Pemphigus Vulgaris IgG Cause Loss of Desmoglein-Mediated Adhesion and Keratinocyte Dissociation Independent of Epidermal Growth Factor Receptor
2009; Elsevier BV; Volume: 174; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.080392
ISSN1525-2191
AutoresWolfgang-Moritz Heupel, Peter Engerer, Enno Schmidt, Jens Waschke,
Tópico(s)Urticaria and Related Conditions
ResumoAutoantibody-induced cellular signaling mechanisms contribute to the pathogenesis of autoimmune blistering skin disease pemphigus vulgaris (PV). Recently, it was proposed that epidermal growth factor receptor (EGFR) might be involved in PV signaling pathways. In this study, we investigated the role of EGFR by comparing the effects of epidermal growth factor (EGF) and PV-IgG on the immortalized human keratinocyte cell line HaCaT, and primary normal human keratinocytes. In contrast to EGF treatment, PV-IgG neither caused the canonical activation of EGFR via phosphorylation at tyrosine (Y)1173 followed by internalization of EGFR nor the phosphorylation of the EGFR at the c-Src-dependent site Y845. Nevertheless, both PV-IgG and EGF led to cell dissociation and cytokeratin retraction in keratinocyte monolayers. Moreover, the effects of EGF were blocked by inhibition of EGFR and c-Src whereas the effects of PV-IgG were independent of both signaling pathways. Similarly, laser tweezer experiments revealed that impaired bead binding of epidermal cadherins desmoglein (Dsg) 3 and Dsg 1 in response to PV-IgG was not affected by inhibition of either EGFR or c-Src. In contrast, EGF treatment did not interfere with Dsg bead binding. Taken together, our study indicates that the loss of Dsg-mediated adhesion and keratinocyte dissociation in pemphigus is independent of EGFR. Moreover, the mechanisms by which both EGF and PV-IgG lead to keratinocyte dissociation and cytokeratin retraction appear to be different. Autoantibody-induced cellular signaling mechanisms contribute to the pathogenesis of autoimmune blistering skin disease pemphigus vulgaris (PV). Recently, it was proposed that epidermal growth factor receptor (EGFR) might be involved in PV signaling pathways. In this study, we investigated the role of EGFR by comparing the effects of epidermal growth factor (EGF) and PV-IgG on the immortalized human keratinocyte cell line HaCaT, and primary normal human keratinocytes. In contrast to EGF treatment, PV-IgG neither caused the canonical activation of EGFR via phosphorylation at tyrosine (Y)1173 followed by internalization of EGFR nor the phosphorylation of the EGFR at the c-Src-dependent site Y845. Nevertheless, both PV-IgG and EGF led to cell dissociation and cytokeratin retraction in keratinocyte monolayers. Moreover, the effects of EGF were blocked by inhibition of EGFR and c-Src whereas the effects of PV-IgG were independent of both signaling pathways. Similarly, laser tweezer experiments revealed that impaired bead binding of epidermal cadherins desmoglein (Dsg) 3 and Dsg 1 in response to PV-IgG was not affected by inhibition of either EGFR or c-Src. In contrast, EGF treatment did not interfere with Dsg bead binding. Taken together, our study indicates that the loss of Dsg-mediated adhesion and keratinocyte dissociation in pemphigus is independent of EGFR. Moreover, the mechanisms by which both EGF and PV-IgG lead to keratinocyte dissociation and cytokeratin retraction appear to be different. Pemphigus is an autoimmune blistering skin disease caused by antibodies against keratinocyte surface antigens.1Waschke J The desmosome and pemphigus.Histochem Cell Biol. 2008; 130: 21-54Crossref PubMed Scopus (162) Google Scholar, 2Stanley JR Amagai M Pemphigus bullous impetigo, and the staphylococcal scalded-skin syndrome.N Engl J Med. 2006; 355: 1800-1810Crossref PubMed Scopus (361) Google Scholar, 3Anhalt GJ Labib RS Voorhees JJ Beals TF Diaz LA Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.N Engl J Med. 1982; 306: 1189-1196Crossref PubMed Scopus (594) Google Scholar Particularly, pathogenic autoantibodies are directed against epidermal cadherins desmoglein (Dsg) 3 and Dsg 1.4Amagai M Klaus-Kovtun V Stanley JR Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.Cell. 1991; 67: 869-877Abstract Full Text PDF PubMed Scopus (886) Google Scholar, 5Stanley JR Koulu L Thivolet C Distinction between epidermal antigens binding pemphigus vulgaris and pemphigus foliaceus autoantibodies.J Clin Invest. 1984; 74: 313-320Crossref PubMed Scopus (212) Google Scholar, 6Amagai M Karpati S Prussick R Klaus-Kovtun V Stanley JR Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.J Clin Invest. 1992; 90: 919-926Crossref PubMed Scopus (312) Google Scholar In the mucosal-dominant form of pemphigus vulgaris (PV), antibodies against Dsg 3 are produced, whereas Dsg 1 is an additional target when epidermal involvement occurs. For pemphigus foliaceus (PF) and the Brazilian endemic variant fogo selvagem, Dsg 1 is the major autoantigen. However, non-Dsg targets have also been identified.7Nguyen VT Ndoye A Shultz LD Pittelkow MR Grando SA Antibodies against keratinocyte antigens other than desmogleins 1 and 3 can induce pemphigus vulgaris-like lesions.J Clin Invest. 2000; 106: 1467-1479Crossref PubMed Scopus (164) Google Scholar Among those, pemphaxin, cholinergic receptors and E-cadherin are the best-studied so far.8Nguyen VT Ndoye A Grando SA Pemphigus Vulgaris Antibody Identifies Pemphaxin. A novel keratinocyte annexin-like molecule binding acetylcholine.J Biol Chem. 2000; 275: 29466-29476Crossref PubMed Scopus (118) Google Scholar, 9Nguyen VT Ndoye A Grando SA Novel human {alpha}9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity.Am J Pathol. 2000; 157: 1377-1391Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 10Evangelista F Dasher DA Diaz LA Prisayanh PS Li N E-cadherin is an additional immunological target for pemphigus autoantibodies.J Invest Dermatol. 2008; 128: 1710-1718Crossref PubMed Scopus (53) Google Scholar In addition, pathogenic non-autoantibody factors in pemphigus patients' sera such as Fas ligand are discussed.11Puviani M Marconi A Cozzani E Pincelli C Fas ligand in pemphigus sera induces keratinocyte apoptosis through the activation of caspase-8.J Invest Dermatol. 2003; 120: 164-167Crossref PubMed Scopus (119) Google Scholar Nevertheless, there is an ongoing debate whether acantholysis—the cellular hallmark of pemphigus pathogenicity—is induced by Dsg antibodies directly interfering with Dsg transinteraction or by cellular signaling mechanisms triggered by Dsg or non-Dsg autoantibodies.12Amagai M Ahmed AR Kitajima Y Bystryn JC 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 Are desmoglein autoantibodies essential for the immunopathogenesis of pemphigus vulgaris, or just ‘witnesses of disease’?.Exp Dermatol. 2006; 15: 815-831Crossref PubMed Scopus (1) Google Scholar At least for PF, cellular signaling seems to be important since no direct inhibition of Dsg 1-mediated binding by PF-IgG was observed by atomic force microscopy under conditions where autoantibodies caused keratinocyte dissociation.13Waschke J Bruggeman P Baumgartner W Zillikens D Drenckhahn D Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction.J Clin Invest. 2005; 115: 3157-3165Crossref PubMed Scopus (140) Google Scholar Nevertheless, recently we have demonstrated that PV-IgG directly interfere with Dsg 3 transinteraction.14Heupel WM Zillikens D Drenckhahn D Waschke J Pemphigus vulgaris IgG directly inhibit desmoglein 3-mediated transinteraction.J Immunol. 2008; 181: 1825-1834PubMed Google Scholar Over the past years, the involvement of several signaling pathways has been studied. However, the mechanisms involved in outside-in signaling as well as the interplay of the several pathways leading to acantholysis remain unclear.15Muller EJ Williamson L Kolly C Suter MM Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation?.J Invest Dermatol. 2008; 128: 501-516Crossref PubMed Scopus (138) Google ScholarIn vitro and in vivo studies have shown activation of p38 mitogen activated protein kinase (MAPK) by pemphigus IgG.16Berkowitz P Hu P Liu Z Diaz LA Enghild JJ Chua MP Rubenstein DS Desmosome signaling. Inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization.J Biol Chem. 2005; 280: 23778-23784Crossref PubMed Scopus (212) Google Scholar, 17Berkowitz P Hu P Warren S Liu Z Diaz LA Rubenstein DS p38MAPK inhibition prevents disease in pemphigus vulgaris mice.Proc Natl Acad Sci USA. 2006; 103: 12855-12860Crossref PubMed Scopus (194) Google Scholar Blocking p38 MAPK prevented cell dissociation and cytokeratin retraction. In addition, activation of the small GTPase RhoA completely antagonized pemphigus IgG-mediated effects in cultured human epidermis and keratinocyte monolayers.18Waschke J Spindler V Bruggeman P Zillikens D Schmidt G Drenckhahn D Inhibition of Rho A activity causes pemphigus skin blistering.J Cell Biol. 2006; 175: 721-727Crossref PubMed Scopus (131) Google Scholar Pemphigus IgG-induced Rho A inactivation was also p38 MAPK-dependent. Furthermore, plakoglobin depletion by pemphigus IgG is supposed to lead to diminished cell adhesion via c-Myc overexpression, which has been shown to result in keratinocyte hyperproliferation.19Caldelari R de Bruin A Baumann D Suter MM Bierkamp C Balmer V Muller E A central role for the armadillo protein plakoglobin in the autoimmune disease pemphigus vulgaris.J Cell Biol. 2001; 153: 823-834Crossref PubMed Scopus (165) Google Scholar, 20Williamson L Raess NA Caldelari R Zakher A de Bruin A Posthaus H Bolli R Hunziker T Suter MM Mller EJ Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin.EMBO J. 2006; 25: 3298-3309Crossref PubMed Scopus (159) Google Scholar Promotion of cell-cycle progression by PV-IgG-mediated upregulation of cyclin-dependent kinase 2 is another mechanism believed to cause acantholysis via continuing keratinocyte proliferation.21Lanza A Cirillo N Rossiello R Rienzo M Cutillo L Casamassimi A de Nigris F Schiano C Rossiello L Femiano F Gombos F Napoli C Evidence of key role of CDK-2 overexpression in pemphigus vulgaris.J Biol Chem. 2008; 283: 8736-8745Crossref PubMed Scopus (49) Google Scholar The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that activates a complex cellular signaling network involving the classical MAPK cascade (leading to activation of Erk and Akt), signal transducer and activator of transcription, phospholipase Cγ, and RhoA.22Schlessinger J Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3588) Google Scholar, 23Yarden Y Sliwkowski MX Untangling the ErbB signalling network.Nat Rev Mol Cell Biol. 2001; 2: 127-137Crossref PubMed Scopus (5749) Google Scholar EGFR can be activated by extracellular ligands like EGF, by intracellular kinases such as c-Src or by G protein coupled receptors.24Simeonova PP Wang S Hulderman T Luster MI c-Src-dependent activation of the epidermal growth factor receptor and mitogen-activated protein kinase pathway by arsenic. Role in carcinogenesis.J Biol Chem. 2002; 277: 2945-2950Crossref PubMed Scopus (108) Google Scholar, 25Carpenter G Employment of the epidermal growth factor receptor in growth factor-independent signaling pathways.J Cell Biol. 2002; 146: 697-702Crossref Scopus (247) Google Scholar Over a decade ago first work highlighted the interdependence of cell adhesion and EGFR function.26Hoschuetzky H Aberle H Kemler R Beta-catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor.J Cell Biol. 1994; 127: 1375-1380Crossref PubMed Scopus (675) Google Scholar Stimulation of EGFR resulted in phosphorylation of catenins (cadherin family adapter proteins) and colocalization of EGFR with the cadherin-catenin complex. Moreover, epidermal growth factor (EGF)-mediated phosphorylation of plakoglobin caused depletion of desmoplakin from desmosomes as well as reduced cell adhesion.27Gaudry CA Palka HL Dusek RL Huen AC Khandekar MJ Hudson LG Green KJ Tyrosine-phosphorylated plakoglobin is associated with desmogleins but not desmoplakin after epidermal growth factor receptor activation.J Biol Chem. 2001; 276: 24871-24880Crossref PubMed Scopus (65) Google Scholar, 28Yin T Getsios S Caldelari R Godsel LM Kowalczyk AP Mller EJ Green KJ Mechanisms of plakoglobin-dependent adhesion: desmosome-specific functions in assembly and regulation by epidermal growth factor receptor.J Biol Chem. 2005; 280: 40355-40363Crossref PubMed Scopus (81) Google Scholar Activation of EGFR following PV-IgG treatment was speculated to up-regulate Fas receptor signaling resulting in apoptosis and finally in acantholysis.29Frusic-Zlotkin M Raichenberg D Wang X David M Michel B Milner Y Apoptotic mechanism in pemphigus autoimmunoglobulins-induced acantholysis–possible involvement of the EGF receptor.Autoimmunity. 2006; 39: 563-575Crossref PubMed Scopus (65) Google Scholar Another study explored c-Src-dependent EGFR activation in pemphigus.30Chernyavsky AI Arredondo J Kitajima Y Sato-Nagai M Grando SA Desmoglein VS. non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens.J Biol Chem. 2007; 282: 13804-13812Crossref PubMed Scopus (125) Google Scholar Blocking of c-Src diminished activation of EGFR as well as of p38 MAPK and also reduced pathogenic effects of PV-IgG. Taken together, EGFR activation could explain various aspects of acantholysis in pemphigus. Therefore, in the present work we aimed to further evaluate the role of EGFR in pemphigus by the following approaches: (i) comparing the effects of EGF and PV-IgG on human keratinocytes, (ii) investigating PV-IgG-mediated EGFR phosphorylation and (iii) examining the requirement of EGFR for PV-IgG-mediated effects by inhibition of EGFR or of c-Src. The immortalized human keratinocyte cell line HaCaT31Boukamp P Petrussevska RT Breitkreutz D Hornung J Markham A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3522) Google Scholar was grown in Dulbecco's modified Eagles medium (Life technologies) that was supplemented with 50 U/ml penicillin-G, 50 μg streptomycin and 10% fetal calf serum (Biochrom) in a humidified atmosphere (95% air/5% CO2) at 37°C. Normal human epidermal keratinocytes (NHEK) derived from juvenile skin were purchased from PromoCell GmbH (Heidelberg, Germany). Cells were grown in Keratinocyte Growth Medium 2 (PromoCell, Heidelberg, Germany) supplemented with 50 U/ml penicillin-G, 50 μg streptomycin and supplement mix (PromoCell). NHEK were grown in low Ca2+ (0.15 mmol/L), which was changed to high Ca2+ (1.2 mmol/L) one day before experiments were started. For experiments, EGF (Sigma-Aldrich, Taufkirchen, Germany) was used at 20 ng/ml. Pharmacological inhibitor of EGFR (GW2974) (Sigma-Aldrich) at 10 μmol/L and neutralizing antibody against EGFR (LA-1) (Millipore, Schwalbach, Germany) at 1 μg/ml. c-Src inhibitor PP2 (Calbiochem, Darmstadt, Germany) was applied at 10 μmol/L. All inhibitors were pre-incubated for 2 hours. CNF-1 and CNFy were used at 300 ng/ml and 900 ng/ml, respectively. Purification was performed as described previously.13Waschke J Bruggeman P Baumgartner W Zillikens D Drenckhahn D Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction.J Clin Invest. 2005; 115: 3157-3165Crossref PubMed Scopus (140) Google Scholar Sera from two patients with mucosal-dominant PV and three patients suffering from a mucocutaneous form of PV whose diagnoses were confirmed clinically, histologically, and serologically and from a volunteer without any skin disease (control IgG) were used for the present study. Patients' sera were tested by enzyme-linked immunosorbent assay (ELISA, Medical and Biological Laboratories, Nagoya, Japan) according to the manufacturer's protocols for reactivity against Dsg 1 and Dsg 3, respectively (see Table 1). The cut-off value was 14 U/ml for Dsg 1 and 7 U/ml for Dsg 3. All samples were run in duplicate. PV-IgG 1 and 5 contained Dsg 3 but not Dsg 1 autoantibodies. PV-IgG 2, 3, and 4 contained both Dsg 1 and Dsg 3 antibodies. IgG fractions were purified by affinity chromatography using protein A agarose. Final concentrations of IgG fractions were adjusted to 500 to 1000 μg/ml for all experiments.Table 1Antibody Profile of Pemphigus Patients' IgGELISADsg 1 (U/ml)Dsg 3 (U/ml)PV-IgG 1—1874PV-IgG 2601000PV-IgG 35351098PV-IgG 4541239PV-IgG 5—1177 Open table in a new tab HaCaT cells were grown on coverslips to confluence and incubated with pemphigus IgG and indicated reagents for 24 hours or the indicated times at 37°C. After incubation, culture medium was removed, monolayers were fixed, permeabilized with ice-cold acetone for 2 minutes and washed with PBS. Afterward, HaCaT cells were incubated for 30 minutes with 10% normal goat serum and 1% bovine serum albumin in PBS at room temperature and incubated for 16 hours at 4°C with mouse monoclonal antibody directed against Dsg 3 (Zytomed, Berlin, Germany; dilution 1:100 in PBS), mouse monoclonal antibody against EGFR (Millipore; 1:100), rabbit polyclonal antibody against Y845 phospho-EGFR (Abcam, Cambridge, USA; 1:50), or mouse monoclonal cytokeratin 5 antibody (Santa Cruz, Heidelberg, Germany; 1:100). After several rinses with PBS (3 × 5 minutes), monolayers were incubated for 60 minutes at room temperature with Cy3-labeled goat anti-mouse or goat anti-rabbit IgG (Dianova). For visualization of filamentous actin (F-actin) or nuclear staining, ALEXA 488-phalloidin (Mobitec, Göttingen, Germany; diluted 1:60 in PBS, incubation for 1 hour at room temperature) and 4,6-diamidino-2-phenylindole (Roche, 1:3000 for 10 minutes at room temperature) were used, respectively. Cells were rinsed with PBS (3 × 5 minutes) and finally mounted on glass slides with 60% glycerol in PBS, containing 1.5% n-propyl gallate (Serva, Heidelberg, Germany). Monolayers were examined using a LSM 510 (Zeiss, Oberkochen, Germany). Images were processed using Adobe Photoshop 7.0 software (Adobe, München, Germany). For evaluation of PV-IgG binding to the surface of keratinocytes, images were quantitatively measured using ImageJ (National Institutes of Health, Bethesda, MD) software analysis. Therefore, after incubation of PV-IgG on HaCaT keratinocytes for the indicated times, cells were fixed and incubated with Cy3-labeled goat anti-human antibody to detect surface-bound PV-IgG. After washing, mean fluorescent intensities were measured in 3 independent areas of each condition with ImageJ. Each condition was repeated three times. After incubation with pemphigus patients' IgG or reagents for the indicated times, HaCaT cells were dissolved in sample buffer, heated at 95°C for 5 minutes and finally subjected to sodium dodecyl sulfate 7.5% or 10% polyacrylamide gel electrophoresis and immunoblotting to Hybond nitrocellulose membranes (Amersham, Buckinghamshire, UK). Membranes were blocked with 5% low fat milk for 1 hour at room temperature in PBS and incubated with the respective primary antibody overnight at 4°C. The rabbit antibodies against EGFR (Santa Cruz) and Y845 phospho-EGFR (Abcam) as well as the mouse Y1173 phospho-EGFR (Millipore) antibody were used at 1:400. As secondary antibodies HRP-labeled goat anti-mouse or goat anti-rabbit (both from Dianova, Hamburg, Germany) were used. Visualization was achieved using the enhanced chemiluminescence technique (Amersham). Phosphorylation of EGFR at Y845 was detected using chemiluminescence-based FACE EGFR (Y845) ELISA Kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. HaCaT cells were seeded on 96-well plates and grown to confluence before treatment with EGF or pemphigus patients' IgG in the absence or presence of various reagents for 1 hour. The assay was performed as described in the literature with the following modifications.19Caldelari R de Bruin A Baumann D Suter MM Bierkamp C Balmer V Muller E A central role for the armadillo protein plakoglobin in the autoimmune disease pemphigus vulgaris.J Cell Biol. 2001; 153: 823-834Crossref PubMed Scopus (165) Google Scholar, 32Ishii K Harada R Matsuo I Shirakata Y Hashimoto K Amagai M In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.J Invest Dermatol. 2005; 124: 939-946Crossref PubMed Scopus (107) Google Scholar HaCaT cells were seeded on 12-well plates and grown to confluence. After incubation for 24 hours under various conditions, cells were washed with Hanks' buffered salt solution and treated for 30 minutes with 0.3 ml dispase II (2.4 U/ml, Sigma) at 37°C. Afterward, dispase solution was carefully removed and cells dissolved in 0.5 ml Hank's buffered salt solution (HBSS). Mechanical stress was then applied by pipetting 10 times with a 1 ml pipette. Finally, dissociation was quantified by counting and averaging cell fragments in three defined areas of each condition under a binocular microscope. Every condition was repeated at least four times. Expression and purification of recombinant Dsg 3 and Dsg 1, coating of polystyrene beads and the laser tweezer set-up were described previously in detail.13Waschke J Bruggeman P Baumgartner W Zillikens D Drenckhahn D Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction.J Clin Invest. 2005; 115: 3157-3165Crossref PubMed Scopus (140) Google Scholar Coated beads (10 μl of stock solution) were suspended in 200 μl of culture medium and allowed to interact with HaCaT monolayers for 30 minutes at 37°C before measuring the number of bound beads (= control values). Beads were considered tightly bound when resisting laser displacement at 42 mW setting. For every condition 100 beads were counted. Afterward, EGF or PV-IgG with or without test reagents (pre-incubated for 2 hours) were applied for 30 minutes or the indicated times. Percentage of beads resisting laser displacement under various experimental conditions was normalized to control values. Differences in ELISA values or bead adhesion between different protocols have been assessed using two-tailed Student's t-test. Mann-Whitney U-test was used for comparison of dispase-based dissociation assay experiments. Values throughout are expressed as mean ± SE. Statistical significance is assumed for P < 0.05. In our study, we compared the effects of PV-IgG versus EGF treatment in various experimental setups to gain insight into the role of EGFR in pemphigus. First, we tested the effects of EGF and PV-IgG on desmosomes and the intermediate filament cytoskeleton in cultured human keratinocytes (HaCaT) using immunostaining. In addition, Alexa 488-phalloidin staining of filamentous actin (F-actin) was applied to sensitively detect keratinocyte dissociation, especially under conditions when Dsg 3 staining was altered and thus cell borders were hard to identify. Under control conditions, Dsg 3 was distributed along cell-cell borders together with F-actin (Figure 1, A and B). To examine pathogenic effects on the cytokeratin filament network, we stained the cells for cytokeratin 5 (CK5). In controls, CK5 was detected as a meshwork throughout the cytoplasm except of the cell periphery where staining was weak (Figure 1C). Incubation for 24 hours with control IgG yielded a similar phenotype (Figure 1, D–F). In contrast, EGF caused formation of intercellular gaps and keratinocyte dissociation after 24 hours, paralleled by disorganization of the actin cytoskeleton including the generation of stress fibers and short F-actin aggregates (Figure 1, G–H). More significantly, CK5 staining revealed a strong retraction of keratin filaments from the cell periphery to the perinuclear area (Figure 1I). Theses effects already appeared after 2 hours (not shown), but were more prominent after 24 hours EGF treatment. Similarly, PV-IgG incubation caused keratinocyte dissociation, actin reorganization and cytokeratin retraction (Figure 1, J–L). However, in contrast to EGF, Dsg 3 staining shifted from the membrane to the cytoplasm and was fragmented along cell borders in response to PV-IgG indicating loss and aggregation of desmosomes. Experiments were reproduced with PV-IgG 2–4 (data not shown). Thus, prominent cytokeratin retraction and intercellular gap formation were induced by both EGF and PV-IgG. All experiments were repeated with NHEK cells, which yielded similar results (data not shown). These data suggested that EGFR signaling may account for at least some of the autoantibody-mediated effects, a conclusion that led us to further explore whether EGFR was activated by PV-IgG under these conditions. Next we tested if PV-IgG were capable to activate EGFR via the canonical EGF pathway. It is known that EGFR is classically activated via ligand binding leading to dimerization and intracellular EGFR kinase activation.22Schlessinger J Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3588) Google Scholar This finally results in Y1173 EGFR phosphorylation, subsequent initiation of the MAP kinase cascade as well as in EGFR endocytosis and termination of EGFR signaling. We incubated EGF or PV-IgG on HaCaT cells for 1 hour and subsequently probed for Y1173 phospho-EGFR in Western blotting studies (Figure 2A) because previous studies from the literature reported significant phosphorylation of EGFR within 1 hour.29Frusic-Zlotkin M Raichenberg D Wang X David M Michel B Milner Y Apoptotic mechanism in pemphigus autoimmunoglobulins-induced acantholysis–possible involvement of the EGF receptor.Autoimmunity. 2006; 39: 563-575Crossref PubMed Scopus (65) Google Scholar, 30Chernyavsky AI Arredondo J Kitajima Y Sato-Nagai M Grando SA Desmoglein VS. non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens.J Biol Chem. 2007; 282: 13804-13812Crossref PubMed Scopus (125) Google Scholar As expected, EGF treatment resulted in strong phosphorylation of EGFR at Y1173. However, no enhanced signal was seen after PV-IgG 1–4 treatment. Comparable results were obtained when longer incubation periods for up to 24 hours were used. To exclude that EGFR activation is impaired in immortalized HaCaT keratinocytes, experiments were repeated with NHEK cells (Figure 2A). However, Western blotting studies using the Y1173 phospho-EGFR antibody similarly showed that PV-IgG did not activate EGFR by phosphorylation at Y1173. Immunostaining revealed EGFR to be localized continuously along cell borders in HaCaT cells (Figure 2B). EGF treatment for 1 hour resulted in punctuated EGFR staining within the cytoplasm of HaCaT cells likely representing endocytosed vesicles containing EGFR (Figure 2C). After PV-IgG treatment, localization of EGFR was not altered (Figure 2, D–G). This was also confirmed in EGFR-overexpressing A431 cells (data not shown). Therefore, canonical activation with phosphorylation of EGFR at Y1173 leading to EGFR endocytosis was not observed in response to PV-IgG, which is in contrast to previous findings from the literature.29Frusic-Zlotkin M Raichenberg D Wang X David M Michel B Milner Y Apoptotic mechanism in pemphigus autoimmunoglobulins-induced acantholysis–possible involvement of the EGF receptor.Autoimmunity. 2006; 39: 563-575Crossref PubMed Scopus (65) Google Scholar, 30Chernyavsky AI Arredondo J Kitajima Y Sato-Nagai M Grando SA Desmoglein VS. non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens.J Biol Chem. 2007; 282: 13804-13812Crossref PubMed Scopus (125) Google Scholar As a next step, we looked for other modes of EGFR activation by PV-IgG. Y845 is not only an autophosphorylation site in the kinase domain of EGFR but also phosphorylated by c-Src.33Biscardi JS Maa M-C Tice DA Cox ME Leu T-H Parsons SJ c-Src-mediated Phosphorylation of the Epidermal Growth Factor Receptor on Tyr845 and Tyr1101 Is Associated with Modulation of Receptor Function.J Biol Chem. 1999; 274: 8335-8343Crossref PubMed Scopus (571) Google Scholar Since it was recently reported that c-Src is activated after PV-IgG treatment,30Chernyavsky AI Arredondo J Kitajima Y Sato-Nagai M Grando SA Desmoglein VS. non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens.J Biol Chem. 2007; 282: 13804-13812Crossref PubMed Scopus (125) Google Scholar it was important to test if EGFR was phosphorylated at Y845 in response to PV-IgG. We measured phosphorylation of EGFR at Y845 using a commercial ELISA kit (Figure 3A). Control IgG did not lead to an enhanced signal in ELISA experiments. EGF induced a strong phosphorylation (ELISA score 1585% ± 244% of control). This signal could be efficiently blocked to 42% ± 5%, 45% ± 10% and 61% ± 2% of EGF values using neutralizing EGFR antibody (LA-1), pharmacological inhibitiors of EGFR (GW2974) or c-Src kinase (PP2) (Figure 3B). Surprisingly, all PV-IgG tested also resulted in strong and significant EGFR phosphorylation (ELISA scores 590% ± 16%, 748% ± 55%, 680% ± 67%, Figure 3A). These signals, however, were also detected when the respective secondary antibody was applied without Y845 phospho-EGFR primary antibody (ELISA scores 686% ± 23%, 937% ± 38%, 476% ± 66%, respectively). This indicated that the ELISA signals were due to cross-reactivity of the ELISA secondary goat anti-rabbit (garb) antibody with pemphigus IgG bound to the k
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