RESUMEN
Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB), a skin fragility disorder that, unexpectedly, manifests also with elevated colonization of commensal bacteria and frequent wound infections. Here, we describe an unprecedented systemic function of collagen VII as a member of a unique innate immune-supporting multiprotein complex in spleen and lymph nodes. In this complex, collagen VII specifically binds and sequesters the innate immune activator cochlin in the lumen of lymphoid conduits. In genetic mouse models, loss of collagen VII increased bacterial colonization by diminishing levels of circulating cochlin LCCL domain. Intraperitoneal injection of collagen VII, which restored cochlin in the spleen, but not in the skin, reactivated peripheral innate immune cells via cochlin and reduced bacterial skin colonization. Systemic administration of the cochlin LCCL domain was alone sufficient to diminish bacterial supercolonization of RDEB mouse skin. Human validation demonstrated that RDEB patients displayed lower levels of systemic cochlin LCCL domain with subsequently impaired macrophage response in infected wounds. This study identifies an intrinsic innate immune dysfunction in RDEB and uncovers a unique role of the lymphoid extracellular matrix in systemic defense against bacteria.
Asunto(s)
Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/inmunología , Proteínas de la Matriz Extracelular/metabolismo , Inmunidad Innata , Tejido Linfoide/metabolismo , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/inmunología , Humanos , Ratones Noqueados , Piel/microbiologíaRESUMEN
Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Polaridad Celular , Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos CD , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Técnicas de Cocultivo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Queratinocitos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Trasplante de Neoplasias , Transportadores de Anión Orgánico Sodio-Independiente/genética , Regiones Promotoras Genéticas , Transporte de Proteínas , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Transcripción Genética , Células Tumorales Cultivadas , beta Catenina/genética , beta Catenina/metabolismo , KalininaRESUMEN
The replacement of a defective gene with a fully functional copy is the goal of the most basic gene therapy. Recessive dystrophic epidermolysis bullosa (RDEB) is characterised by a lack of adhesion of the epidermis to the dermis. It is an ideal target for gene therapy as all variants of hereditary RDEB are caused by mutations in a single gene, COL7A1, coding for type VII collagen, a key component of anchoring fibrils that secure attachment of the epidermis to the dermis. RDEB is one of the most severe variants in the epidermolysis bullosa (EB) group of heritable skin diseases. Epidermolysis bullosa is defined by chronic fragility and blistering of the skin and mucous membranes due to mutations in the genes responsible for production of the basement membrane proteins. This condition has a high personal, medical and socio-economic impact. People with RDEB require a broad spectrum of medications and specialised care. Due to this being a systemic condition, most research focus is in the area of gene therapy. Recently, preclinical works have begun to show promise. They focus on the virally mediated ex vivo correction of autologous epithelium. These corrected cells are then to be expanded and grafted onto the patient following the lead of the first successful gene therapy in dermatology being a grafting of corrected tissue for junctional EB treatment. Current progress, outstanding challenges and future directions in translating these approaches in clinics are reviewed in this article.
Asunto(s)
Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/terapia , Terapia Genética/métodos , Adhesividad , Animales , Autoinjertos , Colágeno Tipo VII/deficiencia , Colágeno Tipo VII/genética , Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/fisiopatología , Genes Recesivos , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/trasplante , RatonesRESUMEN
Dominant-negative interference by glycine substitution mutations in the COL7A1 gene causes dominant dystrophic epidermolysis bullosa (DDEB), a skin fragility disorder with mechanically induced blistering. Although qualitative and quantitative alterations of the COL7A1 gene product, collagen VII, underlie DDEB, the lack of direct correlation between mutations and the clinical phenotype has rendered DDEB less amenable to therapeutic targeting. To delineate the molecular mechanisms of DDEB, we used recombinant expression of wild-type (WT) and mutant collagen VII, which contained a naturally occurring COL7A1 mutation, G1776R, G2006D, or G2015E, for characterization of the triple helical molecules. The mutants were co-expressed with WT in equal amounts and could form heterotrimeric hybrid triple helices, as demonstrated by affinity purification and mass spectrometry. The thermal stability of the mutant molecules was strongly decreased, as evident in their sensitivity to trypsin digestion. The helix-to-coil transition, T(m), of the mutant molecules was 31-34 degrees C, and of WT collagen VII 41 degrees C. Co-expression of WT with G1776R- or G2006D-collagen VII resulted in partial intracellular retention of the collagen, and mutant collagen VII had reduced ability to support cell adhesion. Intriguingly, controlled overexpression of WT collagen VII gradually improved the thermal stability of the collective of collagen VII molecules. Co-expression in a ratio of 90% WT:10% mutant increased the T(m) to 41 degrees C for G1776R-collagen VII and to 39 degrees C for G2006D- and G2015E-collagen VII. Therefore, increasing the expression of WT collagen VII in the skin of patients with DDEB can be considered a valid therapeutic approach.
Asunto(s)
Colágeno Tipo VII/genética , Proteínas Mutantes/fisiología , Mutación Missense , Adhesión Celular , Línea Celular , Colágeno Tipo VII/química , Colágeno Tipo VII/fisiología , Humanos , Proteínas Mutantes/química , Conformación Proteica , Estabilidad Proteica , Piel/química , Temperatura de TransiciónAsunto(s)
Carcinoma de Células Escamosas/fisiopatología , Colágeno Tipo VII/genética , Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/genética , Queratinocitos/metabolismo , Neoplasias Cutáneas/fisiopatología , Animales , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Moléculas de Adhesión Celular/metabolismo , Transformación Celular Neoplásica , Colágeno Tipo VII/química , Susceptibilidad a Enfermedades , Epidermólisis Ampollosa Distrófica/complicaciones , Epidermólisis Ampollosa Distrófica/metabolismo , Epidermólisis Ampollosa Distrófica/patología , Genes ras , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Queratinocitos/patología , Ratones , Mutación , Inhibidor NF-kappaB alfa , Invasividad Neoplásica , Estructura Terciaria de Proteína , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Transducción Genética , Factor de Crecimiento Transformador beta/metabolismo , KalininaRESUMEN
Type VII collagen defects cause recessive dystrophic epidermolysis bullosa (RDEB), a blistering skin disorder often accompanied by epidermal cancers. To study the role of collagen VII in these cancers, we examined Ras-driven tumorigenesis in RDEB keratinocytes. Cells devoid of collagen VII did not form tumors in mice, whereas those retaining a specific collagen VII fragment (the amino-terminal noncollagenous domain NC1) were tumorigenic. Forced NC1 expression restored tumorigenicity to collagen VII-null epidermis in a non-cell-autonomous fashion. Fibronectin-like sequences within NC1 (FNC1) promoted tumor cell invasion in a laminin 5-dependent manner and were required for tumorigenesis. Tumor-stroma interactions mediated by collagen VII thus promote neoplasia, and retention of NC1 sequences in a subset of RDEB patients may contribute to their increased susceptibility to squamous cell carcinoma.
Asunto(s)
Carcinoma de Células Escamosas/fisiopatología , Colágeno Tipo VII/genética , Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/genética , Genes ras , Queratinocitos/metabolismo , Neoplasias Cutáneas/fisiopatología , Adolescente , Adulto , Animales , Anticuerpos/inmunología , Apoptosis , Carcinoma de Células Escamosas/etiología , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica , Niño , Colágeno Tipo VII/química , Colágeno Tipo VII/inmunología , Susceptibilidad a Enfermedades , Epidermólisis Ampollosa Distrófica/complicaciones , Epidermólisis Ampollosa Distrófica/metabolismo , Epidermólisis Ampollosa Distrófica/patología , Femenino , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Mutación , Inhibidor NF-kappaB alfa , Invasividad Neoplásica , Estructura Terciaria de Proteína , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , KalininaRESUMEN
Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders that are caused by mutations in the type VII collagen gene and for which ex vivo gene therapy has been considered. To develop a simpler approach for treating DEB, we evaluated the feasibility of protein-based therapy by intradermally injecting human recombinant type VII collagen into mouse skin and a DEB human skin equivalent transplanted onto mice. The injected collagen localized to the basement membrane zone of both types of tissues, was organized into human anchoring fibril structures and reversed the features of DEB disease in the DEB skin equivalent.
Asunto(s)
Colágeno Tipo VII/administración & dosificación , Epidermólisis Ampollosa Distrófica/tratamiento farmacológico , Animales , Colágeno Tipo VII/genética , Colágeno Tipo VII/fisiología , Humanos , Ratones , Ratones Pelados , Proteínas Recombinantes/administración & dosificaciónRESUMEN
Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders caused by mutations in the human type VII collagen gene (COL7A1). Individuals with DEB lack type VII collagen and anchoring fibrils, structures that attach epidermis and dermis. The current lack of treatment for DEB is an impetus to develop gene therapy strategies that efficiently transfer and stably express genes delivered to skin cells in vivo. In this study, we delivered and expressed full-length type VII collagen using a self-inactivating minimal lentivirus-based vector. Transduction of lentiviral vectors containing the COL7A1 transgene into recessive DEB (RDEB) keratinocytes and fibroblasts (in which type VII collagen was absent) resulted in persistent synthesis and secretion of type VII collagen. Unlike RDEB parent cells, the gene-corrected cells had normal morphology, proliferative potential, matrix attachment and motility. We used these gene-corrected cells to regenerate human skin on immune-deficient mice. Human skin regenerated by gene-corrected RDEB cells had restored expression of type VII collagen and formation of anchoring fibrils at the dermal-epidermal junction in vivo. These studies demonstrate that it is possible to restore type VII collagen gene expression in RDEB skin in vivo.