 min incubation with PV IgG. This may be explained by the interconversion between phosphocholine and choline via phosphatase and kinase, but not via the PLD pathway. It is well known that DAG elevation causes the activation of conventional PKC (α, βI, βII, and γ) and novel PKC (δ and ε) [35–38]. The total PKC activity in the particulate/cytoskeleton (p/c) fraction is increased after PV IgG exposure, with a peak at 1 min, and is sustained for at least 30 min . Furthermore, PV IgG induces translocation of PKCα from the cytosol to the p/c fraction within 30 min, with a peak at 1 min that lasts at least 30 min. PKCδ is also translocated within 1 min, reaches a peak at 5 min and reduces to basal levels by 30 min. PKCη translocation to the p/c fraction is induced slowly over more than 5 min and is reduced to approximately half-maximum at 30 s, whereas PKCζ translocation reaches a maximum at 30 s, rapidly returning to baseline by 5 min after PV IgG stimulation . The sustained activation of PKC and the unique activation profile of PKC isomers may be caused by the second sustained phase of DAG formation, because PKCα is activated by DAG and PV IgG-induced PKCα translocation is sustained for more than 30 min. These findings suggest that this biphasic DAG generation may be involved in the long-term intracellular signaling events induced by PV IgG binding to Dsg 3.
Phosphoinositide hydrolysis by PI-PLC is accompanied by an increase in the DAG level and the transient activation of PKC in keratinocytes as observed in various other cells [19, 28]. Although it has been proposed that the prolonged DAG elevation may cause the sustained activation of PKC in P-IgG-bound keratinocytes, the physiological significance of the sustained DAG generation from PC hydrolysis and the signals to induce the PC-PLC pathway remain to be explored.
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