RESUMEN
Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis produced as a consequence of desmoglein (Dsg) and non-Dsg autoantibodies binding to several targeting molecules localized on the membrane of keratinocytes. Nitric oxide (NO) may exert a pathogenic function in several immunological processes. We have previously demonstrated that neural nitric oxide synthase (nNOS) plays part in PV acantholysis. Also, our group has described a relevant role for HER [human epidermal growth factor receptor (EGFR) related] isoforms and several kinases such as Src (Rous sarcoma), mammalian target of rapamycin (mTOR) and focal adhesion kinase (FAK), as well as caspases in PV development. Using a passive transfer mouse model of PV, we aimed to investigate the relationship between the increase in nNOS and EGFR, Src, mTOR and FAK kinase upregulation observed in PV lesions. Our results revealed a new function for nNOS, which contributes to EGFR-mediated PV acantholysis through the upregulation of Src, mTOR and FAK. In addition, we found that nNOS participates actively in PV at least in part by increasing caspase-9 and caspase-3 activities. These findings underline the important issue that in PV acantholysis, caspase activation is a nNOS-linked process downstream of Src, mTOR and FAK kinase upregulation.
Asunto(s)
Acantólisis/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Pénfigo/enzimología , Serina-Treonina Quinasas TOR/metabolismo , Familia-src Quinasas/metabolismo , Animales , Biopsia , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.
Asunto(s)
Acantólisis/enzimología , Pénfigo/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acantólisis/patología , Animales , Apoptosis/efectos de los fármacos , Autoanticuerpos/metabolismo , Caspasa 3/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patología , Desmogleína 1/metabolismo , Desmogleína 3/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Inmunoglobulina G/metabolismo , Ratones , Pénfigo/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Técnicas de Cultivo de Tejidos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Focal adhesion kinase is a protein-tyrosine kinase that is found in cellular contact sites and is phosphorylated in response to cell attachment. It is possible that the immunohistochemical detection of this enzyme might be increased in keratinocytes involved in an acantholytic process. Normal skin, pemphigus vulgaris and foliaceus, Darier's disease, Hailey--Hailey disease, warty dyskeratoma, Grover's disease and spongiotic dermatitis were assayed for the immunohistochemical expression of focal adhesion kinase. Focal adhesion kinase was not observed in normal epidermis. This antigen was observed in keratinocytes adjacent to acantholytic spaces and in acantholytic cells in pemphigus vulgaris and foliaceus. Focal adhesion kinase was not detected in keratinocytes involved in focal acantholytic dyskeratoses such as Darier's disease, Grover's disease and warty dyskeratoma but was weakly detected in Hailey--Hailey disease. One consequence of immunologically mediated acantholysis is the upregulation of focal adhesion kinase possibly as a component of biochemical pathways that reconstruct the process of adhesion or respond to the process of acantholysis.
Asunto(s)
Acantólisis/enzimología , Moléculas de Adhesión Celular/análisis , Queratinocitos/enzimología , Pénfigo/enzimología , Proteínas Tirosina Quinasas/análisis , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Técnicas para Inmunoenzimas , Piel/enzimologíaRESUMEN
BACKGROUND: The formation of lacunae and acantholysis as well as dyskeratosis are characteristic features of Hailey-Hailey disease (HHD) and Darier's disease (DD). Matrix metalloproteinases (MMPs) and their inhibitors like tissue inhibitors of metalloproteinases (TIMPs) have been thought to play major roles in the tissue metabolism. OBJECTIVE: The aim of this study was to investigate the role of MMP-9 and TIMP-1 in HHD and DD. METHODS: We examined localizations of these two molecules by immunostaining using specific monoclonal antibodies. RESULTS: MMP-9 was positively stained in dyskeratotic or detaching cells around lacunae in HHD and DD. TIMP-1 showed a positive staining pattern throughout the epidermis. CONCLUSION: MMP-9 might be involved in the pathophysiological process of HHD and DD in the presence of TIMP-1.
Asunto(s)
Colagenasas/análisis , Enfermedad de Darier/patología , Glicoproteínas/análisis , Inhibidores de la Metaloproteinasa de la Matriz , Pénfigo Familiar Benigno/patología , Inhibidores de Proteasas/análisis , Acantólisis/enzimología , Acantólisis/patología , Anticuerpos Monoclonales , Western Blotting , Adhesión Celular , Células Cultivadas , Colorantes , Enfermedad de Darier/enzimología , Enfermedad de Darier/fisiopatología , Electroforesis en Gel de Poliacrilamida , Epidermis/enzimología , Epidermis/patología , Humanos , Inmunohistoquímica , Queratinocitos/patología , Queratosis/enzimología , Queratosis/patología , Metaloproteinasa 9 de la Matriz , Pénfigo Familiar Benigno/enzimología , Pénfigo Familiar Benigno/fisiopatología , Dodecil Sulfato de Sodio , Inhibidores Tisulares de MetaloproteinasasAsunto(s)
Acantólisis/inducido químicamente , Erupciones por Medicamentos/etiología , Pénfigo/inducido químicamente , Compuestos de Sulfhidrilo/efectos adversos , Acantólisis/enzimología , Acantólisis/patología , Técnicas de Cultivo , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Humanos , Queratinocitos/enzimología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activadores Plasminogénicos/metabolismo , Piel/efectos de los fármacos , Piel/patología , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Pemphigus may be an idiopathic disease or a syndrome induced by drugs, mainly thiol drugs. Autoantibodies, always present in the idiopathic form but often lacking in the drug-induced one, may cause acantholysis by activating endogenous proteolytic enzymes. Pathogenesis of drug-induced pemphigus when antibodies are absent has not been elucidated yet. Extracts of skin tissues cultured for 4 days with penicillamine, captopril or thiopronine were assayed for the presence of plasminogen activator (PA) and plasminogen activator inhibitors (PAI) on agar fibrin plates. Moreover, uPA, tPA, and PAI-1 were identified in the extracts by immunoenzymatic assay. The results have shown progressively decreasing amounts of PAI-1 in penicillamine, thiopronine and captopril-cultured tissue extracts, respectively. This suggests that the acantholytic potential of thiol drugs is directly correlated to their capability of reducing PAI-1 in the epidermal cells leading to increased PA activity. This PA-PAI imbalance may be itself the cause of intraepidermal splitting.
Asunto(s)
Acantólisis/inducido químicamente , Acantólisis/metabolismo , Activadores Plasminogénicos/análisis , Inactivadores Plasminogénicos/análisis , Compuestos de Sulfhidrilo/efectos adversos , Acantólisis/enzimología , Acantólisis/patología , Captopril/farmacología , Medios de Cultivo , Técnicas de Cultivo , Epidermis/enzimología , Epidermis/patología , Humanos , Penicilamina/farmacología , Inhibidor 1 de Activador Plasminogénico/análisis , Activadores Plasminogénicos/metabolismo , Inactivadores Plasminogénicos/metabolismo , Tiopronina/farmacología , Activador de Tejido Plasminógeno/análisis , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/análisis , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresAsunto(s)
Pénfigo , Acantólisis/enzimología , Corticoesteroides/uso terapéutico , Erupciones por Medicamentos/etiología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Mucosa Bucal/patología , Pénfigo/inducido químicamente , Pénfigo/tratamiento farmacológico , Pénfigo/inmunología , Pénfigo/patologíaRESUMEN
Using a method which allowed us to study the morphological consequences of the expression and the inhibition of proteases in living tissues, we demonstrated that the primary detectable cellular event in cantharide acantholysis is the dissolution of the dense plaque, leading to the detachment of tonofilaments from desmosomes. This process is inhibited by neutral serine protease inhibitors. This suggests that the desmosome-tonofilament complex, more precisely the desmosomal dense plaque, is the primary target of activated proteases during cantharide acantholysis, and can be disrupted by a specific epidermal protease-anti protease system. Cantharide acantholysis may be useful model for studying desmosomal turnover.