RESUMO
The mechanisms by which catenins regulate cadherin function are not fully understood, and the precise function of p120 catenin (p120ctn) has remained particularly elusive. In microvascular endothelial cells, p120ctn colocalized extensively with cell surface VE-cadherin, but failed to colocalize with VE-cadherin that had entered intracellular degradative compartments. To test the possibility that p120ctn binding to VE-cadherin regulates VE-cadherin internalization, a series of approaches were undertaken to manipulate p120ctn availability to endogenous VE-cadherin. Expression of VE-cadherin mutants that competed for p120ctn binding triggered the degradation of endogenous VE-cadherin. Similarly, reducing levels of p120ctn using siRNA caused a dramatic and dose-related reduction in cellular levels of VE-cadherin. In contrast, overexpression of p120ctn increased VE-cadherin cell surface levels and inhibited entry of cell surface VE-cadherin into degradative compartments. These results demonstrate that cellular levels of p120ctn function as a set point mechanism that regulates cadherin expression levels, and that a major function of p120ctn is to control cadherin internalization and degradation.
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Adesão Celular/genética , Endotélio Vascular/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico/genética , Antígenos CD , Caderinas/genética , Cateninas , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Linhagem Celular , Endocitose/genética , Retroalimentação Fisiológica/genética , Humanos , Mutação/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Interferente Pequeno/farmacologia , delta CateninaRESUMO
Human papillomavirus infection is one of the most common and most distressing cutaneous diseases in patients with HIV infection. It is also a common, and often therapeutically challenging, infection in individuals who are immunologically competent. A wide range of therapeutic options exists for treating cutaneous human papillomavirus infections, but none is uniformly effective. In this report we describe a man with HIV-1 infection and disfiguring facial verruca vulgaris who demonstrated complete clinical response to intravenous cidofovir. Our report provides further support for the use of intravenous cidofovir as therapy for treatment-resistant and/or widespread cutaneous human papillomavirus infection.
Assuntos
Antivirais/administração & dosagem , Citosina/análogos & derivados , Organofosfonatos/administração & dosagem , Infecções por Papillomavirus/tratamento farmacológico , Dermatopatias Virais/tratamento farmacológico , Adulto , Antivirais/uso terapêutico , Cidofovir , Citosina/administração & dosagem , Citosina/uso terapêutico , Humanos , Injeções Intravenosas , Masculino , Organofosfonatos/uso terapêutico , Infecções por Papillomavirus/patologia , Dermatopatias Virais/patologia , Resultado do TratamentoRESUMO
Pemphigus vulgaris (PV) is an epidermal blistering disorder caused by antibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). The mechanism by which PV IgG disrupts adhesion is not fully understood. To address this issue, primary human keratinocytes (KCs) and patient IgG were used to define the morphological, biochemical, and functional changes triggered by PV IgG. Three phases of desmosome disassembly were distinguished. Analysis of fixed and living KCs demonstrated that PV IgG cause rapid Dsg3 internalization, which likely originates from a non-junctional pool of Dsg3. Subsequently, Dsg3 and other desmosomal components rearrange into linear arrays that run perpendicular to cell contacts. Dsg3 complexes localized at the cell surface are transported in a retrograde manner along with these arrays before being released into cytoplasmic vesicular compartments. These changes in Dsg3 distribution are followed by depletion of detergent-insoluble Dsg3 pools and by the loss of cell adhesion strength. Importantly, this process of disassembly can be prevented by expressing exogenous Dsg3, thereby driving Dsg3 biosynthesis and desmosome assembly. These data support a model in which PV IgG cause the loss of cell adhesion by altering the dynamics of Dsg3 assembly into desmosomes and the turnover of cell surface pools of Dsg3 through endocytic pathways.
Assuntos
Desmogleína 3/farmacologia , Desmossomos/efeitos dos fármacos , Imunoglobulina G/farmacologia , Queratinócitos/efeitos dos fármacos , Pênfigo/imunologia , Anticorpos/sangue , Anticorpos/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Desmogleína 3/metabolismo , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Endocitose/efeitos dos fármacos , Humanos , Imunoglobulina G/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Microscopia Imunoeletrônica , Pênfigo/sangueRESUMO
Pemphigus vulgaris (PV) is a life-threatening autoimmune disease characterized by oral mucosal erosions and epidermal blistering. The autoantibodies generated target the desmosomal cadherin desmoglein-3 (Dsg3). Previous studies demonstrate that upon PV IgG binding, Dsg3 is internalized and enters an endo-lysosomal pathway where it is degraded. To define the endocytic machinery involved in PV IgG-induced Dsg3 internalization, human keratinocytes were incubated with PV IgG, and various tools were used to perturb distinct endocytic pathways. The PV IgG.Dsg3 complex failed to colocalize with clathrin, and inhibitors of clathrin- and dynamin-dependent pathways had little or no effect on Dsg3 internalization. In contrast, cholesterol binding agents such as filipin and nystatin and the tyrosine kinase inhibitor genistein dramatically inhibited Dsg3 internalization. Furthermore, the Dsg3 cytoplasmic tail specified sensitivity to these inhibitors. Moreover, inhibition of Dsg3 endocytosis with genistein prevented disruption of desmosomes and loss of adhesion in the presence of PV IgG. Altogether, these results suggest that PV IgG-induced Dsg3 internalization is mediated through a clathrin- and dynamin-independent pathway and that Dsg3 endocytosis is tightly coupled to the pathogenic activity of PV IgG.
Assuntos
Clatrina/química , Desmogleína 3/química , Dinaminas/química , Imunoglobulina G/química , Pênfigo/imunologia , Adesão Celular , Células Cultivadas , Citoplasma/metabolismo , Endocitose , Regulação da Expressão Gênica , Genisteína/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Ligação Proteica , Proteínas Tirosina Quinases/metabolismoRESUMO
Human skin diseases have revealed fundamental mechanisms by which cytoskeletal proteins contribute to tissue architecture and function. In particular, the analysis of epidermal blistering disorders and the role of keratin gene mutations in these diseases has led to significant increases in our understanding of intermediate filament biology. The major cell-surface attachment site for intermediate filament networks is the desmosome, an adhesive intercellular junction prominent in the epidermis and the heart. During the past decade, substantial progress has been made in understanding the molecular basis of a variety of epidermal autoimmune diseases, skin fragility syndromes, and disorders that involve a combination of heart and skin defects caused by perturbations in desmosome structure and function. These human diseases reveal key roles for desmosomes in maintaining tissue integrity, but also suggest functions for desmosomal components in signal transduction pathways and epidermal organization.
Assuntos
Desmossomos , Dermatopatias , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Caderinas de Desmossomos/imunologia , Caderinas de Desmossomos/metabolismo , Desmossomos/química , Desmossomos/imunologia , Desmossomos/patologia , Humanos , Dermatopatias/imunologia , Dermatopatias/patologiaRESUMO
VE-cadherin is an endothelial-specific cadherin that plays important roles in vascular morphogenesis and growth control. To investigate the mechanisms by which endothelial cells regulate cadherin cell surface levels, a VE-cadherin mutant containing the non-adhesive interleukin-2 (IL-2) receptor extracellular domain and the VE-cadherin cytoplasmic tail (IL-2R-VE-cadcyto) was expressed in microvascular endothelial cells. Expression of the IL-2R-VE-cadcyto mutant resulted in the internalization of endogenous VE-cadherin and in a dramatic decrease in endogenous VE-cadherin levels. The internalized VE-cadherin co-localized with early endosomes, and the lysosomal inhibitor chloroquine dramatically inhibited the down-regulation of VE-cadherin in cells expressing the IL-2R-VE-cadcyto mutant. Chloroquine treatment also resulted in the accumulation of a VE-cadherin fragment lacking the beta-catenin binding domain of the VE-cadherin cytoplasmic tail. The formation of the VE-cadherin fragment could be prevented by treating endothelial cells with proteasome inhibitors. Furthermore, inhibition of the proteasome prevented VE-cadherin internalization and inhibited the disruption of endothelial intercellular junctions by the IL-2RVE-cadcyto mutant. These results provide new insights into the mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells.