RESUMEN
Keloids are wounding-induced fibroproliferative human tumor-like skin scars of complex genetic makeup and poorly defined pathogenesis. To reveal dynamic epigenetic and transcriptome changes of keloid fibroblasts, we performed RNA-seq and ATAC-seq analysis on an early passage keloid fibroblast cell strain and its paired normal control fibroblasts. This keloid strain produced keloid-like scars in a plasma clot-based skin equivalent humanized keloid animal model. RNA-seq analysis reveals gene ontology terms including hepatic fibrosis, Wnt-ß-catenin, TGF-ß, regulation of epithelial-mesenchymal transition (EMT), STAT3 and adherens junction. ATAC-seq analysis suggests STAT3 signalling is the most significantly enriched gene ontology term in keloid fibroblasts, followed by Wnt signalling (Wnt5) and regulation of the EMT pathway. Immunohistochemistry confirms that STAT3 (Tyr705 phospho-STAT3) is activated and ß-catenin is up-regulated in the dermis of keloid clinical specimens and keloid skin equivalent implants from the humanized mouse model. A non-linear dose-response of cucurbitacin I, a selective JAK2/STAT3 inhibitor, in collagen type I expression of keloid-derived plasma clot-based skin equivalents implicates a likely role of STAT3 signalling in keloid pathogenesis. This work also demonstrates the utility of the recently established humanized keloid mouse model in exploring the mechanism of keloid formation.
Asunto(s)
Queloide/etiología , Queloide/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Humanos , Ratones , Transcriptoma , Vía de Señalización WntRESUMEN
Keloids are wounding-induced tumor-like human scars. Unclear etiology and lack of animal models to reveal disease mechanisms and invent therapies deepen the grievous health and psychosocial state of vulnerable individuals. Epitomizing the injury-repair environment which triggers and fosters keloid formation and essential dermal/epidermal interactions in disease development, the novel animal model was established by implanting porous polyethylene ring-supported plasma/fibrin-based epidermal-dermal skin constructs on the dorsum of athymic NU/J mice. The implants were stable to 18 weeks, contained abundant human cells, and remodeled to yield scar architecture characteristic of keloid fibrosis compared with normal implants and clinical specimens: (1) macroscopic convex or nodular scar morphology; (2) morphogenesis and accumulation of large collagen bundles from collagen-null initial constructs; (3) epidermal hyperplasia, aberrant epidermal-dermal patency, and features of EMT; (4) increased vasculature, macrophage influx, and aggregation; and (5) temporal-spatial increased collagen-inducing PAI-1 and its interactive partner uPAR expression. Development of such pathology in the NU/J host suggests that T-cell participation is less important at this stage than at keloid initiation. These accessible implants also healed secondary excisional wounds, enabling clinically relevant contemporaneous wounding and treatment strategies, and evaluation. The model provides a robust platform for studying keloid formation and testing knowledge-based therapies.
Asunto(s)
Dermis/citología , Células Epidérmicas , Fibroblastos/citología , Fibrosis/patología , Queloide/patología , Cicatrización de Heridas/fisiología , Animales , Células Cultivadas , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Fibrina/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Trasplante HeterólogoRESUMEN
Wound healing is the inherent ability of an organism to protect itself against injuries. Cumulative evidence indicates that the healing process patterns in part embryonic morphogenesis and may result in either organ regeneration or scarring, phenomena that are developmental stage- or age-dependent. Skin is the largest organ. Its morphogenesis and repair mechanisms have been studied extensively due not only to its anatomical location, which allows easy access and observation, but also to its captivating structure and vital function. Thus, this review will focus on using skin as a model organ to illustrate new insights into the mechanisms of wound healing that are developmentally regulated in mammals, with special emphasis on the role of the Wnt signaling pathway and its crosstalk with TGF-ß signaling. Relevant information from studies of other organs is discussed where it applies, and the clinical impact from such knowledge and emerging concepts on regenerative medicine are discussed in perspective.
Asunto(s)
Mamíferos/crecimiento & desarrollo , Morfogénesis/fisiología , Medicina Regenerativa/métodos , Fenómenos Fisiológicos de la Piel , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/fisiología , Cicatrización de Heridas/fisiología , Factores de Edad , Animales , Humanos , Miofibroblastos/fisiología , Especificidad de la Especie , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The molecular mechanisms coordinating cell cycle exit with cell differentiation and organogenesis are a crucial, yet poorly understood, aspect of normal development. The mammalian cyclin-dependent kinase inhibitor p27(Kip1) is required for the correct timing of cell cycle exit in developing tissues, and thus plays a crucial role in this process. Although studies of p27(Kip1) regulation have revealed important posttranscriptional mechanisms regulating p27(Kip1) abundance, little is known about how developmental patterns of p27(Kip1) expression, and thus cell cycle exit, are achieved. Here, we show that during inner ear development transcriptional regulation of p27(Kip1) is the primary determinant of a wave of cell cycle exit that dictates the number of postmitotic progenitors destined to give rise to the hair cells and supporting cells of the organ of Corti. Interestingly, transcriptional induction from the p27(Kip1) gene occurs normally in p27(Kip1)-null mice, indicating that developmental regulation of p27(Kip1) transcription is independent of the timing of cell cycle exit. In addition, cell-type-specific patterns of p27(Kip1) transcriptional regulation are observed in the mature organ of Corti and retina, suggesting that this mechanism is important in differential regulation of the postmitotic state. This report establishes a link between the spatial and temporal pattern of p27(Kip1) transcription and the control of cell number during sensory organ morphogenesis.
Asunto(s)
Ciclo Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Órgano Espiral/embriología , Transcripción Genética , Animales , Secuencia de Bases , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Cartilla de ADN , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Ratones Transgénicos , Órgano Espiral/citología , Reacción en Cadena de la PolimerasaRESUMEN
Sensory hair cells of the mammalian organ of Corti in the inner ear do not regenerate when lost as a consequence of injury, disease, or age-related deafness. This contrasts with other vertebrates such as birds, where the death of hair cells causes surrounding supporting cells to re-enter the cell cycle and give rise to both new hair cells and supporting cells. It is not clear whether the lack of mammalian hair cell regeneration is due to an intrinsic inability of supporting cells to divide and differentiate or to an absence or blockade of regenerative signals. Here we show that post-mitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, we show that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate the cyclin-dependent kinase inhibitor p27(Kip1) (also known as Cdkn1b). These results indicate that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.
Asunto(s)
Diferenciación Celular , Cóclea/citología , Células Ciliadas Auditivas Internas/citología , Animales , Ciclo Celular , Células Cultivadas , Técnicas de Cocultivo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Citometría de Flujo , Ratones , Órgano Espiral/citología , Receptor de Factor de Crecimiento Nervioso/biosíntesisRESUMEN
Expression of the cyclin-dependent kinase inhibitor p27(Kip1) defines a post-mitotic population of cells in the embryonic mammalian cochlea that constitutes the nascent organ of Corti. Here, we describe techniques to purify these precursors using a transgenic p27/GFP reporter and fluorescence activated cell sorting (FACS). We demonstrate that these cells express other markers of the sensory lineage, such as Sox2, and when placed in dissociated cell culture differentiate as hair cells and supporting cells. The purified sensory progenitors thus obtained provide a means of studying the process of hair cell and supporting cell differentiation in vitro, as well as providing a means of analyzing the molecular and physiological properties of this unique population of cells.