Desmoglein Versus Non-desmoglein Signaling in Pemphigus Acantholysis
2007; Elsevier BV; Volume: 282; Issue: 18 Linguagem: Inglês
10.1074/jbc.m611365200
ISSN1083-351X
AutoresAlex I. Chernyavsky, Juan Arredondo, Yasuo Kitajima, Miki Sato-Nagai, Sergei A. Grando,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAlthough it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces “desmosomal” signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by ∼45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by ∼50% and TUNEL positivity by ∼25%. The p38 MAPK inhibitor PD169316 inhibited these effects by ∼15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked ∼50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies. Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces “desmosomal” signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by ∼45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by ∼50% and TUNEL positivity by ∼25%. The p38 MAPK inhibitor PD169316 inhibited these effects by ∼15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked ∼50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies. Pemphigus vulgaris (PV) 2The abbreviations used are: PV, pemphigus vulgaris; KCs, keratinocytes; MAPK, mitogen-activated protein kinase; RTK, receptor tyrosine kinase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; EGFRK, epidermal growth factor receptor kinase; Dsg, desmoglein. is an immunoblistering disease associated with IgG autoantibodies that react with desmoglein (Dsg) 1 and/or 3 as well as non-Dsg target molecules on the cell membrane of keratinocytes (KCs) (reviewed in Ref. 1Stanley J.R. Amagai M. N. Engl. J. Med. 2006; 355: 1800-1810Crossref PubMed Scopus (362) Google Scholar). It has been convincingly demonstrated that PVIgG binding to KCs evokes an array of intracellular biochemical events resulting in cell detachment and death. The binding of PVIgG to KCs activates phosphatidylcholine-specific phospholipase C, increases inositol 1,4,5-trisphosphate and diacylglycerol production, elevates the intracellular free calcium level, and leads to activation of various kinases, including protein kinase C and p38 MAP kinase (MAPK) (reviewed in Ref. 2Lanza A. Cirillo N. Femiano F. Gombos F. J. Cutan. Pathol. 2006; 33: 401-412Crossref PubMed Scopus (57) Google Scholar). Among the known substrates of PVIgG-dependent phosphorylation are the adhesion molecules E-cadherin, Dsg3, β-catenin, and γ-catenin (a.k.a. plakoglobin) (3Nguyen V.T. Arredondo J. Chernyavsky A.I. Kitajima Y. Pittelkow M. Grando S.A. J. Biol. Chem. 2004; 279: 2135-2146Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and also heat shock protein (Hsp)27 (4Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). PVIgG binding to KCs leads to depletion of Dsg3 from desmosomes, nuclear trafficking of plakoglobin, urokinase-type plasminogen activator secretion, and c-Myc overexpression (reviewed in Ref. 5Kitajima Y. Aoyama Y. Clin. Rev. Allergy Immunol. 2007; (in press)PubMed Google Scholar). DNA microarray assays demonstrated that PVIgG down-regulates expression of the genes encoding keratinocyte adhesion molecules, antigen-processing proteins, regulators of cell cycle and apoptosis, differentiation markers, Na+,K+-ATPase, protein kinases, phosphatases, and others (5Kitajima Y. Aoyama Y. Clin. Rev. Allergy Immunol. 2007; (in press)PubMed Google Scholar). Overall, PVIgG decreased transcription of 198 genes and increased that of 31 genes. The pathobiologic outcome of the PVIgG-induced signaling is induction of the apoptotic and/or oncotic pathways, associated with collapse of the cytoskeleton, because of keratin filament retraction and actin reorganization, cell shrinkage, caused by cell volume decrease, and, finally, cell-cell dyshesion (acantholysis) (reviewed in Ref. 6Bystryn J.-C. Grando S.A. J. Am. Acad. Dermatol. 2006; 55: 513-516Abstract Full Text Full Text PDF Scopus (67) Google Scholar). Elucidation of the signaling pathways mediating PVIgG-induced acantholysis has salient clinical implications. The in vitro and in vivo studies revealed not only the complexity of PVIgG signaling, but also clearly demonstrated the therapeutic potential of kinase inhibitors and pathway modifiers. PVIgG-dependent acantholysis in experimental animals could be ameliorated by inhibitors of phospholipase C, protein kinase C, p38 MAPK, other tyrosine kinases, calmodulin as well as cholinergic agonists (7Nguyen V.T. Arredondo J. Chernyavsky A.I. Pittelkow M.R. Kitajima Y. Grando S.A. Arch. Dermatol. 2004; 140: 327-334Crossref PubMed Scopus (81) Google Scholar, 8Sanchez-Carpintero I. Espana A. Pelacho B. Lopez Moratalla N. Rubenstein D.S. Diaz L.A. Lopez-Zabalza M.J. Br. J. Dermatol. 2004; 151: 565-570Crossref PubMed Scopus (77) Google Scholar, 9Berkowitz P. Hu P. Warren S. Liu Z. Diaz L.A. Rubenstein D.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12855-12860Crossref PubMed Scopus (197) Google Scholar). The triggering events, however, remain obscure. The outside-in signaling of PVIgG may be elicited through both cholinergic receptors and desmosomal proteins, as well as other yet unknown PV antigens/receptors ligated on the cell membrane of KCs by an array of anti-keratinocyte autoantibodies produced in each PV patient, as explained by the “Multiple Hit” hypothesis (10Grando S.A. Dermatology. 2000; 201: 290-295Crossref PubMed Scopus (117) Google Scholar). The major downstream signaling events resulting from activation/blockade of cholinergic receptors expressed in KCs have been characterized (11Grando S.A. Pittelkow M.R. Schallreuter K.U. J. Investig. Dermatol. 2006; 126: 1948-1965Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 12Grando S.A. Exp. Dermatol. 2006; 15: 265-282Crossref PubMed Scopus (113) Google Scholar), but only a little is known about the reputed “desmosome” signaling pathway (4Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Recent studies have demonstrated activation of receptor tyrosine kinases (RTKs) at an early step of PVIgG-induced signaling (13Frusic-Zlotkin M. Raichenberg D. Wang X. David M. Michel B. Milner Y. Autoimmunity. 2006; 39: 563-576Crossref PubMed Scopus (65) Google Scholar), suggesting that such receptors are either novel specific targets of pemphigus autoimmunity or “innocent victims” of the “collateral damage” produced because of PVIgG binding to the keratinocyte plasma membrane, or both. The purpose of this study was to identify the primary pathways mediating PVIgG-induced acantholysis that can provide a promising target for development of novel therapies. We performed a time course correlation study of biochemical and morphologic events induced by PVIgG binding to human KCs. The results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving c-Src kinase (i.e. the protein encoded by the cellular counterpart of the translation product of the src gene of Rous sarcoma virus) and epidermal growth factor receptor kinase (EGFRK). To dissect out the signaling pathways originating from of Dsg 1/3 and from those elicited due to ligation of non-Dsg antigens, we used KCs with the knocked-down expression of the DSG1 and/or DSG3 genes by small interfering RNA (siRNA). The earliest acantholytic events appeared to be triggered by non-Dsg antibodies. Chemicals and Tissue Culture Reagents—A potent, cell-permeable, and selective p38 MAPK inhibitor PD169316, the inhibitor of Src family of protein tyrosine kinase PP2, and PhosphoDetect™ p38 MAPK ELISA kit were from Calbiochem-Novabiochem Corp. (EMD Biosciences, Inc. La Jolla, CA). The FACE™ c-Src and EGFR kits were purchased from Active Motif (Carlsbad, CA). The predesigned and tested siRNA-targeting human DSG1 (NM_001942) mRNA (ONTARGETplus SMARTpool reagent L-011644-00) (siRNA-Dsg1) and siRNA-targeting human DSG3 (NM_001944) mRNA (L-011646-00) (siRNA-Dsg3) were purchased from Dharmacon (Lafayette, CO). The negative control siRNA targeting luciferase gene with the target sequence 5′-CGTACGCGGAATACTTCGA-3′ that was employed in all RNA inhibition experiments was also from Dharmacon. The DeadEnd™ Fluorometric TUNEL System was from Promega (Madison, WI). Mouse monoclonal antibodies to Dsg1 and Dsg3 were purchased from R&D Systems (Minneapolis, MN) and secondary, FITC-labeled anti-mouse IgG antibody, from Sigma-Aldrich, Inc. Pemphigus and Normal Human IgG Fractions—The results reported herein were obtained in experiments utilizing pooled IgG fractions isolated from sera of 6 PV patients with active lesions on both oral mucosa and the skin, and sera of healthy people purchased from Sigma-Aldrich, Inc. This study had been approved by the University of California Davis Human Subjects Review Committee. The diagnosis of PV was made based on the results of comprehensive clinical and histological examinations, and immunological studies, which included direct immunofluorescence of skin biopsies, indirect immunofluorescence of patient sera on various epithelial substrates, and immunoblotting following standard protocols. The titer of “intercellular” antibodies determined on monkey esophagus ranged from 1/640 to 1/2560. The presence of anti-Dsg1 and -Dsg3 antibodies in each serum was established using the MESACUP Dsg-1 & Dsg-3 ELISA test system (MBL, Nagoya, Japan). The index values for Dsg1 antibodies ranged from 64 to 136 and those for Dsg3 antibodies, from 82 to 176, i.e. were unequivocally positive. The IgG fractions were isolated by FPLC protein G affinity chromatography using the FPLC System purchased from Amersham Biosciences and following the manufacturer’s protocol, as detailed by us elsewhere (14Arredondo J. Chernyavsky A.I. Karaouni A. Grando S.A. Am. J. Pathol. 2005; 167: 1531-1544Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Keratinocyte Culture Experiments—Human keratinocyte cultures were started from normal neonatal foreskins (15Grando S.A. Kist D.A. Qi M. Dahl M.V. J. Investig. Dermatol. 1993; 101: 32-36Abstract Full Text PDF PubMed Scopus (237) Google Scholar). The cells were grown in 75-cm2 flasks (Corning Glass Works, Coring, NY) in serum-free keratinocyte growth medium containing 5 ng/ml epidermal growth factor and 50 μg/ml bovine pituitary extract (Invitrogen, Cambridge, MA) at 37 °C in a humid, 5% CO2 incubator. The keratinocyte cultures used in experiments were between passages 2 and 4, ∼80% confluent, grown from at least three different foreskin donors. The concentration of calcium in culture medium was adjusted to either 0.2 mm or 1.6 mm (see below). The IgG fractions were diluted in culture medium and added to the monolayers at the final concentration of 1 mg/ml. Prior to exposures, some monolayers were pretreated with kinase inhibitors (see “Results”). The control monolayers were left intact. All monolayers were incubated in culture media containing 1.6 mm calcium at 37 °C and 5% CO2 for different periods of time (see “Results”), and then used in the Src, EGFRK, and p38 MAPK assays in accord with manufacturer’s protocols, quantitation of numbers of TUNEL (i.e. the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling)-positive cells, or for analyses of cell volume and keratin filament aggregation. The cytosolic volume was quantified by computing the cell diameter as detailed elsewhere (16Orlov M.D. Chernyavsky A.I. Arredondo J. Grando S.A. Autoimmunity. 2006; 39: 557-562Crossref PubMed Scopus (24) Google Scholar), and the result expressed as % control values determined in intact culture. KCs exhibiting keratin filament aggregation were visualized by indirect immunofluorescence in at least three microscopic fields at magnification ×100, using anti-pan keratin antibody (BioLegend, San Diego, CA). siRNA Transfection Experiments—For transfection with siRNAs, we followed the standard protocol described in detail elsewhere (17Chernyavsky A.I. Arredondo J. Karlsson E. Wessler I. Grando S.A. J. Biol. Chem. 2005; 280: 39220-39228Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Briefly, KCs were seeded at a density of 2.5 × 105 cells per well of a 6-well plate, and incubated for 16-24 h to achieve ∼70% confluence. To each well, increasing concentrations of siRNA in the transfection solution with the DharmaFECT™ 1 siRNA Transfection Reagent (Dharmacon) were added, and the transfection was continued for 16 h at 37 °C in a humid, 5% CO2 incubator. The siRNA transfection efficiency was assayed using FITC-labeled luciferase GL2 duplex (Dharmacon). After transfection, the cells were grown for 24 h in culture with 0.09 mm calcium, and then switched to the medium containing higher concentration of calcium and incubated for additional time points (up to 72 h) to achieve maximum inhibition of the Dsg protein expression, as was experimentally determined by immunoblotting and immunofluorescence. For immunoblotting experiments, KCs were dissolved in a sample buffer, separated via SDS-PAGE, and electroblotted onto a 0.2-μm nitrocellulose membrane (Bio-Rad). The membranes were developed using the ECL + Plus chemiluminescent detection system (Amersham Biosciences) and scanned with Storm™/FluorImager (Molecular Dynamics, Mountain View, CA). The PVIgG exposure experiments of transfected KCs were performed at 1.6 mm extracellular Ca2+. Some KCs transfected with siRNA-Dsg3 were cultured and exposed to PVIgG at 0.2 mm extracellular Ca2+. By immunoblotting and indirect immunofluorescence, at this concentration of Ca2+ KCs did not express Dsg1. Electron Microscopic Experiments—Normal human foreskin KCs seeded on glass coverslips were transfected with normal control siRNA (siRNA-NC), siRNA-Dsg1, or siRNA-Dsg3, grown for 48 h in keratinocyte growth medium with 0.09 mm calcium, and then switched to the medium containing 2 mm calcium overnight. On the next day, the cells were fixed in 2% glutaraldehyde at 4 °C for 30 min, washed in cacodylate buffer (pH 7.4) and post fixed in 2% osmium tetroxide at 4 °C for 1 h, followed by embedding in epoxy resins. The ultrathin sections were stained with uranyl acetate and lead citrate, followed by observation under an electron microscope (JEM-100S, JEOL LTD, Tokyo, Japan) as detailed elsewhere (18Sato M. Aoyama Y. Kitajima Y. Lab. Investig. 2000; 80: 1583-1592Crossref PubMed Scopus (75) Google Scholar). The traversing distance of the extracellular core domain (intercellular gap) of desmosome was calculated as follows: ∼50 electron micrographs were taken from each sample at a magnification of ×30,000 and developed at ×2.5. From 30-60 desmosomes were analyzed. The distance was measured on the right-angled line with the line of clear plasma membrane, illustrating trilamellar structure. Statistical Analysis—All experiments were performed in triplicate, and the results were expressed as mean ± S.D. Statistical significance was determined using the Student’s t test. Differences were deemed significant if the calculated p value was <0.05. Time Course Study of Kinase Activities in KCs Exposed to PV IgG—To identify the effectors mediating intracellular signaling of PVIgG at the earliest stages of acantholysis, we studied the activities of Src, EGFRK, and p38 MAPK. The time course studies demonstrated that the activity of Src peaked at 30 min, EGFRK at 60 min, and p38 MAPK at 240 min (Fig. 1, A-C). To elucidate the hierarchy of signaling events, we used the Src inhibitor PP2, because the peak of Src activity was observed at the earliest point in time after addition of PVIgG, i.e. before that of EGFRK and p38 MAPK. The cells were pretreated with 10 μm PP2, incubated with PVIgG, and the activities of EGFRK and p38 MAPK were measured at 60 and 240 min of incubation, respectively, when the activities of these kinases peaked. Preincubation with PP2 decreased PVIgG-dependent EGFRK and p38 MAPK activities by ∼45 and 30% (p < 0.05), respectively (Fig. 1, D and E), indicating that: 1) Src activation is upstream of both EGFRK and p38 MAPK; and 2) in addition to Src, binding of PVIgG to KCs engages other primary signaling pathways. Correlation between Biochemical and Phenotypic Changes in KCs Treated with PVIgG—We have previously reported that PVIgG-treated KCs shrink, detach from neighboring cells, and die through a mixed apoptotic/oncotic pathway, featuring TUNEL positivity (14Arredondo J. Chernyavsky A.I. Karaouni A. Grando S.A. Am. J. Pathol. 2005; 167: 1531-1544Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 16Orlov M.D. Chernyavsky A.I. Arredondo J. Grando S.A. Autoimmunity. 2006; 39: 557-562Crossref PubMed Scopus (24) Google Scholar). Keratinocyte shrinkage upon exposure to PVIgG (Fig. 2, A and B) is associated with collapse of the tonofilament scaffold supporting the polygonal shape of these epithelial cells (Fig. 2, C and D). To identify functional links between specific signaling events and alterations of keratinocyte morphology and viability resulting from PVIgG binding, we assayed changes in cell volume, keratin filament aggregation, and number of TUNEL+ cells in the monolayers at different time points after addition of PVIgG. The shrinkage of KCs became significant (p < 0.05) at 120 min, keratin filament aggregation at 240 min, and an increase of TUNEL+ cells at 360 min (Fig. 3, A-C).FIGURE 3Correlation between biochemical and phenotypic changes in KCs treated with PVIgG. Measurements of the cytosolic volume (A and D), keratin filament aggregation (B and E) and TUNEL positivity (C and F) were performed in KCs exposed to PVIgG without (A-C) or with (D-F) pretreatment with the Src inhibitor PP2, 10 μm, or the p38 MAPK inhibitor PD169316, 10 μm. A-C, time course analysis of keratinocyte cytosolic volume (A), keratin filament aggregation (B), and TUNEL positivity (C) in KCs treated with either PVIgG (- -• - -) or normal human IgG (—▪—). D-F, Src and p38 MAPK inhibitors alter PVIgG effects on KCs. Cell volume (D) was measured 120 min, keratin aggregation (E) 240 min, and TUNEL positivity (F) 360 min after exposure of KCs to PVIgG. Asterisks indicate significant (p < 0.05) differences compared with control KCs treated with normal IgG (control), and the pound signs indicate significant (p < 0.05) differences compared with the effect of PVIgG given alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To establish correlation between biochemical events and phenotypic changes developing in KCs upon PVIgG binding to their plasma membrane, we selected inhibitors of both “early” and “late” kinases, Src and p38 MAPK, respectively. Pretreatment of KCs with the Src inhibitor PP2, 10 μm, blocked PVIgG-dependent cell shrinkage and keratin aggregation by ∼50% (p < 0.05), and TUNEL positivity by ∼25% (p > 0.05). The p38 MAPK inhibitor PD169316, 10 μm, decreased these PVIgG effects by ∼15% (p > 0.05), 22% (p > 0.05), and 70% (p < 0.05), respectively (Fig. 3, D-F). These results indicated that Src was mainly responsible for early events in PVIgG-induced acantholysis, such as collapse of the cytoskeleton, whereas p38 MAPK, for late events, such as induction of apoptosis. Because neither kinase inhibitor could completely normalize studied parameters, as all remained significantly (p < 0.05) different from control values, PVIgG binding to KCs apparently engaged other signaling pathways contributing to the phenotypic changes. The Effects of Silencing of the DSG1 and DSG3 Genes on KCs and Their Response to PVIgG—To elucidate the roles of desmosomal and non-desmosomal targets of pemphigus autoimmunity, we asked whether silencing of the DSG1 or DSG3 genes would affect the biochemical events elicited because of PV IgG binding to KCs. The efficacy of knocking down the expression of Dsg1 or Dsg3 proteins in KCs with the Dharmacon-predesigned siRNAs was controlled by immunoblotting and immunofluorescence. Already 24 h after transfection with either siRNA-Dsg1 or siRNA-Dsg3, the KCs contained no measurable amounts of Dsg1 or Dsg3, respectively (Fig. 4, A and B). siRNA-NC did not affect Dsg1 or Dsg3 expression. Transfection with either siRNA did not alter keratinocyte viability, evaluated by the trypan blue dye exclusion test and TUNEL staining (data not shown). The morphology of the monolayers comprised by KCs transfected with siRNA-Dsg1 or siRNA-Dsg3 or both was indistinguishable from that of intact cells or KCs transfected with siRNA-NC (not shown). The KCs transfected with either type of siRNA formed desmosomes visualized by electron microscopy. To determine if gene silencing of Dsg1 and Dsg3 affected desmosome ultrastructure, we compared the ultrastructure of KCs transfected with siRNA-NC, siRNA-Dsg1 and siRNA-Dsg3 (Fig. 4C). KCs transfected with siRNA-NC exhibited normal ultrastructural features of desmosomes. Their desmosomes were comprised by the plasma membranes with the outer dense plaque and the inner dense plaque overlapped with keratin intermediate-sized filaments, and a ∼22 nm-wide gap at the extracellular core domain (intercellular gap) without electron-dense midline. Similar desmosome structure was observed in desmosome produced by KCs transfected with siRNA-Dsg1. In Dsg3-deficient KCs, we observed fine filaments traversing the extracellular core domain of the desmosome with widened intercellular distance. The traversing distance of the extracellular core domain of desmosomes in KCs transfected with siRNA-NC was 22.5 ± 4.4 nm. In the KCs with knocked-down expression of Dsg1, this distance was not altered, 19.9 ± 4.4 nm. However, silencing of the DSG3 gene significantly (p < 0.0001) increased the distance to 31.2 ± 11.8 nm (n = 37), illustrating the widening of desmosomes. Transfection with siRNAs that inhibited expression of either Dsg1 and Dsg 3 alone or together in all cases blocked ∼50% of p38 MAPK activity (p < 0.05), but only insignificantly (p > 0.05) altered PVIgG-dependent raise in Src and EGFRK activities (Fig. 5), indicating that anti-Dsg1 and -Dsg3 antibodies contributed predominantly to the pathobiologic events resulting from activation of p38 MAPK, such as development of keratinocyte apoptosis. The experiments with the siRNA-Dsg3-transfected KCs that were performed at 0.2 mm of extracellular Ca2+ brought similar results (data not shown). Treatment of transfected cells with normal IgG did not elicit any measurable kinase activities at either concentration of extracellular Ca2+ tested, indicating that the signaling events observed in cells exposed to PVIgG occurred because of ligation of pemphigus autoantigen(s), rather than transfection with siRNAs. The results of this study revealed the sequence of major intracellular biochemical events elicited due to PVIgG binding to the keratinocyte plasma membrane and their correlation with morphologic changes characteristic of acantholysis, and also defined relative contribution of both anti-Dsg and non-Dsg autoantibodies. The early signaling events were triggered predominantly by non-Dsg PVIgG, and involved activation of Src and EGFRK associated with collapse of the cytoskeleton. Anti-Dsg1 and Dsg3 antibodies contributed predominantly to the pathobiologic events associated with p38 MAPK activation, a late signaling step linked to keratinocyte apoptosis. The exclusive roles in pemphigus immunopathogenesis of anti-Dsg 1/3 antibodies, which can cause steric hindrance at the adhesion points of KCs, has been largely reconsidered (19Amagai M. Ahmed A.R. Kitajima Y. Bystryn J.C. Milner Y. Gniadecki R. Hertl M. Pincelli C. Fridkis-Hareli M. Aoyama Y. Frusic-Zlotkin M. Muller E. David M. Mimouni D. Vind-Kezunovic D. Michel B. Mahoney M. Grando S. Exp. Dermatol. 2006; 15: 815-831Crossref PubMed Scopus (1) Google Scholar). Recent development of the “signaling” paradigm of the pathobiologic action of autoantibodies in PV has set the stage for well-controlled studies toward identification of the primary signaling pathway(s) responsible for keratinocyte detachment and death during acantholysis. This study was designed to help answer the central question in elucidation of PV pathogenesis that has immediate clinical implications. To develop adequate anti-acantholytic therapies using kinase inhibitors and pathways modifies, it is essential to determine which pathway: 1) leads directly to a loss of cell-cell adhesion (primary pathway), 2) is being activated secondary to release of autocrine and paracrine cytokines and/or shrinkage and detachment of damaged KCs (secondary pathway), 3) contributes to utilization of altered proteins and organelles (scavenging pathway), and 4) represents a cell defense/survival effort (protective pathway) (20Grando S.A. Autoimmunity. 2006; 39: 521-530Crossref PubMed Scopus (32) Google Scholar). Src and RTKs are known to act either independently or in concert in regulation of cell adhesion (21Mariner D.J. Davis M.A. Reynolds A.B. J. Cell Sci. 2004; 117: 1339-1350Crossref PubMed Scopus (85) Google Scholar). Activation of EGFR can occur by ligand-dependent and ligand-independent mechanisms (22Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (549) Google Scholar). It has been recently demonstrated that the specific tyrosine kinase inhibitor AG1478 abrogated PVIgG-induced EGFR autophosphorylation, acantholysis, and keratinocyte death (13Frusic-Zlotkin M. Raichenberg D. Wang X. David M. Michel B. Milner Y. Autoimmunity. 2006; 39: 563-576Crossref PubMed Scopus (65) Google Scholar). In this study, we demonstrated that the peak of Src activity preceded that of EGFRK. The stimulation of Src is known to cause transactivation of EGFR, with Src binding to the EGFR (23Haas M. Wang H. Tian J. Xie Z. J. Biol. Chem. 2002; 277: 18694-18702Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Because Src is not expressed to the cell surface, one of the self-antigens targeted by PVIgG on the keratinocyte plasma membrane apparently relayed the signal to Src. Thus, engagement of Src/EGFR may be a key step that relays the signal emanating from interaction of PVIgG with various self-antigens on the keratinocyte plasma membrane structures to the pathway affecting the cytoskeleton and adhesion structures. Likewise, PVIgG induces Fas-ligand production in KCs (14Arredondo J. Chernyavsky A.I. Karaouni A. Grando S.A. Am. J. Pathol. 2005; 167: 1531-1544Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and Fas-ligand acts synergistically with PVIgG and TNFα in acantholysis induction (16Orlov M.D. Chernyavsky A.I. Arredondo J. Grando S.A. Autoimmunity. 2006; 39: 557-562Crossref PubMed Scopus (24) Google Scholar). Fas signaling leads to apoptosis through the p38 MAPK step (24Farley N. Pedraza-Alva G. Serrano-Gomez D. Nagaleekar V. Aronshtam A. Krahl T. Thornton T. Rincon M. Mol. Cell Biol. 2006; 26: 2118-2129Crossref PubMed Scopus (74) Google Scholar), which may provide a mechanism fo
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