RESUMO
Desmoglein 3 (Dsg3) is a desmosomal cadherin mediating cell adhesion within desmosomes and is the antigen of the autoimmune blistering skin disease pemphigus vulgaris. Therefore, understanding of the complex desmosome turnover process is of high biomedical relevance. Recently, super resolution microscopy was used to characterize desmosome composition and turnover. However, studies were limited because adhesion measurements on living cells were not possible in parallel. Before desmosomal cadherins are incorporated into nascent desmosomes, they are not bound to intermediate filaments but were suggested to be associated with the actin cytoskeleton. However, direct proof that adhesion of a pool of desmosomal cadherins is dependent on actin is missing. Here, we applied single-molecule force spectroscopy measurements with the novel single molecule hybrid-technique STED/SMFS-AFM to investigate the cytoskeletal anchorage of Dsg3 on living keratinocytes for the first time. By application of pharmacological agents we discriminated two different Dsg3 pools, only one of which is anchored to actin filaments. We applied the actin polymerization inhibitor Latrunculin B to modify the actin cytoskeleton and the PKCα activator PMA to modulate intermediate filament anchorage. On the cellular surface Dsg3 adhesion was actin-dependent. In contrast, at cell-cell contacts, Dsg3 adhesion was independent from actin but rather is regulated by PKC which is well established to control desmosome turn-over via intermediate filament anchorage. Taken together, using the novel STED/SMFS-AFM technique, we demonstrated the existence of two Dsg3 pools with different cytoskeletal anchorage mechanisms.
Assuntos
Doenças Autoimunes , Pênfigo , Humanos , Desmogleína 3/metabolismo , Actinas/metabolismo , Desmossomos/metabolismo , Queratinócitos/metabolismo , Pênfigo/metabolismo , Caderinas/metabolismo , Adesão Celular , Doenças Autoimunes/metabolismoRESUMO
Plakophilins (Pkp) are desmosomal plaque proteins crucial for desmosomal adhesion and participate in the regulation of desmosomal turnover and signaling. However, direct evidence that Pkps regulate clustering and molecular binding properties of desmosomal cadherins is missing. Here, keratinocytes lacking either Pkp1 or 3 in comparison to wild type (wt) keratinocytes were characterized with regard to their desmoglein (Dsg) 1- and 3-binding properties and their capability to induce Dsg3 clustering. As revealed by atomic force microscopy (AFM), both Pkp-deficient keratinocyte cell lines showed reduced membrane availability and binding frequency of Dsg1 and 3 at cell borders. Extracellular crosslinking and AFM cluster mapping demonstrated that Pkp1 but not Pkp3 is required for Dsg3 clustering. Accordingly, Dsg3 overexpression reconstituted cluster formation in Pkp3- but not Pkp1-deficient keratinocytes as shown by AFM and STED experiments. Taken together, these data demonstrate that both Pkp1 and 3 regulate Dsg membrane availability, whereas Pkp1 but not Pkp3 is required for Dsg3 clustering.
Assuntos
Adesão Celular , Desmogleína 1/metabolismo , Desmogleína 3/metabolismo , Placofilinas/genética , Animais , Anisomicina/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Desmogleína 1/genética , Desmogleína 3/genética , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Microscopia de Força Atômica , Placofilinas/deficiência , Placofilinas/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
RATIONALE: The sympathetic nervous system is a major mediator of heart function. Intercalated discs composed of desmosomes, adherens junctions, and gap junctions provide the structural backbone for coordinated contraction of cardiac myocytes. OBJECTIVE: Gap junctions dynamically remodel to adapt to sympathetic signaling. However, it is unknown whether such rapid adaption also occurs for the adhesive function provided by desmosomes and adherens junctions. METHODS AND RESULTS: Atomic force microscopy revealed that ß-adrenergic signaling enhances both the number of desmoglein 2-specific interactions along cell junctions and the mean desmoglein 2-mediated binding forces, whereas N-cadherin-mediated interactions were not affected. This was accompanied by increased cell cohesion in cardiac myocyte cultures and murine heart slices. Enhanced desmoglein 2-positive contacts and increased junction length as revealed by immunofluorescence and electron microscopy reflected cAMP-induced reorganization of intercellular contacts. The mechanism underlying cAMP-mediated strengthening of desmoglein 2 binding was dependent on expression of the intercalated disc plaque protein plakoglobin (Pg) and direct phosphorylation at S665 by protein kinase A: Pg deficiency as well as overexpression of the phospho-deficient Pg-mutant S665A abrogated both cAMP-mediated junctional remodeling and increase of cohesion. Moreover, Pg knockout hearts failed to functionally adapt to adrenergic stimulation. CONCLUSIONS: Taken together, we provide first evidence for positive adhesiotropy as a new cardiac function of sympathetic signaling. Positive adhesiotropy is dependent on Pg phosphorylation at S665 by protein kinase A. This mechanism may be of high medical relevance because loss of junctional Pg is a hallmark of arrhythmogenic cardiomyopathy.
Assuntos
Adesão Celular , Comunicação Celular , Junções Comunicantes/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Desmogleína 2/metabolismo , Imunofluorescência , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/ultraestrutura , Genótipo , Técnicas In Vitro , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Fenótipo , Fosforilação , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , gama Catenina/genética , gama Catenina/metabolismoRESUMO
Desmosomal cadherins are transmembrane adhesion molecules that provide cell adhesion by interacting in the intercellular space of adjacent cells. In keratinocytes, several desmoglein (Dsg1-4) and desmocollin (Dsc1-3) isoforms are coexpressed. We have shown previously that Dsg2 is less important for keratinocyte cohesion compared with Dsg3 and that the latter forms a complex with p38 MAPK. In this study, we compared the involvement of Dsg2 and Dsg3 in the p38 MAPK-dependent regulation of keratinocyte cohesion. We show that loss of cell adhesion and keratin filament retraction induced by Dsg3 depletion is ameliorated by specific p38 MAPK inhibition. Furthermore, in contrast to depletion of Dsg2, siRNA-mediated silencing of Dsg3 induced p38 MAPK activation, which is in line with immunoprecipitation experiments demonstrating the interaction of activated p38 MAPK with Dsg3 but not with Dsg2. Cell fractionation into a cytoskeleton-unbound and a cytoskeleton-anchored desmosome-containing pool revealed that Dsg3, in contrast to Dsg2, is present in relevant amounts in the unbound pool in which activated p38 MAPK is predominantly detectable. Moreover, because loss of cell adhesion by Dsg3 depletion was partially rescued by p38 MAPK inhibition, we conclude that, besides its function as an adhesion molecule, Dsg3 is strengthening cell cohesion via modulation of p38 MAPK-dependent keratin filament reorganization. Nevertheless, because subsequent targeting of Dsg3 in Dsg2-depleted cells led to drastically enhanced keratinocyte dissociation and Dsg2 was enhanced at the membrane in Dsg3 knockout cells, we conclude that Dsg2 compensates for Dsg3 loss of function.
Assuntos
Desmogleína 2/metabolismo , Desmogleína 3/metabolismo , Queratinócitos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Desmogleína 2/genética , Desmogleína 3/genética , Desmossomos/metabolismo , Humanos , Queratinócitos/fisiologia , CamundongosRESUMO
Vascular permeability is mediated by Cortactin (Cttn) and regulated by several molecules including cyclic-adenosine-monophosphate, small Rho family GTPases and the actin cytoskeleton. However, it is unclear whether Cttn directly interacts with any of the junctional components or if Cttn intervenes with signaling pathways affecting the intercellular contacts and the cytoskeleton. To address these questions, we employed immortalized microvascular myocardial endothelial cells derived from wild-type and Cttn-knock-out mice. We found that lack of Cttn compromised barrier integrity due to fragmented membrane distribution of different junctional proteins. Moreover, immunoprecipitations revealed that Cttn is within the VE-cadherin-based adherens junction complex. In addition, lack of Cttn slowed-down barrier recovery after Ca2+ repletion. The role of Cttn for cAMP-mediated endothelial barrier regulation was analyzed using Forskolin/Rolipram. In contrast to Cttn-KO, WT cells reacted with increased transendothelial electrical resistance. Absence of Cttn disturbed Rap1 and Rac1 activation in Cttn-depleted cells. Surprisingly, despite the absence of Cttn, direct activation of Rac1/Cdc42/RhoA by CN04 increased barrier resistance and induced well-defined cortical actin and intracellular actin bundles. In summary, our data show that Cttn is required for basal barrier integrity by allowing proper membrane distribution of junctional proteins and for cAMP-mediated activation of the Rap1/Rac1 signaling pathway.
Assuntos
Junções Aderentes , Antígenos CD , Células Endoteliais , Camundongos , Animais , Junções Aderentes/metabolismo , Células Endoteliais/metabolismo , Actinas/metabolismo , Cortactina/genética , Cortactina/metabolismo , Caderinas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3',5'-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised.
Assuntos
Pênfigo , Humanos , Pênfigo/metabolismo , Pênfigo/patologia , Desmossomos/metabolismo , Adesão Celular/fisiologia , Caderinas/metabolismo , Caderinas/farmacologia , Miocárdio/metabolismo , Epitélio/metabolismo , Endotélio/metabolismoRESUMO
Pemphigus vulgaris is a life-threatening blistering skin disease caused by autoantibodies destabilizing desmosomal adhesion. Current therapies focus on suppression of autoantibody formation and thus treatments directly stabilizing keratinocyte adhesion would fulfill an unmet medical need. We here demonstrate that apremilast, a phosphodiesterase 4 inhibitor used in psoriasis, prevents skin blistering in pemphigus vulgaris. Apremilast abrogates pemphigus autoantibody-induced loss of keratinocyte cohesion in ex-vivo human epidermis, cultured keratinocytes in vitro and in vivo in mice. In parallel, apremilast inhibits keratin retraction as well as desmosome splitting, induces phosphorylation of plakoglobin at serine 665 and desmoplakin assembly into desmosomal plaques. We established a plakoglobin phospho-deficient mouse model that reveals fragile epidermis with altered organization of keratin filaments and desmosomal cadherins. In keratinocytes derived from these mice, intercellular adhesion is impaired and not rescued by apremilast. These data identify an unreported mechanism of desmosome regulation and propose that apremilast stabilizes keratinocyte adhesion and is protective in pemphigus.
Assuntos
Pênfigo , Humanos , Camundongos , Animais , Pênfigo/tratamento farmacológico , gama Catenina , Adesão Celular , Queratinócitos , Epiderme , Vesícula , Autoanticorpos , Queratinas , DesmossomosRESUMO
Adducin (Add) is an actin binding protein participating in the stabilization of actin/spectrin networks, epithelial junctional turnover and cardiovascular disorders such as hypertension. Recently, we demonstrated that Add is required for adherens junctions (AJ) integrity. Here we hypothesized that Add regulates tight junctions (TJ) as well and may play a role in cAMP-mediated barrier enhancement. We evaluated the role of Add in MyEnd cells isolated from WT and Add-Knock-Out (KO) mice. Our results indicate that the lack of Add drastically alters the junctional localization and protein levels of major AJ and TJ components, including VE-Cadherin and claudin-5. We also showed that cAMP signaling induced by treatment with forskolin and rolipram (F/R) enhances the barrier integrity of WT but not Add-KO cells. The latter showed no junctional reorganization upon cAMP increase. The absence of Add also led to higher protein levels of the small GTPases Rac1 and RhoA. In vehicle-treated cells the activation level of Rac1 did not differ significantly when WT and Add-KO cells were compared. However, the lack of Add led to increased activity of RhoA. Moreover, F/R treatment triggered Rac1 activation only in WT cells. The function of Rac1 and RhoA per se was unaffected by the total ablation of Add, since direct activation with CN04 was still possible in both cell lines and led to improved endothelial barrier function. In the current study, we demonstrate that Add is required for the maintenance of endothelial barrier by regulating both AJ and TJ. Our data show that Add may act upstream of Rac1 as it is necessary for its activation via cAMP.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Neuropeptídeos/metabolismo , Junções Íntimas , Proteínas rac1 de Ligação ao GTP/metabolismo , Junções Aderentes/metabolismo , Animais , Caderinas/metabolismo , Camundongos , Junções Íntimas/metabolismoRESUMO
The autoimmune dermatosis pemphigus foliaceus (PF) is predominantly caused by IgG autoantibodies against the desmosomal cadherin desmoglein (Dsg) 1. The exact mechanisms that lead to the characteristic epidermal blistering are not yet fully understood. In the present study, we used a variety of biophysical methods to examine the fate of membrane-bound Dsg1 after incubation with PF patients' IgG. Dispase-based dissociation assays confirmed that PF-IgG used for this study reduced intercellular adhesion in a manner dependent on phospholipase C (PLC)/Ca2+ and extracellular signal-regulated kinase (ERK) 1/2 signaling. Atomic force microscopy (AFM) revealed that Dsg1 binding on single molecule level paralleled effects on keratinocyte adhesion under the different conditions. Stimulated emission depletion (STED) super-resolution microscopy was used to investigate the localization of Dsg1 after PF-IgG incubation for 24 h. Under control conditions, Dsg1 was found to be in part co-localized with desmoplakin and thus inside of desmosomes as well as extra-desmosomal along the cell border. Incubation with PF-IgG reduced the extra-desmosomal Dsg1 fraction. In line with this, fluorescence recovery after photobleaching (FRAP) experiments demonstrated a strongly reduced mobility of Dsg1 in the cell membrane after PF-IgG treatment indicating remaining Dsg1 molecules were primarily located inside desmosomes. Mechanistically, experiments confirmed the involvement of PLC/Ca2+ since inhibition of PLC or 1,4,5-trisphosphate (IP3) receptor to reduce cytosolic Ca2+ reverted the effects of PF-IgG on Dsg1 intra-membrane mobility and localization. Taken together, our findings suggest that during the first 24 h PF-IgG induce redistribution predominantly of membrane-bound extradesmosomal Dsg1 in a PLC/Ca2+ dependent manner whereas Dsg1-containing desmosomes remain.
Assuntos
Pênfigo , Autoanticorpos , Membrana Celular/metabolismo , Desmogleína 1 , Humanos , Imunoglobulina GRESUMO
The short chain fatty acid (SCFA) butyrate, a product of fermentation of dietary fiber in the human colon, is found to exert multiple regulatory processes in colon carcinogenesis. The aim of this study was to find out whether butyrate affects the tumor-promoting genes osteopontin (OPN) and cyclooxygenase (COX)-2, their respective proteins and/or their functional activity in matched normal, adenoma and tumor colon tissues obtained from 20 individuals at colon cancer surgery. Quantitative real-time polymerase chain reaction experiments showed increased levels of OPN and COX-2 messenger RNA in tumor tissues when compared with the adjacent normal samples (P < 0.001). The addition of butyrate reduced OPN and COX-2 mRNA expression in all tissue types compared with the related medium controls (tumor: P < 0.05). In tumor samples, a downregulation of up to median 35% (COX-2) and 50% (OPN) was observed, respectively. Thereby, tumors with lower levels of OPN basal expression were more sensitive to inhibition and vice versa for COX-2 in normal tissue. At the protein and enzyme level, which were determined by using western blot and enzyme immunometric assays, the impact of the SCFA was not clearly visible anymore. The active proteins of OPN and COX-2 (determined by prostaglandin E(2)) were found to correlate with their respective mRNA expression only in 50-63% of analyzed donors. For the first time, our data reveal new insights into the chemoprotective potential of butyrate by showing the suppression of OPN and COX-2 mRNA in primary human colon tissue with the strongest effects observed in tumors.
Assuntos
Adenoma/genética , Butiratos/farmacologia , Neoplasias Colorretais/genética , Ciclo-Oxigenase 2/genética , Osteopontina/genética , RNA Mensageiro/antagonistas & inibidores , Adenoma/tratamento farmacológico , Adenoma/metabolismo , Idoso , Western Blotting , Carcinógenos/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Masculino , Osteopontina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: It is known that alpha-defensin expression is enhanced in colon cancer. However, the expression of human alpha defensin 6 (DEFA 6) in earlier stages, such as adenoma, has so far not yet been studied in a patient resolved manner. METHODS: By using quantitative Real Time-PCR, the gene expression pattern of DEFA 1-3 and DEFA 6 was analyzed in tissue of different stages of carcinogenesis, derived from colorectal cancer patients. In addition to paired normal and tumor tissue, matched normal near tumor and adenoma tissue samples were examined. RESULTS: The median gene expression of human defensin alpha 6 (DEFA 6) has been found to be moderately increased (~ 5 fold) in tumor samples derived from individuals with colorectal cancer (CRC) when compared to their normal counterparts. However, when the data were analyzed in a patient-wise manner, a large expression variation among individual patients is found, making the use of DEFA 6 for individual diagnosis of fully blown colon carcinoma difficult. Surprisingly, in adenoma the gene expression analysis revealed a 100 fold increased median expression of DEFA 6 relative to normal colon tissue. 13/18 samples had an individual overexpression of more than 60 fold in adenoma but only 3/17 in carcinoma. In each of the individual patients, at least either the adenoma or the carcinoma showed strong DEFA 6 overexpression. CONCLUSIONS: We suggest that the expression of DEFA 6 preferably can be used as a potential diagnostic marker for adenoma and not as a marker for fully blown carcinoma. This is supported by the fact that DEFA 6 is a downstream target of the Wnt pathway, which is mutational active during the earliest stage of cancer development.
Assuntos
Adenoma/diagnóstico , Adenoma/metabolismo , Carcinoma/diagnóstico , Carcinoma/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , alfa-Defensinas/biossíntese , Idoso , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Wnt/metabolismoRESUMO
Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.
Assuntos
Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Miocárdio/metabolismo , Neuropeptídeos/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/efeitos dos fármacos , Animais , Antígenos CD/genética , Caderinas/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , AMP Cíclico/genética , Células Endoteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Miocárdio/patologia , Neuropeptídeos/agonistas , Transdução de Sinais/genética , Ativação Transcricional/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/agonistas , Proteína rhoA de Ligação ao GTP/agonistas , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Pemphigus is an autoimmune blistering disease targeting the desmosomal proteins desmoglein (Dsg) 1 and Dsg3. Recently, a genetic variant of the Suppression of tumorigenicity 18 (ST18) promoter was reported to cause ST18 up-regulation, associated with pemphigus vulgaris (PV)-IgG-mediated increase in cytokine secretion and more prominent loss of keratinocyte cohesion. Here we tested the effects of PV-IgG and the pathogenic pemphigus mouse anti-Dsg3 antibody AK23 on cytokine secretion and ERK activity in human keratinocytes dependent on ST18 expression. Without ST18 overexpression, both PV-IgG and AK23 induced loss of keratinocyte cohesion which was accompanied by prominent fragmentation of Dsg3 immunostaining along cell borders. In contrast, release of pro-inflammatory cytokines such as IL-1α, IL-6, TNFα, and IFN-γ was not altered significantly in both HaCaT and primary NHEK cells. These experiments indicate that cytokine expression is not strictly required for loss of keratinocyte cohesion. Upon ST18 overexpression, fragmentation of cell monolayers increased significantly in response to autoantibody incubation. Furthermore, production of IL-1α and IL-6 was enhanced in some experiments but not in others whereas release of TNF-α dropped significantly upon PV-IgG application in both EV- and ST18-transfected HaCaT cells. Additionally, in NHEK, application of PV-IgG but not of AK23 significantly increased ERK activity. In contrast, ST18 overexpression in HaCaT cells augmented ERK activation in response to both c-IgG and AK23 but not PV-IgG. Because inhibition of ERK by U0126 abolished PV-IgG- and AK23-induced loss of cell cohesion in ST18-expressing cells, we conclude that autoantibody-induced ERK activation was relevant in this scenario. In summary, similar to the situation in PV patients carrying ST18 polymorphism, overexpression of ST18 enhanced keratinocyte susceptibility to autoantibody-induced loss of cell adhesion, which may be caused in part by enhanced ERK signaling.
Assuntos
Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Queratinócitos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Pênfigo/imunologia , Proteínas Repressoras/imunologia , Linhagem Celular , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Humanos , Queratinócitos/patologia , Pênfigo/patologiaRESUMO
Pemphigus vulgaris (PV) is a potentially lethal autoimmune disease characterized by blister formation of the skin and mucous membranes and is caused by autoantibodies against desmoglein (Dsg) 1 and Dsg3. Dsg1 and Dsg3 are linked to keratin filaments in desmosomes, adhering junctions abundant in tissues exposed to high levels of mechanical stress. The binding of the autoantibodies leads to internalization of Dsg3 and a collapse of the keratin cytoskeleton-yet, the relevance and interdependence of these changes for loss of cell-cell adhesion and blistering is poorly understood. In live-cell imaging studies, loss of the keratin network at the cell periphery was detectable starting after 60 min of incubation with immunoglobulin G fractions of PV patients (PV-IgG). These rapid changes correlated with loss of cell-cell adhesion detected by dispase-based dissociation assays and were followed by a condensation of keratin filaments into thick bundles after several hours. Dsg3 internalization started at 90 min of PV-IgG treatment, thus following the early keratin changes. By inhibiting casein kinase 1 (CK-1), we provoked keratin alterations resembling the effects of PV-IgG. Although CK-1-induced loss of peripheral keratin network correlated with loss of cell cohesion and Dsg3 clustering in the membrane, it was not sufficient to trigger the internalization of Dsg3. However, additional incubation with PV-IgG was effective to promote Dsg3 loss at the membrane, indicating that Dsg3 internalization is independent from keratin alterations. Vice versa, inhibiting Dsg3 internalization did not prevent PV-IgG-induced keratin retraction and only partially rescued cell cohesion. Together, keratin changes appear very early after autoantibody binding and temporally overlap with loss of cell cohesion. These early alterations appear to be distinct from Dsg3 internalization, suggesting a crucial role for initial loss of cell cohesion in PV.
Assuntos
Autoanticorpos/imunologia , Desmogleína 3/imunologia , Queratinas/imunologia , Pênfigo/imunologia , Pele/imunologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Adesão Celular/imunologia , Linhagem Celular , Desmogleína 1/imunologia , Desmogleína 3/metabolismo , Endocitose/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Filamentos Intermediários/imunologia , Filamentos Intermediários/metabolismo , Queratinócitos , Queratinas/metabolismo , Pênfigo/sangue , Pênfigo/patologia , Pele/citologia , Pele/patologiaRESUMO
Keratins are crucial for the anchorage of desmosomes. Severe alterations of keratin organization and detachment of filaments from the desmosomal plaque occur in the autoimmune dermatoses pemphigus vulgaris and pemphigus foliaceus (PF), which are mainly caused by autoantibodies against desmoglein (Dsg) 1 and 3. Keratin alterations are a structural hallmark in pemphigus pathogenesis and correlate with loss of intercellular adhesion. However, the significance for autoantibody-induced loss of intercellular adhesion is largely unknown. In wild-type (wt) murine keratinocytes, pemphigus autoantibodies induced keratin filament retraction. Under the same conditions, we used murine keratinocytes lacking all keratin filaments (KtyII k.o.) as a model system to dissect the role of keratins in pemphigus. KtyII k.o. cells show compromised intercellular adhesion without antibody (Ab) treatment, which was not impaired further by pathogenic pemphigus autoantibodies. Nevertheless, direct activation of p38MAPK via anisomycin further decreased intercellular adhesion indicating that cell cohesion was not completely abrogated in the absence of keratins. Direct inhibition of Dsg3, but not of Dsg1, interaction via pathogenic autoantibodies as revealed by atomic force microscopy was detectable in both cell lines demonstrating that keratins are not required for this phenomenon. However, PF-IgG shifted Dsg1-binding events from cell borders toward the free cell surface in wt cells. This led to a distribution pattern of Dsg1-binding events similar to KtyII k.o. cells under resting conditions. In keratin-deficient keratinocytes, PF-IgG impaired Dsg1-binding strength, which was not different from wt cells under resting conditions. In addition, pathogenic autoantibodies were capable of activating p38MAPK in both KtyII wt and k.o. cells, the latter of which already displayed robust p38MAPK activation under resting conditions. Since inhibition of p38MAPK blocked autoantibody-induced loss of intercellular adhesion in wt cells and restored baseline cell cohesion in keratin-deficient cells, we conclude that p38MAPK signaling is (i) critical for regulation of cell adhesion, (ii) regulated by keratins, and (iii) targets both keratin-dependent and -independent mechanisms.
Assuntos
Adesão Celular/genética , Desmogleínas/metabolismo , Queratinócitos/fisiologia , Queratinas/metabolismo , Pênfigo/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anisomicina/metabolismo , Autoanticorpos/metabolismo , Células Cultivadas , Humanos , Queratinas/genética , Camundongos , Camundongos Knockout , Pênfigo/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
During inflammation, the disruption of the endothelial barrier leads to increased microvascular permeability. Whether tension along cell junctions contributes to histamine-induced endothelial barrier disruption remains unknown. Rapid Ca2+ influx induced by both histamine and thrombin was accompanied by endothelial barrier breakdown revealed as drop of transendothelial electric resistance in primary human microvascular endothelial cells. Interestingly, GLISA measurements revealed activation of RhoA but not inactivation of Rac1 at the time-point of barrier breakdown. FRET measurements showed activation of RhoA at intercellular junctions after both thrombin and histamine exposure. Breakdown coincided with increased stress fiber formation but not with translocation of vinculin, which was located along junctions in the resting state similar to postcapillary venules ex vivo. Moreover, increased tension at AJs was indicated by immunostaining with a conformation-sensitive antibody targeting the α18-subunit of α-catenin. Ca2+ chelation by BAPTA-AM and ROCK1 inhibition by Y27632 abolished both increase of tension along AJs as well as barrier dysfunction. Moreover, BAPTA-AM decreased RhoA activation following histamine stimulation, indicating a key role of Ca2+ signaling in barrier breakdown. Taken together, in response to histamine, Ca2+ via RhoA/ROCK activation along endothelial adherens junctions (AJs) appears to be critical for barrier disruption and presumably correlated with enhanced tension. However, vinculin appears not to be critical in this process.
Assuntos
Junções Aderentes/metabolismo , Cálcio/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Histamina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Junções Aderentes/ultraestrutura , Animais , Permeabilidade Capilar , Linhagem Celular , Células Endoteliais/ultraestrutura , Endotélio Vascular/ultraestrutura , Ativação Enzimática , Humanos , RatosRESUMO
Tightly controlled intercellular adhesion is crucial for the integrity and function of the epidermis. The keratin filament cytoskeleton anchors desmosomes, supramolecular complexes required for strong intercellular adhesion. We tested whether keratin filaments control cell adhesion by regulating the adhesive properties of desmosomal cadherins such as desmoglein (Dsg) 3. Atomic force microscopy and fluorescence recovery after photobleaching experiments showed reduced Dsg3 adhesive forces and membrane stability in murine keratinocytes lacking all keratin filaments. Impairment of the actin cytoskeleton also resulted in decreased Dsg3 immobilization but did not affect Dsg3 binding properties, indicating that the latter are exclusively controlled by keratins. Reduced binding forces were dependent on p38 mitogen-activated protein kinase activity, which was deregulated in keratin-deficient cells. In contrast, inhibition of protein kinase C signaling, which is known to be controlled by keratins, promoted and spatially stabilized Dsg3-mediated interactions in the membrane. These results show a previously unreported mechanism for how keratins stabilize intercellular adhesion on the level of single desmosomal adhesion molecules.
Assuntos
Adesão Celular/fisiologia , Desmogleína 3/metabolismo , Desmossomos/metabolismo , Queratinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Desmossomos/ultraestrutura , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinas/genética , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Transdução de SinaisRESUMO
Adducins tightly regulate actin dynamics which is critical for endothelial barrier function. Adducins were reported to regulate epithelial junctional remodeling by controlling the assembly of actin filaments at areas of cell-cell contact. Here, we investigated the role of α-adducin for endothelial barrier regulation by using microvascular human dermal and myocardial murine endothelial cells. Parallel transendothelial electrical resistance (TER) measurements and immunofluorescence analysis revealed that siRNA-mediated adducin depletion impaired endothelial barrier formation and led to severe fragmentation of VE-cadherin immunostaining at cell-cell borders. To further test whether the peripheral localization of α-adducin is functionally linked with the integrity of endothelial adherens junctions, junctional remodeling was induced by a Ca(2+)-switch assay. Ca(2+)-depletion disturbed both linear vascular endothelial (VE)-cadherin and adducin location along cell junctions, whereas their localization was restored following Ca(2+)-repletion. Similar results were obtained for α-adducin phosphorylated at a site typical for PKA (pSer481). To verify that endothelial barrier properties and junction reorganization can be effectively modulated by altering Ca(2+)-concentration, TER measurements were performed. Thus, Ca(2+)-depletion drastically reduced TER, whereas Ca(2+)-repletion led to recovery of endothelial barrier properties resulting in increased TER. Interestingly, the Ca(2+)-dependent increase in TER was also significantly reduced after efficient α-adducin downregulation. Finally, we report that inflammatory mediator-induced endothelial barrier breakdown is associated with loss of α-adducin from the cell membrane. Taken together, our results indicate that α-adducin is involved in remodeling of endothelial adhesion junctions and thereby contributes to endothelial barrier regulation.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Junções Aderentes/metabolismo , Cálcio/metabolismo , Linhagem Celular , Humanos , Junções Intercelulares/metabolismoRESUMO
cAMP-mediated PKA signaling is the main known pathway involved in maintenance of the endothelial barrier. Tight regulation of PKA function can be achieved by discrete compartmentalization of the enzyme via physical interaction with A-kinase anchoring proteins (AKAPs). Here, we investigated the role of AKAPs 220 and 12 in endothelial barrier regulation. Analysis of human and mouse microvascular endothelial cells as well as isolated rat mesenteric microvessels was performed using TAT-Ahx-AKAPis peptide, designed to competitively inhibit PKA-AKAP interaction. In vivo microvessel hydraulic conductivity and in vitro transendothelial electrical resistance measurements showed that this peptide destabilized endothelial barrier properties, and dampened the cAMP-mediated endothelial barrier stabilization induced by forskolin and rolipram. Immunofluorescence analysis revealed that TAT-Ahx-AKAPis led to both adherens junctions and actin cytoskeleton reorganization. Those effects were paralleled by redistribution of PKA and Rac1 from endothelial junctions and by Rac1 inactivation. Similarly, membrane localization of AKAP220 was also reduced. In addition, depletion of either AKAP12 or AKAP220 significantly impaired endothelial barrier function and AKAP12 was also shown to interfere with cAMP-mediated barrier enhancement. Furthermore, immunoprecipitation analysis demonstrated that AKAP220 interacts not only with PKA but also with VE-cadherin and ß-catenin. Taken together, these results indicate that AKAP-mediated PKA subcellular compartmentalization is involved in endothelial barrier regulation. More specifically, AKAP220 and AKAP12 contribute to endothelial barrier function and AKAP12 is required for cAMP-mediated barrier stabilization.